Cytotoxic Activity Against P388 Leukemia Cells and Cellular Antioxidant Capacity of Theobroma cacao L. Pod Husk Extract: A Dual Bioactivity Assessment
Abstract
Cacao pod husk (Theobroma cacao L.), a major agro-industrial by-product representing more than 70% of cocoa fruit weight, is a rich source of bioactive secondary metabolites including flavonoids, polyphenols, tannins, alkaloids, and quinones. This study evaluated the cytotoxic activity of a methanol extract against P388 murine leukemia cells using the MTT assay, cellular antioxidant capacity via superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels in hydrogen peroxide (H2O2)-induced human erythrocytes, and comprehensive phytochemical profiling with thin-layer chromatography (TLC) fingerprinting. Physicochemical characterization of the simplicia, performed to verify compliance with standardization parameters, revealed a moisture content of 2.49±0.70% v/w, total ash of 6.185±0.103% w/w, water-soluble extractive of 12.47±0.03% w/w, and ethanol-soluble extractive of 6.86±0.54% w/w. Phytochemical screening confirmed flavonoids, alkaloids, tannins, polyphenols, monoterpenes, sesquiterpenes, and quinones. Soxhlet extraction produced a higher yield (21.59%) than cold maceration (19.46%), supporting its use for subsequent bioactivity testing. At 53.8 μg/mL, the extract reduced MDA levels to a near-normal value (0.8780±0.0060 nmol/mL) and increased SOD activity (14.8493±0.0001 units/mL) to values higher than those obtained with the quercetin positive comparator, although no formal statistical comparison between groups was performed. The MTT assay yielded an IC50>100 ug/mL (extrapolated estimate 106.64 ug/ mL) against P388 leukemia cells, classifying the crude extract as inactive against this cell line. These findings indicate that T. cacao pod husk possesses notable cellular antioxidant activity accompanied by weak direct cytotoxicity at the crude-extract level, supporting its potential as a candidate for bioactivity-guided fractionation in future studies.
Keywords: Theobroma cacao; cellular antioxidant capacity; superoxide dismutase; malondialdehyde; P388 leukemia cells.
Full Text:
PDFReferences
Aniszewski, T., 2015, Alkaloids: Chemistry, Biology, Ecology, and Applications, 3rd ed. Amsterdam: Elsevier.
Armenian, S.H., Lacchetti, C., Barac, A., Carver, J., Constine, L.S., Denduluri, N., et al., 2017, Prevention and monitoring of cardiac dysfunction in survivors of adult cancers: American Society of Clinical Oncology clinical practice guideline, Journal of Clinical Oncology, 35(8), 893-911.
Arts, I.C.W. and Hollman, P.C.H., 2005, Polyphenols
and disease risk in epidemiologic studies, The American Journal of Clinical Nutrition, 81(1 Suppl), 317S-325S.
Atanasov, A.G., Zotchev, S.B., Dirsch, V.M., International Natural Product Sciences Taksforce, and Supuran, C.T., 2021, Natural products in drug discovery: advances and opportunities, Nature Reviews Drug Discovery, 20(3), 200-216.
Ayala, A., Muñoz, M.F., and Argüelles, S., 2014, Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal, Oxidative Medicine and Cellular Longevity, 2014, 360438.
Bautista-Expósito, S., Peñas, E., Frias, J., Dueñas, M., and Martínez-Villaluenga, C., 2021, Alkaloids in health and disease: structure-bioactivity relationships and new analytical approaches, Food Research International, 150, 110775.
Bhakta, N., Force, L.M., Allemani, C., Atun, R., Bray, F., Coleman, M.P., et al., 2019, Childhood cancer burden: a review of global estimates, The Lancet Oncology, 20(1), e42-e53.
Bray, F., Laversanne, M., Sung, H., Ferlay, J., Siegel, R.L., Soerjomataram, I., et al., 2024, Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries, CA: a Cancer Journal for Clinicians, 74(3), 229-263.
Campos-Vega, R., Nieto-Figueroa, K.H., and Oomah, B.D., 2018, Cocoa (Theobroma cacao L.) pod husk: renewable source of bioactive compounds, Trends in Food Science & Technology, 81, 172-184.
Chemat, F., Vian, M.A., and Cravotto, G., 2012, Green extraction of natural products: concept and principles, International Journal of Molecular Sciences, 13(7), 8615-8627.
Cragg, G.M. and Pezzuto, J.M., 2016, Natural products as a vital source for the discovery of cancer chemotherapeutic and chemopreventive agents, Medical principles and practice, 25(Suppl 2), 41-59.
Crozier, S.J., Preston, A.G., Hurst, J.W., Payne, M.J., Mann, J., Hainly, L., et al., 2011, Cacao seeds are a ‘Super Fruit’: a comparative analysis of various fruit powders and products, Chemistry Central Journal, 5, 5.
Dai, J. and Mumper, R.J., 2010, Plant phenolics: extraction, analysis and their antioxidant and anticancer properties, Molecules, 15(10), 7313- 7352.
Do, Q.D., Angkawijaya, A.E., Tran-Nguyen, P.L., Huynh, L.H., Soetaredjo, F.E., Ismadji, S., et al., 2014, Effect of extraction solvent on total phenol content, total flavonoid content, and antioxidant activity of Limnophila aromatica, Journal of Food and Drug Analysis, 22(3), 296- 302.
El-Far, A.H., Al Jaouni, S.K., Li, W., and Mousa, S.A., 2018, Protective roles of thymoquinone by inhibiting VEGF/VEGFR-2 signaling pathway in human hepatocellular carcinoma cells, Environ Sci Pollut Res Int., 25(28), 27899-27908.
Elmore, S., 2007, Apoptosis: a review of programmed cell death, Toxicologic Pathology, 35(4), 495- 516.
Evan, G.I. and Vousden, K.H., 2001, Proliferation, cell cycle and apoptosis in cancer, Nature, 411(6835), 342-348.
Forman, H.J. and Zhang, H., 2021, Targeting oxidative stress in disease: promise and limitations of antioxidant therapy, Nature Reviews Drug Discovery, 20(9), 689-709.
Fraga, C.G., Croft, K.D., Kennedy, D.O., and Tomas-Barberan, F.A., 2019, The effects of polyphenols and other bioactives on human health, Food & Function, 10(2), 514-528.
Frei, B., Stocker, R., and Ames, B.N., 1988, Antioxidant defenses and lipid peroxidation in human blood plasma, Proceedings of the National Academy of Sciences of the United States of America, 85(24), 9748-9752.
Fukai, T. and Ushio-Fukai, M., 2011, Superoxide dismutases: role in redox signaling, vascular function, and diseases, Antioxidants & Redox Signaling, 15(6), 1583-1606.
Galati, G. and O’Brien, P.J., 2004, Potential toxicity of flavonoids and other dietary phenolics: significance for their chemopreventive and anticancer properties, Free Radical Biology & Medicine, 37(3), 287-303.
Gordaliza, M., 2007, Natural products as leads to anticancer drugs, Clinical & Translational Oncology, 9(12), 767-776.
Grasso, S., Brunton, N.P., Lyng, J.G., Lalor, F., and Monahan, F.J., 2014, Healthy processed meat products-regulatory, reformulation and consumer challenges, Trends in Food Science & Technology, 39(1), 4-17.
Harvey, A.L., Edrada-Ebel, R., and Quinn, R.J., 2015, The re-emergence of natural products for drug discovery in the genomics era, Nature Reviews Drug Discovery, 14(2), 111-129.
Hassan, M., Watari, H., AbuAlmaaty, A., Ohba, Y., and Sakuragi, N., 2014, Apoptosis and molecular targeting therapy in cancer, BioMed Research International, 2014, 150845.
Hunger, S.P. and Mullighan, C.G., 2015, Acute lymphoblastic leukemia in children, The New England Journal of Medicine, 373(16), 1541- 1552.
Ighodaro, O.M. and Akinloye, O.A., 2018, First line defence antioxidants-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxi¬dase (GPX): their fundamental role in the entire antioxidant defence grid, Alexandria Journal of Medicine, 54(4), 287-293.
Jomova, K., Raptova, R., Alomar, S.Y., Alwasel, S.H., Nepovimova, E., Kuca, K., et al., 2023, Reactive oxygen species, toxicity, oxidative stress, and antioxidant: chronic, diseases and aging, Archives of Toxicology, 97(10), 2499-2574.
Khader, M. and Eckl, P.M., 2014, Thymoquinone: an emerging natural drug with a wide range of medical applications, Iranian Journal of Basic Medical Sciences, 17(12), 950-957.
Kurata, M., Suzuki, M., and Agar, N.S., 1993, Antioxidant systems and erythrocyte life-span in mammals, Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 106(3), 477-487.
Lambert, J.D., Sang, S., and Yang, C.S., 2007, Possible controversy over dietary polyphenols: benefits vs risks, Chemical Research in Toxicology, 20(4), 583-585.
Lichota, A. and Gwozdzinski, K., 2018, Anticancer activity of natural compounds from plant and marine environment, International Journal of Molecular Sciences, 19(11), 3533.
Lim, T.K., 2013, Edible Medicinal and Non-Medicinal Plants. Vol. 5. Dordrecht: Springer.
Liu, R.H., 2004, Potential synergy of phytochemicals in cancer prevention: mechanism of action, The Journal of Nutrition, 134(12 Suppl), 3479S-3485S.
Maleyki, M.J.A. and Ismail, A., 2010, Antioxidant properties of cocoa powder, Journal of Food Biochemistry, 34(1), 111-128.
Martínez-Pinilla, E., Oñatibia-Astibia, A., and Franco, R., 2015, The relevance of theobromine for the beneficial effects of cocoa consumption, Frontiers in Pharmacology, 6, 30.
Meiyanto, E., Hermawan, A., and Anindyajati, 2012, Natural products for cancer-targeted therapy: citrus flavonoids as potent chemopreventive agents, Asian Pacific Journal of Cancer Prevention, 13(2), 427-436.
Meyer, B.N., Ferrigni, N.R., Putnam, J.E., Jacobsen, L.B., Nichols, D.E., and McLaughlin, J.L., 1982, Brine shrimp: a convenient general bioassay for active plant constituents, Planta Medica, 45(5), 31-34.
Mhaidat, N.M., Abu-Zaiton, A.S., Alzoubi, K.H., et al., 2017, Antioxidant properties of raw and cooked lentils on oxidative damage in human erythrocytes in vitro, Journal of Clinical and Diagnostic Research, 11(4), BC14-BC17.
Mukherjee, P.K., Harwansh, R.K., Bahadur, S., Banerjee, S., Kar, A., Chanda, J., et al., 2017, Development of Ayurveda-tradition to trend, Journal of Ethnopharmacology, 197, 10-24.
Murakami, A., Ashida, H., and Terao, J., 2008, Multitargeted cancer prevention by quercetin, Cancer Letters, 269(2), 315-325.
Newman, D.J. and Cragg, G.M., 2020, Natural products as sources of new drugs over the nearly four decades from 01/1981 to 09/2019, Journal of Natural Products, 83(3), 770-803.
Niki, E., 2009, Lipid peroxidation: physiological levels and dual biological effects, Free Radical Biology & Medicine, 47(5), 469-484.
Nzekoue, F.K., Scheili, E., Caprioli, G., et al., 2020, Cocoa bean shells (Theobroma cacao L.): going from a food by-product to a health-promoting ingredient, Food Chemistry, 310, 125948.
Okiyama, D.C.G., Navarro, S.L.B., and Rodrigues, C.E.C., 2017, Cocoa shell and its compounds: applications in the food industry, Trends in Food Science & Technology, 63, 103-112.
Oo, T.N., Smith, T.J., and Bhatt, D.L., 2021, Tannins and their bioactivities, Molecules, 26(8), 2178.
Othman, A., Ismail, A., Ghani, N.A., and Adenan, I., 2007, Antioxidant capacity and phenolic content of cocoa beans, Food Chemistry, 100(4), 1523-1530.
Panche, A.N., Diwan, A.D., and Chandra, S.R., 2016, Flavonoids: an overview, Journal of Nutritional Science, 5, e47.
Pietta, P.G., 2000, Flavonoids as antioxidants, Journal of Natural Products, 63(7), 1035-1042.
Pizzino, G., Irrera, N., Cucinotta, M., Pallio, G., Mannino, F., Arcoraci, V., et al., 2017, Oxidative stress: harms and benefits for human health, Oxidative Medicine and Cellular Longevity, 2017, 8416763.
Prayong, P., Barusrux, S., and Weerapreeyakul, N., 2008, Cytotoxic activity screening of some indigenous Thai plants, Fitoterapia, 79(7-8), 598-601.
Procházková, D., Boušová, I., and Wilhelmová, N., 2011, Antioxidant and prooxidant properties of flavonoids, Fitoterapia, 82(4), 513-523.
Pui, C.H., Yang, J.J., Bhakta, N., and Rodriguez-Galindo, C., 2018, Global efforts toward the cure of childhood acute lymphoblastic leukaemia, The Lancet Child & Adolescent Health, 2(6), 440-454.
Rifai, L. and Saleh, F.A., 2020, A review on acrylamide in food: occurrence, toxicity, and mitigation strategies, International Journal of Toxicology, 39(2), 93-102.
Riss, T.L., Moravec, R.A., Niles, A.L., Duellman, S., Benink, H.A., Worzella, T.J., et al., 2016, Cell viability assays. In: Sittampalam GS, et al., eds. Assay Guidance Manual. Bethesda: Eli Lilly/NIH.
Rojo de la Vega, M., Krajisnik, A., Zhang, D.D., and Wondrak, G.T., 2017, Targeting NRF2 for improved skin barrier function and photoprotection: Focus on the achiote-derived apocarotenoid bixin, Nutrients, 9(12), 1371.
Russo, M., Milito, A., Spagnuolo, C., Carbone, V., Rosen, A., Monasi, P., et al., 2017, CK2 and PI3K are direct molecular targets of quercetin in chronic lymphocytic leukaemia, Oncotarget, 8(26), 42571-42587.
Saeed, N., Khan, M.R., and Shabbir, M., 2012, Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L., BMC Complementary and Alternative Medicine, 12, 221.
Santi, A., Menezes, C., Duarte, M.M. F., Leitemperger, J., Lopes, T., and Loro, V.L., 2011, Oxidative stress biomarkers and acetylcholinesterase activity in human erythrocytes exposed to clomazone in vitro, Interdisciplinary Toxicology, 4,(3), 149-153.
Schieber, M. and Chandel, N.S., 2014, ROS function in redox signaling and oxidative stress, Current Biology, 24(10), R453-R462.
Smeriglio, A., Barreca, D., Bellocco, E., and Trombetta, D., 2017, Proanthocyanidins and hydrolysable tannins: occurrence, biochemistry and biological activity, Phytochemistry Reviews, 16(2), 337-354.
Spagnuolo, C., Moccia, S., and Russo, G.L., 2018, Anti-inflammatory effects of flavonoids in neurodegenerative disorders, European Journal of Medicinal Chemistry, 153, 105-115.
Spigno, G., Tramelli, L., and De Faveri, D.M., 2007, Effects of extraction time, temperature and solvent on concentration and antioxidant activity of grape marc phenolics, Journal of Food Engineering, 81(1), 200-208.
Stalikas, C.D., 2007, Extraction, separation, and detection methods for phenolic acids and flavonoids, Journal of Separation Science, 30(18), 3268-3295.
Steliarova-Foucher, E., Colombet, M., Ries, L.A.G., Moreno, F., Dolya, A., Bray, F., et al., 2017, International incidence of childhood cancer, 2001-10: a population-based registry study, The Lancet. Oncology, 18(6), 719-731.
Thakkar, A., Sutaria, D., Grandhi, B.K., Wang, J., and Prabhu, S., 2013, The molecular mechanism of action of aspirin, curcumin and sulfora¬phane combinations in the chemoprevention of pancreatic cancer, Oncology Reports, 29(4), 1671-1677.
Tsao, R., 2010, Chemistry and biochemistry of dietary polyphenols, Nutrients, 2(12), 1231-1246.
Tsikas, D., 2017, Assessment of lipid peroxidation by measuring malondialdehyde (MDA) and relatives in biological samples: analytical and biological challenges, Analytical Biochemistry, 524, 13- 30.
Tungmunnithum, D., Thongboonyou, A., Pholboon, A., and Yangsabai, A., 2018, Flavonoids and other phenolic compounds from medicinal plants for pharmaceutical and medical aspects: an overview, Medicines, 5(3), 93.
Vichai, V. and Kirtikara, K., 2006, Sulforhodamine B colorimetric assay for cytotoxicity screening, Nature Protocols, 1(3), 1112-1116.
Wang, Y., Branicky, R., Noë, A., and Hekimi, S., 2018, Superoxide dismutases: dual roles in controlling ROS damage and regulating ROS signaling, The Journal of Cell Biology, 217(6), 1915-1928.
Xu, D., Hu, M.J., Wang, Y.Q., and Cui, Y.L., 2019, Antioxidant activities of quercetin and its complexes for medicinal application, Molecules, 24(6), 1123.
Yin, H., Xu, L., and Porter, N.A., 2011, Free radical lipid peroxidation: mechanisms and analysis, Chemical Reviews, 111(10), 5944-5972.
Yin, S.Y., Wei, W.C., Jian, F.Y., and Yang, N.S., 2013, Therapeutic applications of herbal medicines for cancer patients, Evidence-Based Complementary and Alternative Medicine, 2013, 302426.
Zamorano, J.L., Lancellotti, P., Muñoz, D.R., Aboyans, V., Asteggiano, R., Galderisi, M., et al., 2016, ESC Position Paper on cancer treatments and cardiovascular toxicity developed under the auspices of the ESC Committee for Practice Guidelines: The Task Force for cancer treatments and cardiovascular toxicity of the European Society of Cardiology (ESC), European Heart Journal, 37(36), 2768-2801.
DOI: http://dx.doi.org/10.14499/indonesianjcanchemoprev16iss3pp205-220
Copyright (c) 2025 Indonesian Journal of Cancer Chemoprevention
Indexed by:
Indonesian Society for Cancer Chemoprevention
