Bangladesh Journal of Pharmacology Volume: 20; Number 3; Year 2025 Cite this article as: Gaikwad RG, Shinde AJ, Nadaf SJ. Antiproliferative activity of Tribulus terrestris stem bark extract against A431 cells. Bangladesh J Pharmacol. 2025; 20: 131-33. Antiproliferative activity of Tribulus terrestris stem bark extract against A431 cells Dear Editor, Human epidermoid carcinoma can cause tissue da- mage, metastasis, and destruction of lymph nodes. To treat this, radiation therapy, surgery, chemotherapy, and targeted delivery of EGFR inhibitors have been utilized (Johnston et al., 2006). However, these treat- ments may lead to severe side effects such as skin damage, fatigue, intense pain, and immune suppression (Zheng et al., 2017). Preclinical, in silico, and clinical studies corroborate the usefulness of medicinal plants in managing cancers with fewer adverse effects (Gaikwad and Shinde, 2022). Tribulus terrestris has been utilized in ancient systems, such as Ayurveda and Unani, to treat a variety of dis- eases, including reproductive and urinary system issues (Amanullah et al., 2021). Research on Tribulus terestris has confirmed the presence of diverse phytochemicals, including steroidal saponins, protodioscin, flavonoids, alkaloids, and polyphenolic compounds, thereby vali- dating its therapeutic potential (Chhatre et al., 2014). These phytoconstituents are known to exert their effects by interacting with cell signaling pathways, inhibiting cell proliferation, and inducing apoptosis. Notably, Tribulus terestris exhibits lesser toxicity to normal skin fibroblast cells, confirming its selectivity (Neychev et al., 2022). Previous research validates the cytotoxic potential of T. terestris on MCF-7 cells. This could be attributed to the presence of diverse secondary meta- bolites (Alshabi et al. 2022). In another study, the stem bark extract of T. terestris demonstrated cytotoxic effects on prostate cancer cells (Dabaghkar et al., 2025). Furthermore, the cytotoxic, antiproliferative, and apoptotic effects of the hydroalcoholic extract of the fruit of T. terestris have been reported on different cell lines, such as prostate, colon and fibroblast-like cells (Pourali et al. 2017). The antiglycation, antioxidant, and antiproliferative potential of saponin-rich plant extracts of T. terestris have also been observed against human tumor cell lines (Figueiredo et al. 2021). A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2025; 20: 131-133 Journal homepage: www.banglajol.info; www.bdpsjournal.org Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088; DOI: 10.3329/bjp.v20i3.83249 Letter to the Editor This work is licensed under a Creative Commons Attribution 4.0 International License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor A431 Fluorouracil A431 Control T. terestris 50 100 150 200 250 µg/mL C B A Figure 1: Comparative cell viability of A431 cells on treatment with 5-fluorouracil (standard), untreated control, and T. terrestris stem bark methanolic extract. Bar graph demonstrating comparative cell viability inhibition 50 40 30 20 10 0 % In h ib it io n Concentration (µg/mL) Fluorouracil T. terestris D Thus, in line with the previous reports, we investigated the cytotoxic potential of the methanolic stem bark extract of T. terestris against A431 human epidermoid carcinoma cells. Cytotoxicity was evaluated using the MTT assay (Bahuguna et al., 2017). Cells were seeded at a density of 1 × 10⁴ cells per well in 96-well plates and incubated at 37°C with 5% CO₂ for 24 hours. After 24 hours of treatment with different extract concentrations, each well received 20 µL of MTT reagent (5 mg/mL). After 4-hours incubition period, formazan crystals generated by live cells were dissolved in DMSO, and the absorbance was measured at 570 nm. IC₅₀ value was determined from the dose-response curve (Gai-kwad and Shinde, 2022). As shown in Figure 1, the stem bark methanolic extract of T. terrestris inhibited the viability of 35.26 ± 0.95% A431 cells, corroborating the cytotoxic effect following a 24-hours treatment at the tested dose. In comparison, the standard 5-fluorouracil inhibited the viability of 46.15 ± 0.95% cells. These results indicate that the methanolic extract of the stem bark of T. terrestris could be used as a viable alternative to standard chemothe- rapy, even though the extract exhibited a marginally lesser effect than the standard drug. The moderate cytotoxicity of T. terrestris extract showed increased interest in plant-based treatments. The secon- dary metabolites present in the plant T. terrestris may offer tailored, multi-modal responses with fewer adver- se effects than traditional chemotherapeutics, which fre- quently have wide cytotoxic effects that result in sys- temic toxicity. This has prompted scientists to think about these substances' potential as chemopreventive or adjunctive treatments. Using MTT-based assessments and molecular docking, recent studies have shown the anti-cancer potential of medicinal plant extracts, such Terminalia citrine (Das et al., 2016). The use of A431 cells in our work is relevant given the rising prevalence of skin malignancies other than mela- noma and the shortcomings of existing treatments. Research on natural medicines is appealing, since there is a demand for safer and more economical alternatives. Given its ethnobotanical basis, T. terrestris is a strong contender for more preclinical and clinical investiga- tions. In summary, the methanolic extract of the stem bark of T. terrestris demonstrated moderate antiproliferative activity against A431 human epidermoid carcinoma cells. The consistency of these results with prior literature, coupled with the known phytochemical profile of the plant, suggests its strong potential as a source of anti-cancer agents. We recommend further fractionation, isolation of active components, and mechanistic studies to fully explore the therapeutic benefits of T. terrestris. Conflict of interest: The authors declare that they have no conflicts of interest. Acknowledgments: The authors thankful to Bharati Vidya- peeth College of Pharmacy, Kolhapur for providing necessary facility to research Ravindra G. Gaikwad1, Anilkumar J. Shinde2, Sameer J. Nadaf3 1Department of Pharmaceutical Chemistry, Bharati Vidyapeeth College of Pharmacy, Palus 416310, India; 2Department of Pharmaceutics, Bharati Vidyapeeth College of Pharmacy, Kolhapur 416013, India; 3Department of Pharmaceutics, Bharati Vidyapeeth College of Phar- macy, Palus 416310, India. Corresponding authors: Sameer J. Nadaf, Anilkumar J. Shinde email: sam.nadaf@rediffmail.com; anilkumar.shinde@bharatividyapeeth.edu References Abbas MW, Hussain M, Akhtar S, Ismail T, Qamar M, Shafiq Z, Esatbeyoglu T. Bioactive compounds, antioxidant, anti- inflammatory, anti-cancer, and toxicity assessment of Tribulus terrestris: In vitro and in vivo studies. Antioxidants 2022; 11: 1160. Alshabi AM, Alkahtani SA, Shaikh IA, Orabi MA, Abdel- Wahab BA, Walbi IA, Habeeb MS, Khateeb MM, Shettar AK, Hoskeri JH. Tribulus terrestris cytotoxicity against breast cancer MCF-7 and lung cancer A549 cell lines is mediated via activation of apoptosis, caspase-3, DNA degradation, and suppressing Bcl-2 activity. Separations 2022; 9: 383. Amanullah KZ, Zeenat F, Ahmad W, Firoz S, Naeela A. A comprehensive review on a Unani medicinal plant: Tribulus terrestris Linn. Int J Herb Med. 2021; 9: 23-28. Bahuguna A, Khan I, Bajpai VK, Kang SC. MTT assay to evaluate the cytotoxic potential of a drug. Bangladesh J Pharmacol. 2017; 12: 115-18. Chhatre S, Nesari T, Somani G, Kanchan D, Sathaye S. Phyto- pharmacological overview of Tribulus terrestris. Phcog Rev. 2014; 8: 45. Dabaghkar Y, Eghlima G, Behboudi H, Ebrahimi M, Ghorban- pour M. Agro-morphological characterization and assess- ment of metabolic profiling and anticancer activities in various tribulus (Tribulus terrestris L.) populations. BMC Plant Biol. 2025; 25: 20. Das N, Goshwami D, Hasan MS, Al Mahmud Z, Raihan SZ. Evaluation of antioxidant, antimicrobial and cytotoxic acti- vities of Terminalia citrina leaves. J Pharm Res. 2016; 10: 8-15. Figueiredo CCM, Gomes AC, Granero FO, Bronzel Junior JL, Silva LP, Ruiz ALTG, da Silva RMG. Antiglycation and antitumoral activity of Tribulus terrestris dry extract. Avi- cenna J Phytomed. 2021; 11: 224-37. Gaikwad R, Shinde A. Antiproliferative effect of Memecylon malabaricum leaves methanolic extract against A-431 cell lines. Bangladesh J Pharmacol. 2022; 17: 54-55. Gaikwad RG, Shinde AJ, Hajare AA. Herbal treatment for management of psoriasis: An overview. RJPT. 2022; 15: 1385 -92. Johnston JB, Navaratnam S, Pitz MW, Maniate JM, Wiechec E, Baust H, Gingerich J, Skliris GP, Murphy LC, Los M. Targeting the EGFR pathway for cancer therapy. Curr Med Chem. 2006; 13: 3483-92. 132 Bangladesh J Pharmacol 2025; 20: 131-133 mailto:sam.nadaf@rediffmail.com mailto:anilkumar.shinde@bharatividyapeeth.edu Neychev VK, Nikolova E, Zhelev N, Mitev VI. Saponins from Tribulus terrestris L. are less toxic for normal human fibro- blasts than for many cancer lines: Influence on apoptosis and proliferation. EBM. 2007; 232: 126-33. Pourali M, Yaghoobi MM, Sormaghi MH. Cytotoxic, anti- proliferative and apoptotic effects of Tribulus terrestris L. fruit extract on human prostate cancer Lncap and colon cancer HT-29 Cell Lines. Jundishapur J Nat Pharm Prod. 2017; 1; 12: e33561. Zheng L, He H, Shen X, Sun Y. Centipede scolopendra suppre- sses cell growth in human epidermoid carcinoma cell A431. Bangladesh J Pharmacol. 2017; 12: 299-307. Bangladesh J Pharmacol 2025; 20: 131-133 133