Sains Malaysiana 50(4)(2021): 967-987

http://doi.org/10.17576/jsm-2021-5004-08

Phytochemical Profile, Antioxidant and Anti Proliferative Studies in Different Extracts of Artocarpus kemando Miq. Bark

(Kajian Profil Fitokimia, Antioksidan dan Anti Proliferatif dalam Ekstrak Berbeza Batang Artocarpus kemando Miq.)

NOOR SHAFIFIYAZ MOHD YAZID¹*, NAJIHAH MOHD HASHIM¹, HAPIPAH MOHD AL^I2 & RUSEA GO³

¹Department of Pharmacy, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Federal Territory, Malaysia

²Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Federal Territory, Malaysia

³Department of Biology, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

Diserahkan: 20 September 2019/Diterima: 22 September 2020

ABSTRACT

In this study, the stem bark of Artocarpus kemando was used to find alternative antioxidants from natural sources with fewer side effects. A. kemando was extracted successively using hexane, chloroform and methanol solvents, and evaluated for antioxidant, cytotoxic, and antiproliferative activities. The extracts were investigated for determination of their total phenolic content (TPC) and total flavonoid content (TFC). Then, the antioxidant activities were evaluated using chemical based assays such as ferric reducing antioxidant power (FRAP), total antioxidant capacity, radical scavenging of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and 2,2'-azino-bis (3-ethylbenzothia zoline-6-sulphonic) (ABTS), β-carotene-linoleic acid (BC) assay, oxygen radical absorbance capacity (ORAC), and cell based assay. The cytotoxic study was done using four different cell lines namely human estrogen receptor positive (ER+) breast cancer cell line (MCF7), human ovarian cancer cell line (CAOV-3), human promyelocytic leukemia cell line (HL60), and normal immortalised human ovarian surface epithelial cell line (TI074), and were evaluated using microculture tetrazolium salt (MTT) before morphological change study was done on CAOV-3 cell. In this study, methanol extract displayed the most promising antioxidant activity compared to other extracts when tested with DPPH, FRAP, ABTS, TAOC, BC, ORAC, and cytoprotective assays. The remarkable activity showed by the methanol extract might be due to its high content of phenolic and flavonoid compounds at 855.5 ± 0.01 GAE µg/mL and 145.45 ± 0.06 QAE µg/mL, respectively. Nevertheless, the chloroform extract displayed better scavenging activity compared to other extracts with IC₅₀ value of 618 ± 0.04 µg/mL in DPPH assay. Each extract was analysed using Gas Chromatography Mass Spectrophotometry and the chemical constituents obtained were then analysed. In the cytoprotective activity, the methanol extract showed a comparable cytoprotection with ascorbic acid against the free radicals at the lowest effective concentration (EC₅₀) value of 21.48 µg/mL. However, in the cytotoxicity study, only chloroform extract displayed significant toxicity against the cancer cells with IC₅₀ value of 27.9 ± 0.03, 24.1 ± 0.02 and 9.0 ± 0.04 µg/mL after treatment at 24, 48, and 72 h, respectively. The chloroform extract of A. kemando was found capable of inducing apoptosis as shown with cell membrane blebbing, chromatin condensation and formation of apoptotic bodies. The results obtained from the study showed that A. kemando bark could be a potential antioxidant and antitumor agents particularly on human ovarian cancer cells.

Keywords: Antioxidant; antitumour; Artocapus kemando; cytoprotective; Malaysia

ABSTRAK

Dalam kajian ini, kulit batang Artocarpus kemando digunakan untuk mencari antioksidan alternatif daripada sumber semula jadi dengan kesan sampingan yang kurang. Kulit batang pokok A. kemando diekstrak secara berturutan menggunakan pelarut heksana, kloroform dan metanol serta dinilai untuk aktiviti antioksidan, sitotoksik dan antiproliferatif. Kesemua ekstrak telah dikaji untuk penentuan kandungan jumlah fenolik (TPC) dan jumlah flavonoid (TFC). Kemudian, aktiviti antioksidan telah dinilai melalui ujian berasaskan kimia seperti keupayaan antioksida mengurangkan ferum (FRAP), jumlah kapasiti antioksidan (TAOC), asai pengurangan radikal bebas menggunakan 2,2-difenil-1-pikrilhidrazil (DPPH) dan 2,2'-azino-bis (3-etilbenzotiazolin-6-sulfonik) (ABTS), asai asid β-karoten-linoleik (BC), kapasiti penyerapan oksigen radikal (ORAC), dan ujian berasaskan sel. Kajian sitotoksik dilakukan menggunakan empat titisan sel yang berbeza iaitu titisan sel kanser payudara positif reseptor estrogen (ER +) (MCF7), titisan sel kanser ovari manusia (CAOV-3), titisan sel leukemia promoelositik manusia (HL60) dan titisan sel normal permukaan epitelial ovari manusia (TI074), serta dinilai menggunakan garam mikro tetrazolium (MTT) sebelum kajian perubahan morfologi dilakukan terhadap sel CAOV-3. Dalam kajian ini, ekstrak metanol menunjukkan aktiviti antioksidan yang paling baik berbanding ekstrak lain apabila diuji dengan DPPH, FRAP, ABTS, TAOC, BC, ORAC dan asai sitoprotektif. Aktiviti luar biasa yang ditunjukkan oleh ekstrak metanol mungkin disebabkan oleh kandungan sebatian fenolik dan flavonoid yang tinggi iaitu masing-masing 855.5 ± 0.01 GAE μg/mL dan 145.45 ± 0.06 QAE μg/mL. Walau bagaimanapun, ekstrak kloroform menunjukkan aktiviti pengurangan radikal yang lebih baik berbanding ekstrak lain dengan nilai IC₅₀ 618 ± 0.04 μg/mL dalam asai DPPH. Setiap ekstrak telah dianalisis menggunakan Spektrofotometri Jisim Kromatografi Gas dan komponen kimia yang diperoleh kemudiannya dianalisis. Bagi aktiviti sitoprotektif, ekstrak metanol menunjukkan keupayaan sitoperlindung setanding dengan aktiviti asid askorbik terhadap radikal bebas pada nilai kepekatan berkesan yang paling rendah (EC₅₀) iaitu 21.48 μg/mL. Walau bagaimanapun, dalam kajian sitotoksisiti, hanya ekstrak kloroform yang menunjukkan ketoksikan signifikan terhadap sel kanser dengan nilai IC₅₀ masing-masing sebanyak 27.9 ± 0.03, 24.1 ± 0.02 dan 9.0 ± 0.04 μg/mL selepas rawatan pada 24, 48 dan 72 jam. Ekstrak chloroform A. kemando didapati berupaya mengaruh apoptosis sebagaimana ditunjukkan dengan penunasan membran sel, pengecutan kromatin dan pembentukan jasad apoptosis. Hasil yang diperoleh daripada kajian menunjukkan bahawa batang pokok A. kemando berpotensi sebagai agen antioksidan dan antitumor terutama ke atas sel kanser ovari manusia.

Kata kunci: Antioksidan; antitumor; Artocarpus kemando; Malaysia; sitoperlindung

RUJUKAN

Abu Bakar, M.F., Mohamed, M., Rahmat, A. & Fry, J. 2009. Phytochemicals and antioxidant activity of different parts of bambangan (Mangifera pajang) and tarap (Artocarpus odoratissimus). Food Chemistry 113: 479-483.

Altuntaş, G. & Değer, Y. 2017. The effects of butylated hydroxyl toluene on the total antioxidant status/total oxidant stress and biochemical parameters in rats. World Journal of Pharmacy and Pharmaceutical Science 6: 199-210.

Apak, R., Özyürek, M., Güçlü, K. & Çapanoğlu, E. 2016. Antioxidant activity/capacity measurement: Classification, physicochemical principles, mechanisms, and electron transfer (ET)-based assays. Journal of Agricultural and Food Chemistry 64: 997-1027.

Azwanida, N. 2015. A review on the extraction methods use in medicinal plants, principle, strength and limitation. Medicinal and Aromatic Plants 4(196): 2167-0412.

Benzie, I.F. & Strain, J. 1996. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: The FRAP assay. Analytical Biochemistry 239: 70-76.

Beta, T., Naing, S.K., Nam, S., Mpofu, A. & Therrien, M. 2007. Antioxidant activity in relationship to phenolic content of diverse food barley genotypes. In Antioxidant Measurement and Application, edited by Fereidoon, S. & Chi, T.H. Washington: American Chemical Society. pp. 242-254.

Brown, J.M. & Attardi, L.D. 2005. The role of apoptosis in cancer development and treatment response. Nature Reviews Cancer 5: 231-237.

Cao, G. & Prior, R.L. 1998. Comparison of different analytical methods for assessing total antioxidant capacity of human serum. Clinical Chemistry 44: 1309-1315.

Chan, K., Rajab, N.F., Ishak, M., Ali, A., Yusoff, K., Din, L. & Inayat-Hussain, S. 2006. Goniothalamin induces apoptosis in vascular smooth muscle cells. Chemico-Biological Interactions 159(2): 129-140.

Chang, C.C., Yang, M.H., Wen, H.M. & Chern, J.C. 2002. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Journal of Food and Drug Analysis 10: 178-182.

Dávalos, A., Gómez-Cordovés, C. & Bartolomé, B. 2004. Extending applicability of the oxygen radical absorbance capacity (ORAC− fluorescein) assay. Journal of Agricultural and Food Chemistry 52: 48-54.

David, J.P., Meira, M., David, J.M., Brandão, H.N., Branco, A., de Fátima Agra, M., Barbosa, M.R.V., de Queiroz, L.P. & Giulietti, A.M. 2007. Radical scavenging, antioxidant and cytotoxic activity of Brazilian caatinga plants. Fitoterapia 78: 215-218.

Dudonné, S., Vitrac, X., Coutiere, P., Woillez, M. & Mérillon, J.M. 2009. Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using DPPH, ABTS, FRAP, SOD, and ORAC assays. Journal of Agricultural and Food Chemistry 57: 1768-1774.

Ee, G.C.L., Teo, S.H., Rahmani, M., Lim, C.K., Lim, Y.M. & Go, R. 2011. Artomandin, a new xanthone from Artocarpus kemando (Moraceae). Natural Product Research 25: 995-1003.

Elmore, S. 2007. Apoptosis: A review of programmed cell death. Toxicologic Pathology 35: 495-516.

Govindarajan, R., Rastogi, S., Vijayakumar, M., Shirwaikar, A., Rawat, A.K.S., Mehrotra, S. & Pushpangadan, P. 2003. Studies on the antioxidant activities of Desmodium gangeticum. Biological and Pharmaceutical Bulletin 26: 1424-1427.

Harish, R. & Shivanandappa, T. 2006. Antioxidant activity and hepatoprotective potential of Phyllanthus niruri. Food Chemistry 95: 180-185.

Hashim, N.M., Rahmani, M., Ee, G.C.L., Sukari, M.A., Yahayu, M., Amin, M.A.M., Ali, A.M. & Go, R. 2012. Antioxidant, antimicrobial and tyrosinase inhibitory activities of xanthones isolated from Artocarpus obtusus FM Jarrett. Molecules 17: 6071-6082.

Hashim, N.M., Rahmani, M., Shamaun, S.S., Ee, G.C.L., Sukari, M.A., Ali, A.M. & Go, R. 2011. Dipeptide and xanthones from Artocarpus kemando Miq. Journal of Medicinal Plant Research 5: 4224-4230.

Incani, A., Serra, G., Atzeri, A., Melis, M.P., Serreli, G., Bandino, G., Sedda, P., Campus, M., Tuberoso, C.I. & Deiana, M. 2016. Extra virgin olive oil phenolic extracts counteract the pro-oxidant effect of dietary oxidized lipids in human intestinal cells. Food and Chemical Toxicology 90: 171-180.

Jagtap, U. & Bapat, V. 2010. Artocarpus: A review of its traditional uses, phytochemistry and pharmacology. Journal of Ethnopharmacology 129: 142-166.

Jamil, S., Sirat, H.M., Jantan, I., Aimi, N. & Kitajima, M. 2008. A new prenylated dihydrochalcone from the leaves of Artocarpus lowii. Journal of Natural Medicines 62: 321-324.

Kannan, R.R.R., Arumugam, R. & Anantharaman, P. 2010. In vitro antioxidant activities of ethanol extract from Enhalus acoroides (LF) royle. Asian Pacific Journal of Tropical Medicine 3: 898-901.

Kassim, N.K., Rahmani, M., Ismail, A., Sukari, M.A., Ee, G.C.L., Nasir, N.M. & Awang, K. 2013. Antioxidant activity-guided separation of coumarins and lignan from Melicope glabra (Rutaceae). Food Chemistry 139: 87-92.

Kawase, T., Ichikawa, H., Ohta, T., Nozaki, N., Tashiro, F., Ohki, R. & Taya, Y. 2008. p53 target gene AEN is a nuclear exonuclease required for p53-dependent apoptosis. Oncogene 27: 3797-3810.

Khan, M., Omoloso, A. & Kihara, M. 2003. Antibacterial activity of Artocarpus heterophyllus. Fitoterapia 74: 501-505.

Lü, J.M., Lin, P.H., Yao, Q. & Chen, C. 2010. Chemical and molecular mechanisms of antioxidants: Experimental approaches and model systems. Journal of Cellular and Molecular Medicine 14: 840-860.

Maeura, Y., Weisburger, J.H. & Williams, G.M. 1984. Dose-dependent reduction of N-2-fluorenylacetamide-induced liver cancer and enhancement of bladder cancer in rats by butylated hydroxytoluene. Cancer Research 44: 1604-1610.

Matés, J.M., Pérez-Gómez, C. & De Castro, I.N. 1999. Antioxidant enzymes and human diseases. Clinical Biochemistry 32: 595-603.

Mittal, A., Tandon, S., Singla, S.K. & Tandon, C. 2018. Modulation of lithiatic injury to renal epithelial cells by aqueous extract of Terminalia arjuna. Journal of Herbal Medicine 13: 63-70.

Molyneux, P. 2004. The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin Journal of Science and Technology 26: 211-219.

Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65: 55-63.

Murrell, G.A., Francis, M.J. & Bromley, L. 1990. Modulation of fibroblast proliferation by oxygen free radicals. Biochemical Journal 265: 659-665.

Naczk, M. & Shahidi, F. 2004. Extraction and analysis of phenolics in food. Journal of Chromatography A 1054: 95-111.

Nair, V.D., Panneerselvam, R. & Gopi, R. 2012. Studies on methanolic extract of Rauvolfia species from Southern Western Ghats of India - In vitro antioxidant properties, characterisation of nutrients and phytochemicals. Industrial Crops and Products 39: 17-25.

Oki, T., Masuda, M., Furuta, S., Nishiba, Y., Terahara, N. & Suda, I. 2002. Involvement of anthocyanins and other phenolic compounds in radical‐scavenging activity of purple‐fleshed sweet potato cultivars. Journal of Food Science 67: 1752-1756.

Pinchuk, I., Shoval, H., Dotan, Y. & Lichtenberg, D. 2012. Evaluation of antioxidants: Scope, limitations and relevance of assays. Chemistry and Physics of Lipids 165: 638-647.

Pinteus, S., Silva, J., Alves, C., Horta, A., Fino, N., Rodrigues, A.I., Mendes, S. & Pedrosa, R. 2017. Cytoprotective effect of seaweeds with high antioxidant activity from the Peniche coast (Portugal). Food Chemistry 218: 591-599.

Richter, C. 1993. Pro‐oxidants and mitochondrial Ca²⁺: Their relationship to apoptosis and oncogenesis. FEBS Letters 325: 104-107.

San Tang, K. 2014. Protective effect of arachidonic acid and linoleic acid on 1-methyl-4-phenylpyridinium-induced toxicity in PC12 cells. Lipids in Health and Disease 13: 1.

Sapei, L. & Hwa, L. 2014. Study on the kinetics of vitamin C degradation in fresh strawberry juices. Procedia Chemistry 9: 62-68.

Scalbert, A. & Williamson, G. 2000. Dietary intake and bioavailability of polyphenols. The Journal of Nutrition 130: 2073S-2085S.

Seo, E.K., Lee, D., Shin, Y.G., Chai, H.B., Navarro, H.A., Kardono, L., Rahman, I., Cordell, G.A., Farnsworth, N.R. & Pezzuto, J.M. 2003. Bioactive prenylated flavonoids from the stem bark of Artocarpus kemando. Archives of Pharmacal Research 26: 124-127.

Shahidi, F. & Ho, C.T. 2007. Antioxidant Measurement and Applications. Washington: American Chemical Society. pp. 2-7.

Shier, W.T., Abbas, H. & Mirocha, C. 1991. Toxicity of the mycotoxins fumonisins B 1 and B 2 and Alternaria alternata f. sp. lycopersici toxin (AAL) in cultured mammalian cells. Mycopathologia 116: 97-104.

Singleton, V. & Rossi, J.A. 1965. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American Journal of Enology and Viticulture 16: 144-158.

Stoneman, V.E. & Bennett, M.R. 2004. Role of apoptosis in atherosclerosis and its therapeutic implications. Clinical Science 107: 343-354.

Sun, L., Zhang, J., Lu, X., Zhang, L. & Zhang, Y. 2011. Evaluation to the antioxidant activity of total flavonoids extract from persimmon (Diospyros kaki L.) leaves. Food and Chemical Toxicology 49: 2689-2696.

Tabera, J., Guinda, Á., Ruiz-Rodríguez, A., Señoráns, F.J., Ibáñez, E., Albi, T. & Reglero, G. 2004. Countercurrent supercritical fluid extraction and fractionation of high-added-value compounds from a hexane extract of olive leaves. Journal of Agricultural and Food Chemistry 52: 4774-4779.

Teo, S., Go, R., Lim, C. & Lim, Y. 2012. Free radical scavenging effect of Artocarpus kemando and Artocarpus odoratissimus: Structure-activity relationship of flavonoid derivatives. Asian Journal of Chemistry 24: 231.

Tomosaka, H., Chin, Y.W., Salim, A.A., Keller, W.J., Chai, H. & Kinghorn, A.D. 2008. Antioxidant and cytoprotective compounds from Berberis vulgaris (barberry). Phytotherapy Research 22: 979-981.

Valko, M., Leibfritz, D., Moncol, J., Cronin, M.T., Mazur, M. & Telser, J. 2007. Free radicals and antioxidants in normal physiological functions and human disease. The International Journal of Biochemistry & Cell Biology 39: 44-84.

Videla, L.A. 2010. Cytoprotective and suicidal signaling in oxidative stress. Biological Research 43: 363-369.

Wei, B.L., Weng, J.R., Chiu, P.H., Hung, C.F., Wang, J.P. & Lin, C.N. 2005. Antiinflammatory flavonoids from Artocarpus heterophyllus and Artocarpus communis. Journal of Agricultural and Food Chemistry 53: 3867-3871.

Witschi, H., Malkinson, A.M. & Thompson, J.A. 1989. Metabolism and pulmonary toxicity of butylated hydroxytoluene (BHT). Pharmacology & Therapeutics 42: 89-113.

Wong, S.P., Leong, L.P. & Koh, J.H.W. 2006. Antioxidant activities of aqueous extracts of selected plants. Food Chemistry 99: 775-783.

Xu, W., Saiki, S., Myojin, T., Liu, Y., Zhu, B., Murata, Y., Ashida, H., Tsunenaga, M. & Nakamura, Y. 2018. Lycii fructus extract ameliorates hydrogen peroxide-induced cytotoxicity through indirect antioxidant action. Bioscience, Biotechnology, and Biochemistry 82(10): 1812-1820.

Zubia, M., Fabre, M.S., Kerjean, V., Le Lann, K., Stiger-Pouvreau, V., Fauchon, M. & Deslandes, E. 2009. Antioxidant and antitumoural activities of some Phaeophyta from Brittany coasts. Food Chemistry 116: 693-701.

*Pengarang untuk surat-menyurat; email: shafifiyaz63@gmail.com