Sains Malaysiana 52(10)(2023): 2785-2801

http://doi.org/10.17576/jsm-2023-5210-05

The Phytochemical Components of Kelantan Grown Annona muricata Leaves and Its Anti-Proliferative Properties on MCF-7 Breast Cancer Cells

(Komponen Fitokimia Daun Annona muricata Ditanam di Kelantan dan Sifat Anti-Proliferatifnya terhadap Sel Kanser Payudara MCF-7)

NORZILA ISMAIL¹, HARISHINI RAJARATINAM², KHAIRUNNISA CHE GHAZALI³, SITI FARHANAH MOHD SALLEH¹, WAN SURIYANI FALIQ ADEEBA WAN IBRAHIM⁴, MAYA MAZUWIN YAHYA³ & WAN ZAINIRA WAN ZAIN³

¹Department of Pharmacology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia

²School of Health Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia

³Department of Surgery, Hospital Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia

⁴Faculty of Bioengineering & Technology, Universiti Malaysia Kelantan, Jeli Campus, 17600 Jeli Kelantan, Malaysia

Diserahkan: 13 Jun 2022/Diterima: 26 September 2023

Abstract

Annona muricata leaves exhibit anti-cancer properties against breast cancer. In this study, the phytochemical components, anti-proliferative activity, and cytotoxicity of Kelantan (Malaysia) grown Annona muricata leaves were evaluated using MCF-7 breast cancer cell line. The preparation of Annona muricata leaves extracts was performed with various organic solvents (ethyl acetate, n-hexane, methanol, and aqueous) using Soxhlet method. Each extract was analysed using gas chromatography-mass spectrometry (GCMS) for phytochemical analysis and characterised with Wiley and NIST library searchers. The effects of all extracts on cell proliferation were analysed using MTT assay. The anti-proliferative activity was determined by evaluating the IC₅₀ value. Based on the lowest IC₅₀value, further experiments were done to observe the apoptosis effect using the Annexin V-FITC technique. While for cell cycle analysis, Cycletest Plus DNA Reagent Kit was used. Both were further determined using flow cytometry analysis. The ethyl acetate-based Annona muricata extract contained terpenoids, phenolic compounds, and alkaloids. Ethyl acetate extract of Annona muricata exhibited a significant cytotoxic effect on MCF-7 breast cancer cells. The most potent IC₅₀ value obtained was 21.8 ± 3.85 µg/mL. The changes in morphology were more profound after 72 h of treatment with ethyl acetate extract of Annona muricata, including cell shrinkage. Flow cytometry analysis showed that it was induced in early and late apoptosis in vitro and arrest at G₀/G₁ cell cycle phase in time-dependant manners. In short, ethyl acetate extract of Annona muricata exhibited a range of phytochemical components, anti-proliferative activity and cytotoxic effect on MCF-7 breast cancer cells.

Keywords: Annona muricata; apoptosis; breast cancer; cytotoxicity; MCF-7

Abstrak

Daun Annona muricata menunjukkan sifat anti-kanser terhadap kanser payudara. Dalam kajian ini, komponen fitokimia, aktiviti anti-proliferatif dan kesitotoksikan daun Annona muricata yang ditanam di Kelantan (Malaysia) dinilai menggunakan titisan sel kanser payudara MCF-7. Penyediaan ekstrak daun Annona muricata dilakukan dengan pelbagai pelarut organik (etil asetat, n-heksana, metanol dan akueus) dengan menggunakan kaedah Soxhlet. Setiap ekstrak dianalisis menggunakan spektrometri jisim kromatografi gas (GCMS) untuk analisis fitokimia dan dicirikan dengan pencari perpustakaan Wiley dan NIST. Kesan semua ekstrak pada pertumbuhan sel telah dianalisis menggunakan asai MTT. Aktiviti anti-proliferatif ditentukan melalui nilai IC₅₀. Berdasarkan nilai IC₅₀ yang paling rendah, uji kaji lanjutan telah dilakukan untuk memerhatikan kesan apoptosis menggunakan teknik Annexin V-FITC. Manakala untuk analisis kitaran sel, Cycletest Plus DNA Reagent Kit digunakan. Keputusan daripada kedua-dua uji kaji dikukuhkan dengan kaedah analisis sitometri aliran. Ekstrak Annona muricata berasaskan etil asetat mengandungi terpenoid, sebatian fenol dan alkaloid. Ekstrak etil asetat Annona muricata menunjukkan kesan sitotoksik yang ketara pada sel kanser payudara MCF-7. Nilai IC₅₀ yang paling berkesan diperoleh ialah 21.8 ± 3.85 µg/mL. Perubahan morfologi sel lebih ketara selepas 72 jam rawatan dengan ekstrak etil asetat Annona muricata termasuk pengecutan sel. Analisis sitometri aliran menunjukkan bahawa ia telah diinduksi pada apoptosis awal dan lewat secara in vitro dan perencatan pada fasa kitaran sel G₀/G₁ secara berkadar langsung dengan masa. Ringkasnya, ekstrak etil asetat Annona muricata menunjukkan pelbagai komponen fitokimia, aktiviti anti-proliferatif dan kesan sitotoksik terhadap sel kanser payudara MCF-7.

Kata kunci: Annona muricata; apoptosis; kanser payudara; kesitotoksian; MCF-7

RUJUKAN

Ab Rahman, H.S., Wan-Ibrahim, W.S., Ismail, N., Ismail, T.N.N.T., Mohd-Salleh, S.F., Wong, M.P.K., Samad, M.R.A. & Hashim, M.N.M. 2018. Phytocompounds of Anonna muricata leaves extract and cytotoxic effects on breast cancer cells. Asian Pacific Journal of Tropical Medicine 11(12): 659-665.

Abdul Wahab, S.M., Jantan, I., Haque, M. & Arshad, L. 2018. Exploring the leaves of Annona muricata L. as a source of potential anti-inflammatory and anticancer agents. Frontiers in Pharmacology 9: 661.

Agrawal, M., Bhaskar, A.S.B. & Rao, P.VL. 2015. Involvement of mitogen-activated protein kinase pathway in T-2 toxin-induced cell cycle alteration and apoptosis in human neuroblastoma cells. Molecular Neurobiology 51(3): 1379-1394.

Aguirre-Ghiso, J.A. 2007. Models, mechanisms and clinical evidence for cancer dormancy. Nature Reviews Cancer 7(11): 834-846.

Amaro, F., Silva, D., Reguengo, H., Oliveira, J.C., Quintas, C., Vale, N., Gonçalves, J. & Fresco, P. 2020. β-adrenoceptor activation in breast MCF-10A cells induces a pattern of catecholamine production similar to that of tumorigenic MCF-7 cells. International Journal of Molecular Sciences 21(21): 7968.

Anantharaju, P.G., Gowda, P.C., Vimalambike, M.G. & Madhunapantula, S.V. 2016. An overview on the role of dietary phenolics for the treatment of cancers. Nutrition Journal 15(1): 1-16.

Astirin, O.P., Artanti, A.N., Fitria, M.S., Perwitasari, E.A. & Prayitno, A. 2013. Annona muricata Linn leaf induce apoptosis in cancer cause virus. Journal of Cancer Therapy 4(7): 1244-1250.

Ateba, S.B., Mvondo, M.A., Ngeu, S.T., Tchoumtchoua, J., Awounfack, C.F., Njamen, D. & Krenn, L. 2018. Natural terpenoids against female breast cancer: A 5-year recent research. Current Medicinal Chemistry 25(27): 3162-3213.

Bhardwaj, M., Sali, V.K., Mani, S. & Vasanthi, H.R. 2020. Neophytadiene from Turbinaria ornata suppresses LPS-induced inflammatory response in RAW 264.7 macrophages and Sprague Dawley rats. Inflammation 43(3): 937-950.

Coller, H.A. 2007. What's taking so long? S-phase entry from quiescence versus proliferation. Nature Reviews Molecular Cell Biology 8(8): 667-670.

Department of Statistics Malaysia. 2022. Poket stats negeri Kelantan STI 2022. https://cloud.stats.gov.my/index.php/s/obkiWqCqtKhHo64#pdf Accessed on 27th of August 2023.

Endrini, S., Suherman, S. & Widowati, W. 2014. Annona muricata leaves have strongest cytotoxic activity against breast cancer cells. Universa Medicina 33(3): 179-184.

Farooqi, A.A., Attar, R., Sabitaliyevich, U.Y., Alaaeddine, N., de Sousa, D.P., Xu, B. & Cho, W.C. 2020. The prowess of andrographolide as a natural weapon in the war against cancer. Cancers 12(8): 2159.

Farooqui, M., Hassali, M.A., Shatar, A.K.A., Farooqui, M.A., Saleem, F., ul Haq, N. & Othman, C.N. 2016. Use of complementary and alternative medicines among Malaysian cancer patients: A descriptive study. Journal of Traditional and Complementary Medicine 6(4): 321-326.

Gajalakshmi, S., Vijayalakshmi, S. & Devi, R.V. 2012. Phytochemical and pharmacological properties of Annona muricata: A review. International Journal of Pharmacy and Pharmaceutical Sciences 4(2): 3-6.

Gavamukulya, Y., Abou-Elella, F., Wamunyokoli, F. & AEl-Shemy, H. 2014. Phytochemical screening, anti-oxidant activity and in vitro anticancer potential of ethanolic and water leaves extracts of Annona muricata (Graviola). Asian Pacific Journal of Tropical Medicine 7: S355-S363.

International Agency for Research on Cancer (IARC) 2020. GLOBOCAN Report. https://gco.iarc.fr/ Accessed on 22nd April 2022.

Gull, J., Sultana, B., Anwar, F., Naseer, R., Ashraf, M. & Ashrafuzzaman, M. 2012. Variation in antioxidant attributes at three ripening stages of guava (Psidium guajava L.) fruit from different geographical regions of Pakistan. Molecules 17(3): 3165-3180.

Hadisaputri, Y.E., Habibah, U., Abdullah, F.F., Halimah, E., Mutakin, M., Megantara, S., Abdulah, R. & Diantini, A. 2021. Antiproliferation activity and apoptotic mechanism of soursop (Annona muricata L.) leaves extract and fractions on MCF7 breast cancer cells. Breast Cancer: Targets and Therapy 13: 447-457.

Hanahan, D. & Weinberg, R.A. 2011. Hallmarks of cancer: The next generation. Cell 144(5): 646-674.

He, Y., Zhu, Q., Chen, M., Huang, Q., Wang, W., Li, Q., Huang, Y. & Di, W. 2016. The changing 50% inhibitory concentration (IC₅₀) of cisplatin: A pilot study on the artifacts of the MTT assay and the precise measurement of density-dependent chemoresistance in ovarian cancer. Oncotarget 7(43): 70803-70821.

Hemalatha, G., Sivakumari, K., Rajesh, S. & Shyamala Devi, K. 2020. Phytochemical profiling, anticancer and apoptotic activity of graviola (Annona muricata) fruit extract against human hepatocellular carcinoma (HepG-2) cells. International Journal of Zoology and Applied Biosciences 5(1): 32-47.

Horwitz, K.B., Costlow, M.E. & McGuire, W.L. 1975. MCF-7: A human breast cancer cell line with estrogen, androgen, progesterone, and glucocorticoid receptors. Steroids 26(6): 785-795.

Ilango, S., Sahoo, D.K., Paital, B., Kathirvel, K., Gabriel, J.I., Subramaniam, K., Jayachandran, P., Dash, R.K., Hati, A.K., Behera, T.R., Mishra, P. & Nirmaladewi, R. 2022. A review on Annona muricata and its anticancer activity. Cancers 14(18): 4539.

Isah, T. 2016. Anticancer alkaloids from trees: Development into drugs. Pharmacognosy Reviews 10(20): 90-99.

Mady, M.S., Mohyeldin, M.M., Ebrahim, H.Y., Elsayed, H.E., Houssen, W.E., Haggag, E.G., Soliman, R.F. & El Sayed, K.A. 2016. The indole alkaloid meleagrin, from the olive tree endophytic fungus Penicillium chrysogenum, as a novel lead for the control of c-Met-dependent breast cancer proliferation, migration and invasion. Bioorganic & Medicinal Chemistry 24(2): 113-122.

Malaysian National Cancer Registry (MNCR) report 2012-2016. 2019. https://nci.moh.gov.my/index.php/ms/pengumuman/789-summary-of-malaysian-national-cancer-registry-report-2012-2016 Accessed 22nd of April 2022.

Martínez-Campa, C., Casado, P., Rodríguez, R., Zuazua, P., García-Pedrero, J.M., Lazo, P.S. & Ramos, S. 2006. Effect of vinca alkaloids on ERα levels and Estradiol-induced responses in MCF-7 cells. Breast Cancer Research and Treatment 98(1): 81-89.

Moghadamtousi, S.Z., Karimian, H., Rouhollahi, E., Paydar, M., Fadaeinasab, M. & Kadir, H.A. 2014. Annona muricata leaves induce G1 cell cycle arrest and apoptosis through mitochondria-mediated pathway in human HCT-116 and HT-29 colon cancer cells. Journal of Ethnopharmacology 156: 277-289.

Mohd Mujar, N.M., Dahlui, M., Emran, N.A., Abdul Hadi, I., Wai, Y.Y., Arulanantham, S., Hooi, C.C. & Mohd Taib, N.A. 2017. Complementary and alternative medicine (CAM) use and delays in presentation and diagnosis of breast cancer patients in public hospitals in Malaysia. PLoS ONE 12(4): e0176394.

Muhamad, S., Pihie, A.H.L., Latif, J., Rha, C. & Sambandan, T.G. 2011. Induction of apoptosis in MCF-7 via the Caspase pathway by longilactone from Eurycoma longifolia Jack. Research in Pharmaceutical Biotechnology 3(1): 1-10.

Musini, A., Rao, J.P. & Giri, A. 2015. Phytochemicals of Salacia oblonga responsible for free radical scavenging and antiproliferative activity against breast cancer cell lines (MDA-MB-231). Physiology and Molecular Biology of Plants 21(4): 583-590.

Naik, A.V. & Sellappan, K. 2020. In vitro evaluation of Annona muricata L. (Soursop) leaf methanol extracts on inhibition of tumorigenicity and metastasis of breast cancer cells. Biomarkers 25(8): 701-710.

Nayyab, S., Naureen, H., Maryam, A., Attar, R., Sabitaliyevich, U.Y., Konysbayevna, K.K. & Farooqi, A.A. 2020. Piceatannol mediated regulation of deregulated signaling pathways in different cancers: Tumbling of the ninepins of molecular oncology. Cellular and Molecular Biology 66(6): 157-163.

Pieme, C.A., Kumar, S.G., Dongmo, M.S., Moukette, B.M., Boyoum, F.F., Ngogang, J.Y. & Saxena, A.K. 2014. Antiproliferative activity and induction of apoptosis by Annona muricata (Annonaceae) extract on human cancer cells. BMC Complementary and Alternative Medicine 14(1): 516-525.

Prasad, S.K., Veeresh, P.M., Ramesh, P.S., Natraj, S.M., Madhunapantula, S.V. & Devegowda, D. 2020. Phytochemical fractions from Annona muricata seeds and fruit pulp inhibited the growth of breast cancer cells through cell cycle arrest at G0/G1 phase. Journal of Cancer Research Therapeutics 16(6):1235-1249. doi: 10.4103/jcrt.JCRT_494_19. PMID: 33342779

Rachmani, E.P.N., Suhesti, T.S. & Widiastuti, R. & Aditiyono. 2012. The breast of anticancer from leaf extract of Annona muricata againts cell line in T47D. International Journal of Applied Science Technology 2(1): 157-164.

Sallam, A.A., Ayoub, N.M., Foudah, A.I., Gissendanner, C.R., Meyer, S.A. & El Sayed, K.A. 2013. Indole diterpene alkaloids as novel inhibitors of the Wnt/β-catenin pathway in breast cancer cells. European Journal of Medicinal Chemistry 70: 594-606.

Salleh, S.N.S.M., Farooqui, M., Gnanasan, S. & Karuppannan, M. 2021. Use of complementary and alternative medicines (CAM) among Malaysian cancer patients for the management of chemotherapy related side effects (CRSE). Journal of Complementary and Integrative Medicine 18(4): 805-812.

Solomon-Wisdom, G.O., Ugoh, S.C. & Mohammed, B. 2014. Phytochemical screening and antimicrobial activities of Annona muricata (L) leaf extract. American Journal of Biological, Chemical and Pharmaceutical Sciences 2(1): 1-7.

Suffness, M. & Pezzuto, J.M. 1990. Assays related to cancer drug discovery. Methods in Plant Biochemistry: Assays for Bioactivity 6: 71-133.

Sulistyoningrum, E., Rachmani, E.P.N., Baroroh, H.N. & Rujito, L. 2017. Annona muricata leaves extract reduce proliferative indexes and improve histological changes in rat’s breast cancer. Journal of Applied Pharmaceutical Science 7(1): 149-155.

Syed Najmuddin, S.U.F., Romli, M.F., Hamid, M., Alitheen, N.B. & Nik Abd Rahman, N.M.A. 2016. Anti-cancer effect of Annona muricata Linn leaves crude extract (AMCE) on breast cancer cell line. BMC Complementary and Alternative Medicine 16(1): 1-20.

Taniguchi, K. & Karin, M. 2018. NF-κB, inflammation, immunity and cancer: Coming of age. Nature Reviews Immunology 18(5): 309-324.

Telang, N. 2018. Anti‑proliferative and pro‑apoptotic effects of rosemary and constituent terpenoids in a model for the HER‑2‑enriched molecular subtype of clinical breast cancer. Oncology Letters 16(4): 5489-5497.

Tomko, A.M., Whynot, E.G., Ellis, L.D. & Dupré, D.J. 2020. Anti-cancer potential of cannabinoids, terpenes, and flavonoids present in cannabis. Cancers 12(7): 1985.

Torres, M.P., Rachagani, S., Purohit, V., Pandey, P., Joshi, S., Moore, E.D., Johansson, S.L., Singh, P.K., Ganti, A.K. & Batra, S.K. 2012. Graviola: A novel promising natural-derived drug that inhibits tumorigenicity and metastasis of pancreatic cancer cells in vitro and in vivo through altering cell metabolism. Cancer Letters 323(1): 29-40.

World Health Organization (WHO). 2013. WHO traditional medicine strategy: 2014-2023. https://www.who.int/publications/i/item/9789241506096 Accessed on 22nd of April 2022.

Xu, D. & Xu, Z. 2020. Indole alkaloids with potential anticancer activity. Current Topics in Medicinal Chemistry 20(21): 1938-1949.

Xu, H., Li, Y., Han, B., Li, Z., Wang, B., Jiang, P., Zhang, J., Ma, W., Zhou, D., Li, X. & Ye, X. 2018. Anti-breast-cancer activity exerted by β-Sitosterol-d-glucoside from sweet potato via upregulation of microRNA-10a and via the PI₃K–Akt signaling pathway. Journal of Agricultural and Food Chemistry 66(37): 9704-9718.

Yang, H. & Ping Dou, Q. 2010. Targeting apoptosis pathway with natural terpenoids: Implications for treatment of breast and prostate cancer. Current Drug Targets 11(6): 733-744.

Yip, C.H., Pathy, N.B. & Teo, S.H. 2014. A review of breast cancer research in Malaysia. Medical Journal of Malaysia 69(Suppl A): 8-22.

*Pengarang untuk surat-menyurat; email: zainira@usm.my