Determination of The Potential Antioxidant Activity of Isolated Piperine from White Pepper Using DPPH, ABTS, and FRAP Methods

Nindya Kusumorini(1), Akhmad Kharis Nugroho(2*), Suwijiyo Pramono(3), Ronny Martien(4)

(1) Doctoral Program in Pharmaceutical Science, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta 55281 Indonesia
(2) Department of Pharmaceutics, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta 55281 Indonesia
(3) Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta 55281 Indonesia
(4) Department of Pharmaceutics, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta 55281 Indonesia
(*) Corresponding Author


White pepper (Piper nigrum L) is a native plant of Indonesia that has been used for generations as a spice and traditional medicine. White pepper contains the main alkaloid compound, namely piperine which has antioxidant activity as shown in the previous studies. The purpose of this study was to determine the potential antioxidant activity of isolated piperine compared with n-hexane extract of white pepper using DPPH, ABTS, and FRAP test methods. The isolated piperine was obtained from extraction using Soxhlet with n-hexane as a solvent, followed by purification with cyclohexane. The evaluation of antioxidant activity was carried out using the DPPH method to see free radicals, while the ABTS and FRAP methods evaluated antioxidant activity. Antioxidant activity was expressed as IC50 value and Trolox compound was used as a positive control of antioxidant activity. The analysis results show isolated piperine had antioxidant activity in the ABTS test with the results of 4.35 ± 0.004 mg/mL and 2.53 ± 0.08 mg/mL, respectively  and FRAP test was 10.53 mol TE/g sample and 6.86 mol TE/g sample, respectively. Isolated piperine and n-hexane extract of white pepper did not show their activity as free radical scavengers. The antioxidant activity of isolated piperine was lower than that of n-hexane extract of white pepper. The presence of other compounds in white pepper shows a synergistic interaction in increasing the antioxidant activity of white pepper extract.


white pepper; piperine; antioxidant; DPPH; ABTS; FRAP

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Benzie, I.F.F., and Strain, J.J., 1996. The Ferric Reducing Ability of Plasma (FRAP) as a Measure of “Antioxidant Power”: The FRAP Assay. Analytical Biochemistry, 239 (1): 70–76.

Dayan, N., 2008. Skin Aging Handbook: An Integrated Approach to Biochemistry and Product Development. William Andrew, Norwich, New York.

Dejian, H., Boxin, O., and Prior, R.L., 2005. The Chemistry behind Antioxidant Capacity Assays (world). American Chemical Society. DOI:10.1021/jf030723c

Floegel, A., Kim, D.O., Chung, S.J., Koo, S.I., and Chun, O.K., 2011. Comparison of ABTS/DPPH assays to measure antioxidant capacity in popular antioxidant-rich US foods. Journal of Food Composition and Analysis, 24 (7): 1043–1048. DOI:10.1016/j.jfca.2011.01.008

Gülçin, I., Mshvildadze, V., Gepdiremen, A., and Elias, R., 2004. Antioxidant activity of saponins isolated from ivy: Alpha-hederin, hederasaponin-C, hederacolchiside-E and hederacolchiside-F. Planta Medica, 70 (6): 561–563. DOI:10.1055/s-2004-827158

Kedare, S.B., and Singh, R.P., 2011. Genesis and development of DPPH method of antioxidant assay. Journal of Food Science and Technology, 48 (4): 412–422. DOI:10.1007/s13197-011-0251-1

Koksal, E., and Gülçi̇n, İ., 2008. Antioxidant activity of cauliflower (Brassica oleracea L.). Turkish Journal of Agriculture and Forestry. DOI:10.3906/tar-0707-46

Kumar, S., and Pandey, A.K., 2013. Chemistry and biological activities of flavonoids: An overview. The Scientific World Journal. DOI:10.1155/2013/162750

Kusumorini, N., Nugroho, A.K., Pramono, S., and Martien, R., 2021. Development of New Isolation and Quantification Method of Piperine from White Pepper Seeds (Piper Nigrum L) Using A Validated HPLC. Indonesian Journal of Pharmacy, 158–165. DOI:22146/ijp.866

Litescu, S.C., Eremia, S.A.V., Diaconu, M., Tache, A., and Radu, G.L., 2011. Biosensors Applications on Assessment of Reactive Oxygen Species and Antioxidants, In Environmental Biosensors. Intech Open. DOI:10.5772/16250

Mareček, V., Mikyška, A., Hampel, D., Čejka, P., Neuwirthová, J., Malachová, A., and Cerkal, R., 2017. ABTS and DPPH methods as a tool for studying antioxidant capacity of spring barley and malt. Journal of Cereal Science, 73: 40–45. DOI:10.1016/j.jcs.2016.11.004

Müller, L., Fröhlich, K., and Böhm, V., 2011. Comparative antioxidant activities of carotenoids measured by ferric reducing antioxidant power (FRAP), ABTS bleaching assay (αTEAC), DPPH assay and peroxyl radical scavenging assay. Food Chemistry, 129 (1): 139–148. DOI:1016/j.foodchem.2011.04.045

Nahak, G., and Sahu, R.K., 2011, Phytochemical Evaluation and Antioxidant activity of Piper cubeba and Piper nigrum. Journal of Applied Pharmaceutical Science, 08: 153–157. DOI:10.1016/j.lwt.2012.07.036

Ou, B., Huang, D., Hampsch-Woodill, M., Flanagan, J.A., and Deemer, E.K., 2002. Analysis of antioxidant activities of common vegetables employing oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays: A comparative study. Journal of Agricultural and Food Chemistry, 50 (11): 3122–3128. DOI:10.1021/jf0116606

Pellegrini, N., Serafini, M., Colombi, B., Del, R.D., Salvatore, S., Bianchi, M., and Brighenti, F., 2003. Total Antioxidant Capacity of Plant Foods, Beverages and Oils Consumed in Italy Assessed by Three Different In Vitro Assays. The Journal of Nutrition, 133 (9): 2812–2819. DOI:10.1093/jn/133.9.2812

Purwantiningsih, P., Murwanti, R., and Hakim, L., 2019. Antioxidant Activities of n-Hexane Soluble and Insoluble Fraction, Ethyl Acetate Soluble and Insoluble Fraction from Ethanol Extract of Sambung Nyawa Leaf (Gynura procumbens (Lour.) Merr.). Majalah Obat Tradisional, 24 (2): 91. DOI:10.22146/mot.43743

Ramadan-Hassanien, M.F., 2008. Total antioxidant potential of juices, beverages and hot drinks consumed in Egypt screened by DPPH in vitro assay. Grasas y Aceites, 59 (3): 254–259. DOI:10.3989/gya.2008.v59.i3.516

Rekka, E.A., Kourounakis, A.P., and Kourounakis, P.N., 1996. Investigation of the effect of chamazulene on lipid peroxidation and free radical processes. Research Communications in Molecular Pathology and Pharmacology, 92 (3): 361–364.

Shui, G., and Leong, L.P., 2006. Residue from star fruit as valuable source for functional food ingredients and antioxidant nutraceuticals. Food Chemistry, 97 (2): 277–284. DOI:10.1016/j.foodchem.2005.03.048

Singh, S., Kapoor, I.P.S., Singh, G., Schuff, C., De Lampasona, M.P., and Catalan, C.A.N., 2013. Chemistry, Antioxidant and Antimicrobial Potentials of White Pepper (Piper nigrum L.) Essential Oil and Oleoresins. Proceedings of the National Academy of Sciences. India Section B: Biological Sciences, 83 (3): 357–366. DOI:10.1007/s40011-012-0148-4

Soare, J.R., Dinis, T.C.P., Cunha, A.P., and Almeida, L., 2009. Antioxidant Activities of Some Extracts of Thymus zygis. Free Radical Research, 26: 469–478. DOI:10.3109/10715769709084484

Sun, Y.E., Wang, W.D., Chen, H.W., and Li, C., 2011 Autoxidation of unsaturated lipids in food emulsion. Critical Reviews in Food Science and Nutrition, 51 (5): 453–466. DOI:10.1080/10408391003672086

Thaipong, K., Boonprakob, U., Crosby, K., Cisneros-Zevallos, L., and Hawkins, B.D., 2006 Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts. Journal of Food Composition and Analysis, 19 (6): 669–675. DOI:10.1016/j.jfca.2006.01.003

Tirzitis, G., and Bartosz, G., 2010. Determination of antiradical and antioxidant activity: Basic principles and new insights. Acta Biochimica Polonica, 57 (2): 139–142.

Vijayakumar, R.S., and Nalini, N., 2006. Lipid-Lowering Efficacy of Piperine from Piper Nigrum L. in High-Fat Diet and Antithyroid Drug-Induced Hypercholesterolemic Rats. Journal of Food Biochemistry, 30 (4): 405–421. DOI:10.1111/j.1745-4514.2006.00074.x

Vijayakumar, R.S., Surya, D., and Nalini, N., 2004. Antioxidant efficacy of black pepper (Piper nigrum L.) and piperine in rats with high fat diet induced oxidative stress. Redox Report: Communications in Free Radical Research, 9 (2): 105–110. DOI:10.1179/135100004225004742

Widyastuti, N., 2010. Measurement of Antioxidant Activity by Cuprac, DPPH, and Frap Methods and Their Correlation with Phenols and Flavonoids in Six Plants. [Disertation], Institut Pertanian Bogor, Bogor. [Indonesia]

Yamaguchi, T., Takamura, H., Matoba, T., and Terao, J., 1998. HPLC Method for Evaluation of the Free Radical-scavenging Activity of Foods by Using 1,1-Diphenyl-2-picrylhydrazyl. Bioscience, Biotechnology, and Biochemistry, 62 (6): 1201–1204. DOI:10.1271/bbb.62.1201

Zarai, Z., Boujelbene, E., Ben, S.N., Gargouri, Y., and Sayari, A., 2013. Antioxidant and antimicrobial activities of various solvent extracts, piperine and piperic acid from Piper nigrum. LWT - Food Science and Technology, 50 (2): 634–641. DOI:10.1016/j.lwt.2012.07.036


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