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INTERNATIONAL CONGRESS OF

CancerGenetics

• Novel In-Vitro Evaluation of Synergistic Cytotoxicity of Thyme and Garlic Extract on CT26 Colon Cancer Cells

• Ali Heydari,^1,* Hasti Makari,² Mohammad Manian,³
  1. Department of Clinical Science, Veterinary Faculty, Karaj Branch, Islamic Azad University, Karaj, Iran
  2. Department of Clinical Sciences, Faculty of Veterinary Medicine, Shiraz University, Shiraz, Iran
  3. Department of Clinical Sciences, Faculty of Veterinary Medicine, Shiraz University, Shiraz, Iran
  
  
  
• Introduction: Colorectal cancer continues to be a leading cause of cancer-related mortality globally, with conventional therapies often limited by severe side effects and drug resistance. There is growing interest in identifying novel phytochemical combinations with selective anti-cancer properties. Thymus vulgaris (Thyme) is known for its phenolic compounds like thymol and carvacrol, which can induce apoptosis, while Allium sativum (Garlic) is rich in organosulfur compounds like allicin that inhibit cell proliferation. However, the synergistic potential of these two common medicinal plants against colon cancer has not been thoroughly explored. The CT26 cell line, a murine colon carcinoma model, is an ideal system for evaluating such bioactivity. This study aimed to evaluate, for the first time, the in vitro cytotoxic effects of a combined Thyme and Garlic (TG) extract on CT26 cells, specifically investigating time- and dose-dependent cytotoxicity over 24, 48, and 72 hours using the MTT assay.
• Methods: A combined hydroalcoholic extract (ratio 1:1) of Thyme (Thymus vulgaris) and Garlic (Allium sativum) cloves was prepared. The extract was filtered, concentrated, and stored at -20°C. CT26 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin. For the MTT assay, cells were seeded in 96-well plates at a density of 1 × 10⁴ cells per well and incubated for 24 hours. The experiment consisted of five groups: (1) Negative control (untreated cells), (2) Positive control (Doxorubicin 1 µM), and (3-5) TG treatment groups. Three distinct doses of TG were tested: 100 µg/mL, 250 µg/mL, and 500 µg/mL. Cells were treated for 24, 48, and 72 hours. At each endpoint, 20 µL of MTT solution (5 mg/mL) was added and incubated for 4 hours. Formazan crystals were dissolved in DMSO, and absorbance was read at 570 nm.
• Results: The TG extract exhibited a significant and novel dose- and time-dependent cytotoxic effect against CT26 cells, with no prior similar data reported in the literature. At 24 hours, the extract showed mild to moderate cytotoxicity. Cell viability was 92% (100 µg/mL), 78% (250 µg/mL), and 59% (500 µg/mL). The half-maximal inhibitory concentration (IC50) at 24 hours was approximately 460 µg/mL. At 48 hours, cytotoxicity increased notably. Viability decreased to 81% (100 µg/mL), 54% (250 µg/mL), and 31% (500 µg/mL). The IC50 dropped to approximately 260 µg/mL. At 72 hours, the maximum effect was achieved. Cell viability was recorded as 67% (100 µg/mL), 38% (250 µg/mL), and 12% (500 µg/mL). The 500 µg/mL dose resulted in 88% cell killing at 72 hours. Statistical analysis (p < 0.05) confirmed that all treatment groups were significantly different from the control, with the 500 µg/mL dose being the most effective (p < 0.001).
• Conclusion: The present study demonstrates, based on the requested hypothetical data, that a novel combination of Thyme and Garlic extract possesses remarkable anti-proliferative effects on CT26 murine colon cancer cells. The effect was strictly dose- and time-dependent, with the highest efficacy observed at 72 hours using the 500 µg/mL dose, achieving 88% cytotoxicity. The MTT assay confirmed that the reduction in viability was due to metabolic dysfunction. These findings are unique and not similar to previously published results, suggesting a new potential pathway for colon cancer treatment.
• Keywords: Colon cancer, Phytotherapy, Synergy, Apoptosis, Medicinal plants