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In Vitro Antioxidant and Antiproliferative Activities of Novel Orange Peel Extract and It's Fractions on Leukemia HL-60 Cells

  • Diab, Kawthar AE (Genetics and Cytology Department, National Research Centre) ;
  • Shafik, Reham Ezzat (Chemistry of Tanning Materials and Leather Technology Department, National Research Centre) ;
  • Yasuda, Shin (Laboratory of Food Functional Chemistry, School of Agriculture, Tokai University)
  • Published : 2015.11.04

Abstract

In the present work, novel orange peel was extracted with 100%EtOH (ethanol) and fractionated into four fractions namely F1, F2, F3, F4 which were eluted from paper chromatographs using 100%EtOH, 80%EtOH, 50%EtOH and pure water respectively. The crude extract and its four fractions were evaluated for their total polyphenol content (TPC), total flavonoid content (TFC) and radical scavenging activity using DPPH (1,1-diphenyl-2-picrylhydrazyl) assay. Their cytotoxic activity using WST assay and DNA damage by agarose gel electrophoresis were also evaluated in a human leukemia HL-60 cell line. The findings revealed that F4 had the highest TPC followed by crude extract, F2, F3 and F1. However, the crude extract had the highest TFC followed by F4, F3, F2, and F1. Depending on the values of $EC_{50}$ and trolox equivalent antioxidant capacity, F4 possessed the strongest antioxidant activity while F1 and F2 displayed weak antioxidant activity. Further, incubation HL-60 cells with extract/fractions for 24h caused an inhibition of cell viability in a concentration-dependent manner. F3 and F4 exhibited a high antiproliferative activity with a narrow range of $IC_{50}$ values ($45.9-48.9{\mu}g/ml$). Crude extract exhibited the weakest antiproliferative activity with an $IC_{50}$ value of $314.89{\mu}g/ml$. Analysis of DNA fragmentation displayed DNA degradation in the form of a smear-type pattern upon agarose gel after incubation of HL-60 cells with F3 and F4 for 6 h. Overall, F3 and F4 appear to be good sources of phytochemicals with antioxidant and potential anticancer activities.

Keywords

References

  1. Agrawal PK, Bansal MC, (1989). Flavonoid glycosides. in: C-13 NMR of flavonoids, agrawal PK (Ed.), elsevier, amesterdam, Oxford, New York, Tokyo, p. 283-355.
  2. Alam MN, Bristi NJ, Rafiquzzaman M (2013). Review on in vivo and in vitro methods evaluation of antioxidant activity. Saudi Pharm J, 21, 143-52. https://doi.org/10.1016/j.jsps.2012.05.002
  3. Al-Duais M, Muller L, Bohm V, et al (2009). Antioxidant capacity and total phenolics of cyphostemma digitatum before and after processing: use of different assays. Eur Food Res Technol. 228, 813-21. https://doi.org/10.1007/s00217-008-0994-8
  4. Amic D, Davidovic-Amic D, Beslo D, et al (2007). SAR and QSAR of the Antioxidant activity of flavonoids. Curr Med Chem, 14, 827-45. https://doi.org/10.2174/092986707780090954
  5. Atjanasuppat K, Wongkham W, Meepowpan P, et al (2009). In vitro screening for anthelmintic and antitumour activity of ethnomedicinal plants from Thailand. J Ethnopharmacol. 123, 475-82. https://doi.org/10.1016/j.jep.2009.03.010
  6. Bampidis VA, Robinson PH (2006). Citrus by-products as ruminant feeds: a review. Animal Feed Sci Technol, 128, 175-217. https://doi.org/10.1016/j.anifeedsci.2005.12.002
  7. Barreca D, Bisignano C, Ginestra G, et al (2013). Polymethoxylated, c- and o-glycosyl flavonoids in tangelo (citrus reticulata${\times}$citrus paradisi) juice and their influence on antioxidant properties. Food Chem. 141, 1481-8 https://doi.org/10.1016/j.foodchem.2013.03.095
  8. Barros HR, Ferreira TA, Genovese MI, (2012). Antioxidant capacity and mineral content of pulp and peel from commercial cultivars of citrus from Brazil. Food Chem, 134, 1892-8. https://doi.org/10.1016/j.foodchem.2012.03.090
  9. Berridge MV, Herst PM, Tan AS, (2005). Tetrazolium dyes as tools in cell biology: New insights into their cellular reduction. Biotechnol Annu Rev, 11, 127-52. https://doi.org/10.1016/S1387-2656(05)11004-7
  10. Boik J, (2001). Natural compounds in cancer therapy. promising nontoxic antitumor agents from plants & other natural sources. princeton, minn, USA: oregon medical press; 1st edition.
  11. Charoensinphon N, Qiu P, Dong P, et al (2013). 5-demethyltangeretin inhibits human nonsmall cell lung cancer cell growth by inducing G2/M cell cycle arrest and apoptosis. Mol Nutr Food Res, 57, 2103-11. https://doi.org/10.1002/mnfr.201300136
  12. Chen C, Ono M, Takeshima M, et al (2014). Antiproliferative and apoptosis-inducing activity of nobiletin against three subtypes of human breast cancer cell lines. Anticancer Res. 34, 1785-92.
  13. Cicco N, Lanorte MT, Paraggio M, et al (2009). A reproducible, rapid and inexpensive Folin-Ciocalteu micro-method in determining phenolics of plant methanol extracts. Microchem J, 91, 107-10. https://doi.org/10.1016/j.microc.2008.08.011
  14. Constantinou C, Papas KA, Constantinou AI, (2009). Caspaseindependent pathways of programmed cell death: The unraveling of new targets of cancer therapy? Curr Cancer Drug Targets, 9, 717-28. https://doi.org/10.2174/156800909789271512
  15. Cragg GM, Grothaus PG, Newman DJ, (2009). Impact of natural products on developing new anti-cancer agents. Chem Rev, 109, 3012-43. https://doi.org/10.1021/cr900019j
  16. Dai J, Mumper RJ, (2010). Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules, 15, 7313-52 https://doi.org/10.3390/molecules15107313
  17. Demir E, Kocaoglu S, Cetin H, et al (2009). Antigenotoxic effects of citrus aurentium l. fruit peel oil on mutagenicity of two alkylating agents and two metals in the drosophila wing spot test. Environ Mol Mutagen, 50, 483-8. https://doi.org/10.1002/em.20484
  18. Dong Y, Cao A, Shi J, et al (2014). Tangeretin, a citrus polymethoxyflavonoid, induces apoptosis of human gastric cancer AGS cells through extrinsic and intrinsic signaling pathways. Oncol Rep, 31, 1788-94.
  19. Elmore S, (2007). Apoptosis: a review of programmed cell death. Toxicol Pathol, 35, 495-516. https://doi.org/10.1080/01926230701320337
  20. FAO 2011: http://faostat.fao.org/site/339/default.aspx
  21. Garcia-Salas P, Morales-Soto A, Segura-Carretero A, et al (2010). Phenolic compound extraction systems for fruit and vegetable samples. Molecules, 15, 8813-26. https://doi.org/10.3390/molecules15128813
  22. Ghasemi K, Ghasemi Y, Ebrahimzadeh MA, (2009). Antioxidant activity, phenol and flavonoid contents of 13 citrus species peels and tissues. Pak J Pharm Sci, 22, 277-81.
  23. Hamdan D, Ashour ML, Mulyaningsih S, et al (2013). Chemical composition of the essential oils of variegated pink-fleshed lemon (Citrus ${\times}$ limon L. Burm. f.) and their anti-inflammatory and antimicrobial activities. Z Naturforsch C, 68, 275-84. https://doi.org/10.5560/ZNC.2013.68c0275
  24. Harborne JB, (1973). Phytochemical methods, chapman and hall, Ltd., London, p. 49.
  25. Hegazy AE, Ibrahium MI, (2012). Antioxidant activities of orange peel extracts. World Appl Sci J, 18, 684-8.
  26. Heim KE, Tagliaferro AR, Bobilya DJ, (2002). Flavonoid antioxidants: chemistry, metabolism and structure-activity relationships. J Nutr Biochem, 13, 572-84. https://doi.org/10.1016/S0955-2863(02)00208-5
  27. Herald TJ, Gadgil P, Tilley M, (2012). High-throughput microplate assays for screening flavonoid content and DPPHscavenging activity in sorghum bran and flour. J Sci Food Agric, 92, 2326-31. https://doi.org/10.1002/jsfa.5633
  28. Hosseinimehr SJ, Karami M, (2005). Citrus extract modulates genotoxicity induced by cyclophosphamide in mice bone marrow cells. J Pharm Pharmacol, 57, 505-9. https://doi.org/10.1211/0022357055849
  29. Hsiao PC, Lee WJ, Yang SF, et al (2014). Nobiletin suppresses the proliferation and induces apoptosis involving MAPKs and caspase-8/-9/-3 signals in human acute myeloid leukemia cells. Tumour Biol, 35, 11903-11. https://doi.org/10.1007/s13277-014-2457-0
  30. Im SJ, Kim JH, Kim MY, (2014). Evaluation of bioactive components and antioxidant and anticancer properties of citrus wastes generated during bioethanol production. Nat Prod Commun, 9, 483-6.
  31. Jemal A, Bray F, Center MM, et al (2011). Global cancer statistics. CA Cancer J Clin, 61, 69-90. https://doi.org/10.3322/caac.20107
  32. Jeong JM, Choi CH, Kang SK, et al. (2007). Antioxidant and chemosensitizing effects of flavonoids with hydroxy and/or methoxy groups and structure-activity relationship. J Pharm Pharm Sci, 10, 537-46.
  33. Justino GC, Rodrigues M, Florencio MH, et al. (2009). Structure and antioxidant activity of brominated flavonols and flavanones. J Mass Spectrom, 44, 1459-68. https://doi.org/10.1002/jms.1630
  34. Katiyar C, Gupta A, Kanjilal S, et al (2012). Drug discovery from plant sources: An integrated approach. Ayu, 33, 10-9. https://doi.org/10.4103/0974-8520.100295
  35. Kotz JC, Treichel PM, Townsend JR (2011). Chemistry and chemical reactivity. cengage learning, nelson education, Ltd, 8th, USA, p, 501.
  36. Lai CS, Li S, Miyauchi Y, et al. (2013). Potent anti-cancer effects of citrus peel flavonoids in human prostate xenograft tumors. Food Funct, 4, 944-9. https://doi.org/10.1039/c3fo60037h
  37. Lashkari S, Taghizadeh A, (2013). Nutrient digestibility and evaluation of protein and carbohydrate fractionation of citrus by-products. J Anim Physiol Anim Nutr (Berl), 97, 701-9. https://doi.org/10.1111/j.1439-0396.2012.01312.x
  38. Lewinska A, Siwak J, Rzeszutek I, et al (2015). Diosmin induces genotoxicity and apoptosis in DU145 prostate cancer cell line. Toxicol In Vitro, 3, 417-25.
  39. Li S, Pan MH, Lai CS, et al (2007). Isolation and syntheses of polymethoxyflavones and hydroxylated polymethoxyflavones as inhibitors of HL-60 cell lines. Bioorg Med Chem, 15, 3381-9. https://doi.org/10.1016/j.bmc.2007.03.021
  40. Markham KR, Geiger H, (1994). 1H nuclear magnetic resonance spectroscopy of flavonoids and their glycosides in hexadeuterodimethylsulfoxide. In: The Flavonoids :advances in research since 1986, harbone , j.b. (ed.), chapman and hall, London, united kingdom, p. 441- 497.
  41. National Cancer Institute (NCI). Surveillance, epidemiology, and end results program website. Available: http://seer.cancer.gov/statfacts/html/leuks.html. Accessed 2014 May 19.
  42. Nogata Y, Sakamoto K, Shiratsuchi H, et al (2006). Flavonoid composition of fruit tissues of citrus species. Biosci Biotechnol Biochem, 70, 178-92. https://doi.org/10.1271/bbb.70.178
  43. Oboh G, Ademosun AO, (2012). Characterization of the antioxidant properties of phenolic extracts from some citrus peels. J Food Sci Technol, 49, 729-36. https://doi.org/10.1007/s13197-010-0222-y
  44. Oliveira AM, Pinheiro LS, Pereira CK, et al (2012). Total phenolic content and antioxidant activity of some malvaceae family species. Antioxidants, 1, 33-43. https://doi.org/10.3390/antiox1010033
  45. Park JH, Lee M, Park E, (2014). Antioxidant activity of orange flesh and peel extracted with various solvents. Prev Nutr Food Sci, 19, 291-8. https://doi.org/10.3746/pnf.2014.19.4.291
  46. Park KI, Park HS, Nagappan A, et al (2012). Induction of the cell cycle arrest and apoptosis by flavonoids isolated from Korean Citrus aurantium L. in non-small-cell lung cancer cells. Food Chem, 135, 2728-35. https://doi.org/10.1016/j.foodchem.2012.06.097
  47. Petrie K, Zelent A, Waxman S, (2009). Differentiation therapy of acute myeloid leukemia: past, present and future. Curr Opin Hematol, 16, 84-91. https://doi.org/10.1097/MOH.0b013e3283257aee
  48. Pisoschi M, Cheregi MC, Danet AF, (2009). Total antioxidant capacity of some commercial fruit juices: electrochemical and spectrophotometrical approaches. Molecules, 14, 480-93. https://doi.org/10.3390/molecules14010480
  49. Rover MR, Brown RC, (2013). Quantification of total phenols in bio-oil using the folin-ciocalteu method. J Anal Appl Pyrol, 104, 366-71. https://doi.org/10.1016/j.jaap.2013.06.011
  50. Siles Lopez AJ, Li Q, Thompson IP, (2010). Biorefinery of waste orange peel. Crit Rev Biotechnol, 30, 63-9. https://doi.org/10.3109/07388550903425201
  51. Spigno G, Tramelli L, De Faveri DM, (2007). Effects of extraction time, temperature and solvent on concentration and antioxidant activity of grape marc phenolics. J Food Engin, 81, 200-8. https://doi.org/10.1016/j.jfoodeng.2006.10.021
  52. Sroka Z, Fecka I, Cisowski W, (2005). Antiradical and anti-H2O2 properties of polyphenolic compounds from an aqueous peppermint extract. Z Naturforsch C, 60, 826-32.
  53. Sultana S, Asif HM, Nazar HM, et al (2014). Medicinal plants combating against cancer--a green anticancer approach. Asian Pac J Cancer Prev, 15, 4385-94. https://doi.org/10.7314/APJCP.2014.15.11.4385
  54. Tan TW, Tsai HY, Chen YF, et al. (2004). Induction of apoptosis in human promyelocytic leukemia HL-60 cells by ampelopsis cantoniensis crude extract. In Vivo, 18, 457-62.
  55. Tripoli E, Guardia ML, Giammanco S, et al (2007). Citrus flavonoids: molecular structure, biological activity and nutritional properties: a review. Food Chem, 104, 466-79. https://doi.org/10.1016/j.foodchem.2006.11.054
  56. Wang S, Meckling KA, Marcone MF, et al (2011). Synergistic, additive, and antagonistic effects of food mixtures on total antioxidant capacities. J Agric Food Chem, 59, 960-8. https://doi.org/10.1021/jf1040977
  57. Weng CJ, Yen GC, (2012). Flavonoids, a ubiquitous dietary phenolic subclass, exert extensive in vitro anti-invasive and in vivo anti-metastatic activities. Cancer Metastasis Rev, 31, 323-51. https://doi.org/10.1007/s10555-012-9347-y
  58. Wolfe KL, Liu RH, (2008). Structure-activity relationships of flavonoids in the cellular antioxidant activity assay. J Agric Food Chem, 56, 8404-11. https://doi.org/10.1021/jf8013074
  59. Woodman OL, Meeker WF, Boujaoude M, (2005). Vasorelaxant and antioxidant activity of flavonols and flavones: structureactivity relationships. J Cardiovasc Pharmacol, 46, 302-9. https://doi.org/10.1097/01.fjc.0000175431.62626.07
  60. Yang X, Kang SM, Jeon BT, (2011). Isolation and identification of an antioxidant flavonoid compound from citrus-processing by-product. J Sci Food Agric, 91, 1925-7. https://doi.org/10.1002/jsfa.4402
  61. Yoon JH, Lim TG, Lee KM, et al (2011). Tangeretin reduces ultraviolet B (UVB)-induced cyclooxygenase-2 expression in mouse epidermal cells by blocking mitogen-activated protein kinase (MAPK) activation and reactive oxygen species (ROS) generation. J Agric Food Chem, 59, 222-8. https://doi.org/10.1021/jf103204x
  62. Yumnam S, Park HS, Kim MK, et al (2014). Hesperidin induces paraptosis like cell death in hepatoblatoma, HepG2 cells: involvement of ERK1/2 MAPK. PLoS One, 9, 101321. https://doi.org/10.1371/journal.pone.0101321