Efficacy of Different Solvents in the Extraction of Bioactive Compounds and Anticancer Activities of Thyme (Thymus vulgaris L.) Leaves and Twigs
Thyme contains phytochemicals that exhibit cytotoxic and cytogenic activities. Differ solvents have different abilities in dissolving plant bioactive compounds. The objective of this study is to determine the anticancer properties of thyme leaves and twigs including profile of bioactive compounds, cytotoxicity, ability of apoptosis induction and caspase-3 activation of different solvent extracts on T47D breast cancer cell line. An experimental study was conducted in eight groups. T47D breast cancer cell line was treated with thyme extracts as treatment group, doxorubicin as positive control, while untreated T47D cells were used as negative control. Thyme was extracted by maceration using 6 different solvents: methanol, ethanol, chloroform, ethyl acetate, dichloromethane, and n-hexane. Bioactive compounds of all extracts were analyzed by GC-MS. Cytotoxicity was investigated through MTT assay on T47D breast cancer cell line. The percentage of apoptosis and caspase-3 were detected by flow cytometry using Annexin V-PI and BD Cytofix/Cytoperm, respectively. Thymol as the main bioactive compound in thyme was found in all extracts. MTT results revealed that six solvents extracts had moderate to weak cytotoxicity with dichloromethane had the lowest IC50 values at 120.23 µg/ml. All thyme extracts promoted apoptosis in T47D cells with apoptosis percentage of methanol, chloroform, ethyl acetate, ethanol, dichlorometane, n-Hexane and doxorubicin were 82.67; 80.98; 73.13; 72.28; 60.62; 27.74 and 98.43%, respectively. Among all extracts, methanol extract showed the highest apoptosis and caspase-3 activation percentage. We conclude that all thyme extracts had anticancer properties in T47D breast cancer cell line although efficacy of each solvent extract was differed.
Al-seragy, I. M. H., Kharat, K. R., & Dhabe, A. S. (2019). Cell Cycle Arrest and Induction of Apoptosis in Human Breast Cancer Cells (T-47D) by Annona squamosa L. and Thymus vulgaris L. Ethanolic Extract. Journal of Biologically Active Products from Nature, 9(1), 47–56.
Arul, S., Rajagopalan, H., Ravi, J., & Dayalan, H. (2020). Beta-Caryophyllene Suppresses Ovarian Cancer Proliferation by Inducing Cell Cycle Arrest and Apoptosis. Anti-Cancer Agents in Medicinal Chemistry, 20(13), 1530–1537.
Aydin, S., Başaran, A. A., & Başaran, N. (2005). The effects of thyme volatiles on the induction of DNA damage by the heterocyclic amine IQ and mitomycin C. Mutation Research - Genetic Toxicology and Environmental Mutagenesis, 581(1–2), 43–53.
Ayesh, B. M., Abed, A. A., & Faris, D. M. (2014). In vitro inhibition of human leukemia THP-1 cells by Origanum syriacum L. and Thymus vulgaris L. extracts. BMC Research Notes, 7(1), 1–6.
Bandala, C., Perez-Santos, J. L. M., Lara-Padilla, E., Lopez, M. G. D., & Anaya-Ruiz, M. (2013). Effect of Botulinum Toxin A on Proliferation and Apoptosis in the T47D Breast Cancer Cell Line. Asian Pacific Journal of Cancer Prevention, 14(2), 891–894.
Boice, A., & Bouchier-Hayes, L. (2020). Targeting apoptotic caspases in cancer. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1867(6), 118688.
Brahmi, F., Madani, K., Dahmoune, F., Tiziri, R., Karima, B., Oukhmanou-Bensidhoum, S., & Mohamed, C. (2012). Optimisation of Solvent Extraction of Antioxidants (Phenolic Compounds) From Algerian Mint (Mentha spicata L.). Pharmacognosy Communications, 2(4), 72–86.
Deb, D. D., Parimala, G., Saravana Devi, S., & Chakraborty, T. (2011). Effect of thymol on peripheral blood mononuclear cell PBMC and acute promyelotic cancer cell line HL-60. Chemico-Biological Interactions, 193(1), 97–106.
Fadholly, A., Nur, A., Ansori, M., Proboningrat, A., Patera Nugraha, A., Purnama, R., Iskandar, D., Rantam, F. A., & Sudjarwo, A. (2020). Apoptosis of HeLa Cells via Caspase-3 Expression Induced by Chitosan-Based Nanoparticles of Annona squamosa Leaf Extract: In vitro Study. Indian Journal of Pharmaceutical Education and Research, 54(2), 416–421.
Fathima, H. M., Gayathri, R., & Vishnupriya, V. (2017). Genotoxicity Potential of Thymus vulgaris (Thyme) on Oral Cancer Cell Line. International Journal of Pharmaceutical Sciences Review and Research, 43(2), 80–82.
Global Cancer Observatory. (2020). Retrieved August 7, 2022, from https://gco.iarc.fr/
Ilieva, Y., Dimitrova, L., Zaharieva, M. M., Kaleva, M., Alov, P., Tsakovska, I., Pencheva, T., Tibi, I. P. el, Najdenski, H., & Pajeva, I. (2021). Cytotoxicity and microbicidal activity of commonly used organic solvents: A comparative study and application to a standardized extract from vaccinium macrocarpon. Toxics, 9(5).
Islam, R., & Lam, K. W. (2020). Recent progress in small molecule agents for the targeted therapy of triple-negative breast cancer. European Journal of Medicinal Chemistry, 207, 112812.
Jan, R., & Chaudhry, G. e. S. (2019). Understanding Apoptosis and Apoptotic Pathways Targeted Cancer Therapeutics. Advanced Pharmaceutical Bulletin, 9(2), 205.
Jia, S. S., Xi, G. P., Zhang, M., Chen, Y. B., Lei, B., Dong, X. S., & Yang, Y. M. (2013). Induction of apoptosis by D-limonene is mediated by inactivation of Akt in LS174T human colon cancer cells. Oncology Reports, 29(1), 349–354.
Jung, Y. Y., Hwang, S. T., Sethi, G., Fan, L., Arfuso, F., & Ahn, K. S. (2018). Potential Anti-Inflammatory and Anti-Cancer Properties of Farnesol. Molecules, 23(11), 2827.
Kubatka, P., Uramova, S., Kello, M., Kajo, K., Samec, M., Jasek, K., Vybohova, D., Liskova, A., Mojzis, J., Adamkov, M., Zubor, P., Smejkal, K., Svajdlenka, E., Solar, P., Samuel, S. M., Zulli, A., Kassayova, M., Lasabova, Z., Kwon, T. K., … Büsselberg, D. (2019). Anticancer activities of Thymus vulgaris L. In experimental breast carcinoma in vivo and in vitro. International Journal of Molecular Sciences, 20(7).
Mahrye, Anwar, F., Mehmood, T., Qadir, R., & Riaz, M. (2022). Phenolics profiling and biological activities of different solvent extracts from aerial parts of wild thyme (Thymus vulgaris L.). Journal of Food Measurement and Characterization, 16(1), 610–618.
Majtnerová, P., & Roušar, T. (2018). An overview of apoptosis assays detecting DNA fragmentation. Molecular Biology Reports 2018, 45(5), 1469–1478.
Mishra, S., Verma, S. S., Rai, V., Awasthee, N., Arya, J. S., Maiti, K. K., & Gupta, S. C. (2019). Curcuma raktakanda Induces Apoptosis and Suppresses Migration in Cancer Cells: Role of Reactive Oxygen Species. Biomolecules, 9(4), 159.
Nur Syukriah, A., Liza, M., Harisun, Y., & Fadzillah, A. (2014). Effect of solvent extraction on antioxidant and antibacterial activities from Quercus infectoria (Manjakani). International Food Research Journal, 21(3), 1031–1037.
Park, K. R., Nam, D., Yun, H. M., Lee, S. G., Jang, H. J., Sethi, G., Cho, S. K., & Ahn, K. S. (2011). β-Caryophyllene oxide inhibits growth and induces apoptosis through the suppression of PI3K/AKT/mTOR/S6K1 pathways and ROS-mediated MAPKs activation. Cancer Letters, 312(2), 178–188.
Patil, S. M., Ramu, R., Shirahatti, P. S., Shivamallu, C., & Amachawadi, R. G. (2021). A systematic review on ethnopharmacology, phytochemistry and pharmacological aspects of Thymus vulgaris Linn. Heliyon, 7(5), e07054.
Prša, P., Karademir, B., Biçim, G., Mahmoud, H., Dahan, I., Yalçın, A. S., Mahajna, J., & Milisav, I. (2020). The potential use of natural products to negate hepatic, renal and neuronal toxicity induced by cancer therapeutics. Biochemical Pharmacology, 173, 113551.
Purnamasari, R., Winarni, D., Permanasari, A. A., Agustina, E., Hayaza, S., & Darmanto, W. (2019). Anticancer Activity of Methanol Extract of Ficus carica Leaves and Fruits Against Proliferation, Apoptosis, and Necrosis in Huh7it Cells. Cancer Informatics, 18.
Rababah, T. M., Banat, F., Rababah, A., Ereifej, K., & Yang, W. (2010). Optimization of Extraction Conditions of Total Phenolics, Antioxidant Activities, and Anthocyanin of Oregano, Thyme, Terebinth, and Pomegranate. Journal of Food Science, 75(7), C626–C632.
Rezaie, M., Farhoosh, R., Iranshahi, M., Sharif, A., & Golmohamadzadeh, S. (2015). Ultrasonic-assisted extraction of antioxidative compounds from Bene (Pistacia atlantica subsp. mutica) hull using various solvents of different physicochemical properties. Food Chemistry, 173, 577–583.
Roby, M. H. H., Sarhan, M. A., Selim, K. A. H., & Khalel, K. I. (2013). Evaluation of antioxidant activity, total phenols and phenolic compounds in thyme (Thymus vulgaris L.), sage (Salvia officinalis L.), and marjoram (Origanum majorana L.) extracts. Industrial Crops and Products, 43(1), 827–831.
Salehi, B., Mishra, A. P., Shukla, I., Sharifi-Rad, M., Contreras, M. del M., Segura-Carretero, A., Fathi, H., Nasrabadi, N. N., Kobarfard, F., & Sharifi-Rad, J. (2018). Thymol, thyme, and other plant sources: Health and potential uses. Phytotherapy Research, 32(9), 1688–1706.
Salmani, A., Kosari, A. A., Pirouzi, A., Omidi, M., & Mohsenzadeh, M. (2015). Protective effect of methanolic extracts of Thymus vulgaris L. against cy- clophosphamide-induced DNA damage in mouse bone marrow cells us- ing the micronucleus test. Trends in Pharmaceutical Sciences, 1(4), 243–250.
Sung, B., Chung, H. S., Kim, M., Kang, Y. J., Kim, D. H., Hwang, S. Y., Kim, M. J., Kim, C. M., Chung, H. Y., & Kim, N. D. (2015). Cytotoxic effects of Solvent-Extracted active components of Salvia miltiorrhiza Bunge on human cancer cell lines. Experimental and Therapeutic Medicine, 9(4), 1421–1428.
Taghouti, M., Martins-Gomes, C., Félix, L. M., Schäfer, J., Santos, J. A., Bunzel, M., Nunes, F. M., & Silva, A. M. (2020). Polyphenol composition and biological activity of Thymus citriodorus and Thymus vulgaris: Comparison with endemic Iberian Thymus species. Food Chemistry, 331, 127362.
Tuță-sas, I., Proks, M., Păunescu, V., Pînzaru, I., Sas, I., Coricovac, D., Moacă, A., & Dehelean, C. (2019). Thymus vulgaris Extract Formulated as Cyclodextrin Complexes: Synthesis, Characterization, Antioxidant Activity And In Vitro Cytotoxicity Assessment. Farmacia, 67, 3.
Villanueva Bermejo, D., Angelov, I., Vicente, G., Stateva, R. P., Rodriguez García-Risco, M., Reglero, G., Ibañez, E., & Fornari, T. (2015). Extraction of thymol from different varieties of thyme plants using green solvents. Journal of the Science of Food and Agriculture, 95(14), 2901–2907.
Widiyastuti, Y., Yanti, I., Sholikhah, M., & Haryanti, S. (2019). Cytotoxic activities of ethanolic and dichloromethane extract of leaves, stems, and flowers of Jarong [Stachytarpheta jamaicensis (L.) Vahl.] on HeLa and T47D cancer cell line. AIP Conference Proceedings, 2202(020101), 1–6.
World Health Organization. (2021). Retrieved August 7, 2022, from https://www.who.int/news-room/fact-sheets/detail/breast-cancer
Yu, X., Lin, H., Wang, Y., Lv, W., Zhang, S., Qian, Y., Deng, X., Feng, N., Yu, H., & Qian, B. (2018). d-limonene exhibits antitumor activity by inducing autophagy and apoptosis in lung cancer. OncoTargets and Therapy, 11, 1833.
Yusuf, H., Suryawati, S., & Fahriani, M. (2020). Combination Therapy of Eurycomanone and Doxo as Anticancer on T47D and MCF-7 Cell Line. Sys Rev Pharm, 11(10), 335–341.
Zeng, Q., Che, Y., Zhang, Y., Chen, M., Guo, Q., & Zhang, W. (2020). Thymol Isolated from Thymus vulgaris L. Inhibits Colorectal Cancer Cell Growth and Metastasis by Suppressing the Wnt/β-Catenin Pathway. Drug Design, Development and Therapy, 14, 2535.