Malaysian Journal of Analytical Sciences Vol 23 No 3 (2019): 534 - 547

FRACTIONATION OF AQUEOUS EXTRACT OF Ficus deltoidea VAR. Kunstleri’s LEAVES USING SOLID PHASE EXTRACTION METHOD FOR ANTICANCER ACTIVITY ON DU145 CELL LINE

(Pemeringkatan Ekstrak Akueus dari Daun Ficus deltoidea var. Kunstleri Menggunakan Kaedah Pengestrakan Fasa Pepejal untuk Aktiviti Antikanser Pada Titisan Sel DU145)

Husnul Hanani Soib¹, Harisun Yaakob^1,2*, Mohamad Roji Sarmidi^2,3, Mohamad Norisham Mohamad Rosdi¹

¹Department of Bioprocess Engineering, Faculty of Chemical and Energy Engineering

²Institute of Bioproduct Development

³Innovation Centre in Agritechnology for Advanced Bioprocessing (ICA)

Universiti Teknologi Malaysia, 81310 UTM Skudai, Johor, Malaysia

*Corresponding author: harisun@ibd.utm.my

Received: 25 October 2017; Accepted: 22 January 2019

Abstract

Ficus deltoidea var. kunstleri or also known as ‘Mas Cotek’ belongs to the Moraceae family. It is widely used for postnatal treatment, anti-diabetic, anti-breast cancer, anti-nociceptive and anti-hypertension. In this study, the anticancer effects of flavonoid compounds from aqueous ethanolic fractions of F. deltoidea var. kunstleri leaves against prostate cancer cell lines, DU145 were investigated. The aqueous extract of F. deltoidea var. kunstleri was subjected for fractionation by solid phase extraction method by using various concentration of aqueous - ethanol ranging 20-99.8%. The fractions (fraction I-IV) were screened to obtain the most active fraction through total phenolic and flavonoid contents and MTT assays. The induction of apoptosis was evaluated using Annexin V-FITC/PI while the morphological changes of DU145 cells was assessed using acridine orange/propidium iodide (AO/PI) staining method. HPLC, FTIR-ATR and LC-MS/MS analysis were conducted for identification and confirmation of bioactive compounds. Fraction I which was fractionated using 20% of ethanol exhibited the highest polyphenol contents and highest inhibition against DU145 at 100 mg/mL (45.45%). Furthermore, cell viability of DU145 cells treated with crude extract of F. deltoidea var. kunstleri and its active fraction (I) were also compared using MTT assay. It was revealed that the crude and its active fraction I caused cell reduction through apoptotic machinery which was evidenced by the formation of apoptotic bodies, fragmentation, cell blebbing and cell shrinkage. These findings were confirmed by AO/PI staining method and Annexin V-FITC/PI. The presence of bioactive compounds of vitexin and isovitexin were identified by HPLC, FTIR-ATR and LC-MS/MS analysis. Positive correlations had been established between total phenolic and flavonoid content and anti prostate cancer activities. Therefore, these results suggest that flavonoids content of F. deltoidea var. kunstleri and its fraction I could potentially can be developed as an anti-cancer agent for prostate cancer.

Keyword: anti-cancer, antioxidant, F. deltoidea var. kunstleri, prostate cancer, vitexin

Abstrak

Ficus deltoidea var. kunstleri atau juga dikenali sebagai 'Mas Cotek' tergolong dalam keluarga Moraceae. Ia digunakan secara meluas untuk rawatan selepas bersalin, anti diabetes, anti kanser payudara, anti nosiseptif dan anti hipertensi. Dalam kajian ini, kesan antikanser oleh sebatian flavonoid daripada pemeringkatakan akueus etanol daun F. deltoidea var. kunstleri terhadap titisan sel kanser prostat, DU145 telah dikaji. Ekstrak akueus daripada F. deltoidea var. kunstleri telah dipilih untuk pemeringkatan oleh pengestrakan fasa pepejal menggunakan pelbagai kepekatan akueus - etanol berkadaran 20-99.8%. Semua pemeringkatan (pecahan I-IV) telah disaring untuk mendapatkan pecahan yang paling aktif melalui jumlah kandungan fenol dan flavonoid serta ujian MTT. Induksi melalui apoptosis dinilai menggunakan Annexin V-FITC/PI manakala perubahan morfologi pada sel DU145 juga telah dinilai menggunakan kaedah pewarnaan acridin oren/propidium iodida (AO/PI). HPLC, FTIR-ATR dan LC-MS/MS analisis telah digunakan untuk mengenalpasti dan pengesahan sebatian bioaktif. Pecahan I yang terpecah menggunakan 20% etanol mempamerkan kandungan polifenol tertinggi dan perencatan paling tinggi terhadap DU145 pada 100 mg/mL (45.45%). Tambahan pula, sel hidup DU145 yang telah dirawat oleh ekstrak mentah F. deltoidea var. kunstleri dan pecahan aktifnya (I) juga telah dibandingkan menggunakan ujian MTT. Ia telah membuktikan bahawa ekstrak mentah dan pecahan aktifnya I menyebabkan pengurangan sel melalui mesin apoptosis dimana telah dibuktikan melalui pembentukan badan-badan apoptosis, pemecahan, sel kembung dan pengecutan sel yang disahkan oleh kaedah pewarnaan AO/PI dan Anexin V-FITC/PI. Kehadiran sebatian bioaktif vitexin dan isovitoksin telah dikenal pasti oleh analisis HPLC, FTIR-ATR and LC-MS/MS. Hubungan positif telah wujud di antara jumlah kandungan fenol dan flavonoid dan aktiviti anti-kanser prostat. Oleh itu, keputusan ini mencadangkan bahawa kandungan flavonoid F. deltoidea var. kunstleri dan pecahannya boleh dibangunkan sebagai agen anti-kanser untuk kanser prostat.

Kata kunci: anti-kanser, antioksida, F. deltoidea var. kunstleri, kanser prostat, vitexin

References

1. Li, Y., Ma, D., Sun, D., Wang, C., Zhang, J., Xie, Y. and Guo, T. (2015). Total phenolic, flavonoid content, and antioxidant activity of flour, noodles, and steamed bread made from different colored wheat grains by three milling methods. The Crop Journal, 3(4): 328 - 334.

2. Settharaksa, S., Jongjareonrak, A., Hmadhlu, P., Chansuwan, W. and Siripongvutikorn, S. (2012). Flavonoid, phenolic contents and antioxidant properties of Thai hot curry paste extract and its ingredients as affected of pH, solvent types and high temperature. International Food Research Journal, 19(4): 1581 - 1587.

3. Kefayati, Z., Motamed, S., Shojaii, A., Noori, M. and Ghods, R. (2017). Antioxidant activity and phenolic and flavonoid contents of the extract and subfractions of Euphorbia splendida Mobayen. Pharmacognosy Research, 9(4): 362 - 365.

4. Wahle, K. J., Brown, I., Rotondo, D. and Heys, S. (2010). Plant phenolics in the prevention and treatment of cancer. In M. Giardi, G. Rea and B. Berra (Eds.). Bio-Farms for Nutraceuticals. United State: Springer: pp. 36 - 51.

5. Adefegha, S. A. and Oboh. G. (2011). Cooking enhances the antioxidant properties of some tropical green leafy vegetables. African Journal of Biotechnology, 10(4): 632 - 639.

6. Sahreen, S., Khan, M. R. and Khan, R. A. (2014). Effects of Carissa opaca fruits extracts on oxidative pulmonary damages and fibrosis in rats. BMC Complementary and Alternative Medicine, 14(1): 1 - 9.

7. Islam, S., Akhtar, M., Parvez, S., Alam, J. and Alam, F. M. (2013). Antitumor and antibacterial activity of a crude methanol leaf extract of Vitex negundo L. Archives of Biological Sciences, 65(1): 229 - 238.

8. Jain, N. K., Siddiqi, M. A. and Weisburger J. H. (2006). Protective effects of tea on human health. United Kingdom: Cromwell Press.

9. McConnel, T. H. (2007). The nature of disease: Pathology for the health professions. United States: Lippincott Wiliams & Wilkin.

10. Henry, R.Y. and O’mahony, D. (1999). Treatment of prostate cancer. Journal of Clinical Pharmacy and Therapeutics, 24: 93 - 102.

11. Rang, H. P., Dale, M. M., Ritter, J. M. and Flower, R. J. (2008). Pharmacology. United States: Churchill Livingstone.

12. Farsi, E., Mariam, A. Sook, Y. H., Mohamed B. K. A., Mun, F. Y. and Mohd, Z. A. (2014). Standardized extract of Ficus deltoidea stimulates insulin secretion and blocks hepatic glucose production by regulating the expression of glucose-metabolic genes in streptozitocin induced diabetic rats. BMC Complementary and Alternative Medicine, 14: 220.

13. Corner, E. J. H. (1969). The complex of Ficus deltoidea; a recent invasion of the Sunda Shelf. Philosophical Transactions of the Royal Society of London, 256: 281 - 317.

14. Lee, S. W., Wendy, W., Julius, Y. F. S. and Desy, F. S. (2011). Characterization of antioxidant, antimicrobial, anti cancer property and chemical composition of Ficus deltoidea Jack. Leaf extract. Journal of Biologically Active Products from Nature, 2011: 2231 - 1874.

15. Sulaiman, M. R., Hussain, M. K., Zakaria, Z. A., Somchit, M. N., Moin, S., Mohamad, A. S. and Israf, D. A. (2008). Evaluation of the antinociceptive activity of Ficus deltoidea aqueous extract. Fitoterapia, 79: 557 - 561.

16. Wahid, S. Mahmud, T. M. M., Maziah, M., Yahya, A. and Rahim, M. A. (2010). Total phenolics content and antioxidant activity of hot water extracts from dried Ficus deltoidea leaves. Journal of Tropical Agriculture and Food Science, 38 (1): 115 - 122.

17. Norra I. (2011). Free radical scavenging activity and phenolic content of Ficus deltoidea accessions MFD4 and MFD6 leaves. Journal of Tropical Agriculture and Food Science, 39(1): 000 -000.

18. Zunoliza, A., Khalid, H., Zhari, I. and Rasadah, M. A. (2009). Anti-inflammatory activity of standardised extracts of leaves of three varieties of Ficus deltoidea. International Journal of Pharmaceutical and Clinical Research, 1(3): 100 - 105.

19. Zakaria, Z. A., Hussain, M. K., Mohamad, A. S., Abdullah, F. C. and Sulaiman, M. R. (2011). Anti-inflammatory activity of the aqueous extract of Ficus deltoidea. Biological Research for Nursing, 14(1): 90 - 97.

20. Sahreen, S., Khan, M. R. and Khan, R. A. (2013). Estimation of flavoniods, antimicrobial, antitumor and anticancer activity of carissa opaca fruits. BMC Complementary and Alternative Medicine, 13(1): 372.

21. Hwa, J. C., Jae, S. E., Bang, G. K., Sun, Y. K., Hoon, J. and Yunjo, S. (2006). Vitexin, a HIF-1α inhibitor, has anti-metastatic potential in PC12 cells. Molecules and Cells, 22(3): 291 - 299.

22. Hock, E. K., Azrina, A., Amin, I. and Faridah Abas. (2012). Antioxidative properties of defatted dabai pulp and peel prepared by solid phase extraction. Molecules, 17: 9754 - 9773.

23. Petelinc, T., Polak, T., Demšar, L. and Jamnik, P. (2013). Fractionation of phenolic compounds extracted from propolis and their activity in the yeast saccharomyces cerevisiae. PLoS ONE, 8(2): e56104.

24. Vesna, T. T., Sonja, M. D., Jasna, M. C., Gordana, S. C. and Sladana, M. S. (2007). Solid phase extraction of antioxidant compounds from commercial cranberry extract and its antiradical activity. Acta Periodica Technologica, 38: 157 - 164.

25. Theodoridis, G., Lasakova, M., Sherikova, V., Tegou, A., Gianntsiou, N. and Jandera, P. (2006). Molecular imprinting of natural flavonoid antioxidants: Application in solid-phase extraction for the sample pre-treatment of natural products prior to HPLC analysis. Journal of Separation Science, 29: 2310 - 2321.

26. Sanagi, M. M., Sulaiman, A. and Ibrahim, W. A. W. (2007). Principles of chemical analysis. (2^nd edition). Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia.

27. Ku, Y. R., Liu, Y. C. and Lin, J. H. (2001). Solid-phase extraction and high-performance liquid chromatographic analysis of prednisone adultered in a foreign herbal medicine. Journal of Food and Drug Analysis, 9(3): 150 - 152.

28. Misbah, H., Abdul Aziz, A. and Aminudin, N. (2013). Antidiabetic and antioxidant properties of Ficus deltoidea fruit extracts and fractions. BMC Complementary and Alternative Medicine, 118(13): 1 - 12.

29. Farsi, E., Shafaei, A., Hor, S. Y., Khadeer Ahamed, M. B., Yam, M. F., Attitalla, I. H., Asmawi. M. Z. and Ismail, Z. (2011). Correlation between enzymes inhibitory effects and antioxidant activities of standardized fractions of methanolic extract obtained from Ficus deltoidea leaves. African Journal of Biotechnology, 67(10): 15184 - 15194.

30. Anwar, F. and Przybylski, R. (2012). Effect of solvents extraction on total phenolics and antioxidant activity of extracts from flaxseed (Linum usitatissimum L.). Acta Scientiarum Polonorum Technology Aliment, 11(3): 293 - 301.

31. Stankovic, M. S. (2011). Total phenolic content, flavonoid concentration and antioxidant activity of Marrubium peregrinum L. extracts. Kragujevac Journal Science, 33: 63-72.

32. Soib, H. H., Ware, I., Yaakob, H., Mukrish, H. and Sarmidi. M. R. (2015). Antioxidant and anticancer activity of standardized extracts of three varieties of Ficus deltoidea’ s leaves. Jurnal Teknologi, 77(3): 19 - 25.

33. Singleton, V. L., Orthofer, R. and Lamuela-Raventos, R. M. (1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin Ciocalteau reagent. Method Enzymology, 299: 152 - 178.

34. Atanassova, M., Marinova, D. and Ribarova, F. (2005). Total phenolics and total flavonoids in Bulgarian fruits and vegetables. Journal of the University of Chemical Technology Metallurgy, 40: 255 - 260.

35. Patel, H. D., Zaveri, A. D., Zaveri, D. N., Shah, S. and Solanki, A. (2013). Comparison of the MTT and alamar blue assay for in vitro anti-cancer activity by testing of various chalcone and thiosemicarbazone derivatives. International Journal of Pharmaceutical and Biological Sciences, 4 (2): 707 - 716.

36. Elham, F., Shafie, A., Hor, S. Y., Khadeer Ahamed, M. B., Yam, M. F., Asmawi. M. Z. and Ismail, Z. (2013). Genotoxicity and acute and subchronic toxicity studies of a standardized methanolic extract of Ficus deltoidea leaves. Clinics, 68(6): 865 - 875.

37. Zwir-Ferenc, A. and Biziuk, M. (2006). Solid phase extraction technique–trends, opportunities and applications. Polish Journal of Environmental Studies, 15(5): 677 - 690.

38. Sultana, B., Anwar, F. and Ashraf, M. (2009). Effect of extraction solvent/technique on the antioxidant activity of selected medicinal plant extracts. Molecules, 14: 2167 – 2180.

39. Dai, J. and Mumper, R. J. (2010). Plant phenolics: Extraction, analysis and their antioxidant and anticancer properties. Molecules, 15: 7313 - 7352.

40. Khoo, He., Azlan, A., Ismail, A. and Abas F. (2012). Antioxidative properties of defatted dabai pulp and peel prepared by solid phase extraction. Molecules, 17(8): 9754 - 0773.

41. Camel, C. (2003). Solid phase extraction of elements. Spectrochimica Acta, 58(7): 1177 - 1233.

42. Himmat, S. C. (2011). Prospective study of antioxidants, its mechanism and potential role in cancer. International Journal of Research in Pharmaceutical and Biomedical Science, 2(3): 888 - 894.

43. Yizhong, Cai., Qiong, L., Mei, S. and Harold, C. (2003). Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Sciences, 74: 2157 - 2184.

44. Mariño, G. and Kroemer, G. (2013). Mechanisms of apoptotic phosphatidylserine exposure. Cell Research, 23(11): 1247 - 1248.

45. Fattahi, S., Ardekani, A. M., Zabihi, E., Abedian, Z., Mostafazadeh, A., Pourbagher, R. and Niaki, A. A. (2013). Antioxidant and apoptotic effects of an aqueous extract of Utica dioica on the MCF-7 human breast cancer cell line. Asian Pacific Journal of Cancer Prevention, 14(9): 5317 - 5323.

46. Demchenko, A. P. (2012). The change of cellular membranes on apoptosis: Fluorescence detection. Experimental Oncology, 34(3): 263 - 268

47. Heerde, W. L. V., Robert-Offerman, S., Dumot, E. A. W. J., Hofstra, L., Doevendans, P. A. and Smits, J. (2000). Markers of apoptosis in cardiovascular tissues: Focus on Annexin V. Cardiovascular Research, 45(3): 549 - 559.