Evaluation of antioxidant activity of various parts of broccoli

國立屏東科技大學食品科學國際碩士學位學程 International Master’s Degree Program in Food Science National Pingtung University of Science and Technology 碩士學位論文 Master’s Thesis 青花菜不同部位之抗氧化力評估 Evaluation of antioxidant activity of various parts of broccoli 指導教授 (Advisor): 謝寶全 博士 (Pao-Chuan Hsieh, Ph.D.) 研究生 (Student): 黎青寧 (Le Thanh Ninh) 中華民國 104 年 月 15 日 July 15, 2015 摘要 學號: G10280008 論文題目: 青花菜不同部位之抗氧化力評估 總頁數: 91 學校名稱: 國立屏東科技大學 系所別: 食品科學國際碩士學位學程 畢業時間及摘要別：103學年度第2學期碩士學位論文摘要 研究生: 黎青寧 指導教授: 謝寶全博士 論文摘要內容: 青花菜是一種具有高抗氧化活性的蔬菜。抗氧化劑可以清除自由基並保 護人體不受氧化傷害，而氧化壓力是造成心臟、心血管及慢性疾病還有 各種形式癌症的主要原因，本研究的主要目的是調查青花菜不同部位的 抗氧化活性。取青花菜小花、葉、種子和嫩芽（分 3、5、7、10 和 12 天）四種樣品，乾燥後再經由不同濃度的乙醇和熱水萃取，再使用多種 分析（ABTS、DPPH、還原力測試）來測定其抗氧化活性。本研究還測 定了總酚、總類黃酮及維生素 C 的含量。之後，將四組樣品的萃取液進 行細胞試驗包含肝癌細胞（HepG2 細胞）、人類肺癌細胞（A549）和小 鼠肝臟正常細胞（FL83B）之 MTT 試驗。結果顯示，在抗氧化劑的清 除力中，利用 80% 濃度的乙醇是具有最高能力的萃取溶劑。而在四組樣 品中青花菜嫩芽有最高的抗氧化活性，不論是在 DPPH（91％清除力、 ABTS（6909.5 µmol TE /100g）及還原力測試，也具有較高量的酚類、 類黃酮和維生素 C 含量，其含量分別為 2926.6（GAE），730（CE）和 378.3（AA）mg/100g。在不同濃度（50 - 500 µg/mL）的嫩芽和種子萃 取物中，顯示出樣品可抑制腫瘤細胞的增殖活性（40-50％ cell viability） 和幫助正常細胞生長的能力（約 120％ cell viability）。不同樣品的萃取 物其 IC50 值約在 100 - 400µg/mL 間。這項研究可能對消費者的食品選 I 擇有顯著的影響，如再進行深入的研究後，可對未來相關的商業產品提 供參考資訊。 關鍵字：青花菜、抗氧化活性、增殖、腫瘤細胞 II Abstract Student ID: G10280008 Title of Thesis: Evaluation of antioxidant activity of various parts of broccoli Total Pages: 91 Name of Institute: National Pingtung University of Science and Technology Department: International Master’s Degree Program in Food Science Graduate Date: July 15, 2015 Degree conferred: Master Degree Name of Student: Le Thanh Ninh Advisor: Pao-Chuan Hsieh, Ph.D The Contents of Abstract in This Thesis: Broccoli (Brassica oleracea L) is a potential vegetable with high antioxidant activity Antioxidants can scavenge free radicals and protect the human body from oxidative stress, which is the main cause of heart, cardiovascular and chronic diseases, and various forms of cancer Therefore, the main objective of the study is to investigate antioxidant activity of broccoli Broccoli was used as a testing material and sampled in four groups, including floret, leave, seed and sprout (3, 5, 7, 10 and 12 days) After drying and extraction by different concentrations of ethanol and hot water, the antioxidant activity of four groups were measured using diverse analyses including 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS); 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and reducing power assays The study also estimated total phenolics, total flavonoids and vitamin C contents in samples Thereafter, the effect of broccoli extracts on proliferation of cell lines was determined by MTT test in liver cancer cell (HepG2), lung cancer cell (A549) and liver normal cell (FL83B) The results indicated that 80% ethanol was the solvent with highest ability in antioxidant scavenging assays In four groups of sample, broccoli sprout extracted by 80% ethanol had the highest antioxidant activity analyzed by DPPH (91% scavenging), ABTS (6909.5 µmol TE/100g) and reducing power (3.91 absorbance at 700nm) III assays; and contained higher amounts of phenolic, flavonoid and vitamin C at 2926.6 (GAE), 730 (CE), and 378.3 (AA) mg/100g respectively Sprout and seed extracts showed the highest anti-proliferative activity of tumor cells (4050% cell viability) and the highest proliferative activity of normal cell (around 120% cell viability) in different concentrations (50 – 500 µg/mL) The IC50 values of different sample extracts were from approximately 100 400 µg/mL The study might have a significant impact on consumers’ food selections and provide scientific information for in-depth research about commercial products Keywords: broccoli, antioxidant activity, proliferation, tumor cell IV Acknowledgements Firstly, I would like to take this opportunity to express my sincere appreciation and gratitude to the National Pingtung University of Science and Technology (NPUST) for funding my studies in Taiwan, and the International Master’s Degree Program in Food Science (IMAFS) for providing study facilities and new education experience I would like to sincerely express my appreciation and gratitude to Prof Pao-Chuan Hsieh, my advisor, for his advice, encouragement and valuable suggestions throughout my Master program I also would like to acknowledge my thanks to Dr Jia-Hsin Guo and Dr Chiu-Hsia Chiu for their advice and motivation to studying I am very grateful to all of my laboratory members (FS206) and my friends at “IMAFS” for their friendship, supports, pushing me up and special experiences in Taiwan Finally, I wish to express my deepest appreciation to my beloved family for their unconditional love, support and motivation during the years of study abroad and this thesis is dedicated to them Le Thanh Ninh V Table of Contents 摘要 I Abstract III Acknowledgements V Table of Contents VI List of Tables VIII List of Figures IX Introduction Literature Review 2.1 Broccoli 2.1.1 Origin and taxonomy 2.1.2 Health promoting compounds 2.1.3 Antioxidant properties 12 2.1.3.1 Water-soluble antioxidants 12 2.1.3.2 Lipid-soluble antioxidants 17 2.1.4 Biological activities and health 21 2.1.4.1 Antioxidant activities 23 2.1.4.2 Anticancer activities 27 2.1.4.3 Other activities 28 2.1.5 In vitro Broccoli cell-based studies 29 2.2 Antioxidants and antioxidant activity 31 2.2.1 Free radicals and oxidative stress 31 2.2.2 Natural antioxidants 34 2.2.3 Methodologies for antioxidant activity 37 2.2.3.1 Antioxidant activity screening assays 37 2.2.3.2 Antioxidant compounds analysis 39 2.2.4 In vitro effects of phenolics 41 Materials and Methods 43 3.1 Materials 43 3.1.1 Broccoli 43 VI 3.1.2 Cell lines 43 3.1.3 Chemicals 43 3.2 Methods 46 3.2.1 Preparation and drying of group samples 47 3.2.2 Samples extraction 47 3.2.3 Determination of total phenolic contents (TPC) 48 3.2.4 Determination of total flavonoid contents (TFC) 48 3.2.5 Determination of vitamins C contents 48 3.2.6 DPPH radical scavenging assay 49 3.2.7 ABTS radical cation decolorization assay 49 3.2.8 Reducing power assay 50 3.2.9 Cell lines and culture 50 3.2.10 Cell viability assay 51 3.2.11 Statistical analysis 51 Results and Discussion 52 4.1 Antioxidant compounds analysis 52 4.1.1 Total phenolics 52 4.1.2 Total flavonoids 55 4.1.3 Total vitamins C 58 4.2 Antioxidant capacity analysis 61 4.2.1 DPPH scavenging assay 61 4.2.2 ABTS assay 64 4.2.3 Reducing power assay 67 4.3 Proliferation on cell lines analysis 70 4.3.1 Proliferation on A549 cell line 70 4.3.2 Proliferation on HepG2 cell line 72 4.3.3 Proliferation on FL83B cell line 74 Conclusion 76 References 77 Biosketch of Author 91 VII List of Tables Table Ascorbic acid (AA) content of Brassica vegetables 14 Table Applications in the analysis of vitamins C in broccoli samples 15 Table Applications in the analysis of phenolic compounds in broccoli samples 17 Table Applications in the analysis of carotenoids in broccoli samples 20 Table Summary of works evaluating the antioxidant potential of broccoli 25 Table List of the important assays to screen antioxidant activity 38 Table List of the important techniques used for antioxidants analysis 40 VIII List of Figures Figure Morphology of sprouting broccoli Figure The published works in the last years related to determination of broccoli compounds Figure The extraction techniques used to analyze health promoting compounds of broccoli in the last five years Figure The health promoting compounds analyzed of broccoli in the last five years Figure The characterization techniques used to analyze health promoting compounds of broccoli in the last five years 10 Figure Summary of the different biological activities attributed to the health promoting compounds of broccoli in the last five years 22 Figure ORAC values of selected vegetables 26 Figure Structures of glucosinolate precursor (A) glucoraphanin and isothiocyanate hydrolysis product (B) sulforaphane 27 Figure Targets of free radicals 33 Figure 10 Natural antioxidants separated in classes 36 Figure 11 Morphology of FL83B cell at different magnifications 45 Figure 12 Morphology of HepG2 cell at different magnifications 45 Figure 13 Morphology of A549 cell at different magnifications 45 Figure 14 The flow chart of experimental design 46 Figure 15 Total phenolic content of broccoli fresh weight in solvent extractions of sprout in different sprouting times (a) and different parts (b) 53 Figure 16 Total phenolic content of broccoli dry weight in solvent extractions of sprout in different sprouting times (a) and different parts (b) 54 IX References Abdulah, R., A Faried, K Kobayashi, C Yamazaki, E W Suradji, K Ito, K Suzuki, M Murakami, H Kuwano, and H Koyama 2009 Selenium enrichment of broccoli sprout extract increases chemosensitivity and apoptosis of LNCaP prostate cancer cells BMC Cancer (1):414-425 Aires, A., C Fernandes, R Carvalho, R N Bennett, M J Saavedra, and E A Rosa 2011 Seasonal effects on bioactive compounds and antioxidant capacity of six economically important brassica vegetables Molecules 16 (8):6816-6832 Akanitapichat, P., K Phraibung, K Nuchklang, and S Prompitakkul 2010 Antioxidant and hepatoprotective activities of five eggplant varieties Food and Chemical Toxicology 48 (10):3017-3021 Akhlaghi, M., and B Bandy 2010 Dietary broccoli sprouts protect against myocardial oxidative damage and cell death during ischemiareperfusion Plant Foods for Human Nutrition 65 (3):193-199 Alam, M N., N J Bristi, and M Rafiquzzaman 2013 Review on in vivo and in vitro methods evaluation of antioxidant activity Saudi Pharmaceutical Journal 21 (2):143-152 Alvarez, M V., A G Ponce, and M D R Moreira 2013 Antimicrobial efficiency of chitosan coating enriched with bioactive compounds to improve the safety of fresh cut broccoli LWT-Food Science and Technology 50 (1):78-87 Anwar, F., U Kalsoom, B Sultana, M Mushtaq, T Mehmood, and H Arshad 2013 Effect of drying method and extraction solvent on the total phenolics and antioxidant activity of cauliflower (Brassica oleracea L.) extracts International Food Research Journal 20 (2):653659 Ares, A M., M J Nozal, and J Bernal 2013 Extraction, chemical characterization and biological activity determination of broccoli health promoting compounds Journal of Chromatography A 1313:78-95 77 Arnáiz, E., J Bernal, M Martín, M Nozal, J Bernal, and L Toribio 2012 Supercritical fluid extraction of free amino acids from broccoli leaves Journal of Chromatography A 1250:49-53 Aruoma, O 1994 Nutrition and health aspects of free radicals and antioxidants Food and Chemical Toxicology 32 (7):671-683 Bahadoran, Z., P Mirmiran, F Hosseinpanah, A Rajab, G Asghari, and F Azizi 2012 Broccoli sprouts powder could improve serum triglyceride and oxidized LDL/LDL-cholesterol ratio in type diabetic patients: a randomized double-blind placebo-controlled clinical trial Diabetes Research and Clinical Practice 96 (3):348-354 Banerjee, R., A K Verma, A K Das, V Rajkumar, A Shewalkar, and H Narkhede 2012 Antioxidant effects of broccoli powder extract in goat meat nuggets Meat Science 91 (2):179-184 Beecher, C W 1994 Cancer preventive properties of varieties of Brassica oleracea: a review The American Journal of Clinical Nutrition 59 (5):1166S-1170S Bjelakovic, G., D Nikolova, L L Gluud, R G Simonetti, and C Gluud 2012 Antioxidant supplements for prevention of mortality in healthy participants and patients with various diseases The Cochrane Library 148:373-380 Boivin, D., S Lamy, S Lord-Dufour, J Jackson, E Beaulieu, M Côté, A Moghrabi, S Barrette, D Gingras, and R Béliveau 2009 Antiproliferative and antioxidant activities of common vegetables: A comparative study Food Chemistry 112 (2):374-380 Borowski, J., A Szajdek, E J Borowska, E Ciska, and H Zieliński 2008 Content of selected bioactive components and antioxidant properties of broccoli (Brassica oleracea L.) European Food Research and Technology 226 (3):459-465 Branca, F 2008 Cauliflower and broccoli In Vegetables I: Springer, 151186 78 Buck, P 1956 Origin and taxonomy of broccoli Economic Botany 10 (3):250-253 Cai, Y., Q Luo, M Sun, and H Corke 2004 Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer Life Sciences 74 (17):2157-2184 Carocho, M., and I C Ferreira 2013 A review on antioxidants, prooxidants and related controversy: Natural and synthetic compounds, screening and analysis methodologies and future perspectives Food and Chemical Toxicology 51:15-25 Cartea, M E., M Francisco, P Soengas, and P Velasco 2010 Phenolic compounds in Brassica vegetables Molecules 16 (1):251-280 Cevallos-Casals, B A., and L Cisneros-Zevallos 2010 Impact of germination on phenolic content and antioxidant activity of 13 edible seed species Food Chemistry 119 (4):1485-1490 Chaudhary, A., G Rampal, U Sharma, T S Thind, B Singh, A P Vig, and S Arora 2012 Anticancer, antioxidant activities and GC-MS analysis of glucosinolates in two cultivars of broccoli Medicinal Chemistry & Drug Discovery (2):30-37 Chaudhary, A., U Sharma, A P Vig, B Singh, and S Arora 2014 Free radical scavenging, antiproliferative activities and profiling of variations in the level of phytochemicals in different parts of broccoli (Brassica oleracea italica) Food Chemistry 148:373-380 Cheng, T O 2008 Not only green tea but also green leafy vegetables inhibit warfarin International Journal of Cardiology 125 (1):101-116 Chuanphongpanich, S., S Phanichphant, D Bhuddasukh, M Suttajit, and B Sirithunyalug 2006 Bioactive glucosinolates and antioxidant properties of broccoli seeds cultivated in Thailand Songklanakarin Journal of Science and Technology 28:55-61 Clarke, J D., R H Dashwood, and E Ho 2008 Multi-targeted prevention of cancer by sulforaphane Cancer Lett 269 (2):291-304 79 Dai, J., and R J Mumper 2010 Plant phenolics: extraction, analysis and their antioxidant and anticancer properties Molecules 15 (10):73137352 Dalar, A., Y Guo, and I Konczak 2014 Phenolic composition and potential anti-inflammatory properties of Verbascum cheiranthifolium var cheiranthifolium leaf Journal of Herbal Medicine (4):195-200 Damianaki, A., E Bakogeorgou, M Kampa, G Notas, A Hatzoglou, S Panagiotou, C Gemetzi, E Kouroumalis, P M Martin, and E Castanas 2000 Potent inhibitory action of red wine polyphenols on human breast cancer cells Journal of Cellular Biochemistry 78 (3):429-441 Dominguez-Perles, R., D A Moreno, M Carvajal, and C Garcia-Viguera 2011 Composition and antioxidant capacity of a novel beverage produced with green tea and minimally-processed byproducts of broccoli Innovative Food Science & Emerging Technologies 12 (3):361-368 Domínguez-Perles, R., D A Moreno, and C García-Viguera 2012 Analysis of the tumoral cytotoxicity of green tea-infusions enriched with broccoli Food Chemistry 132 (3):1197-1206 Dominguez‐Perles, R., M C Martinez‐Ballesta, F Riquelme, M Carvajal, C Garcia‐Viguera, and D A Moreno 2011 Novel varieties of broccoli for optimal bioactive components under saline stress Journal Science Food Agriculture 91 (9):1638-1647 Elbarbry, F., and N Elrody 2011 Potential health benefits of sulforaphane: a review of the experimental, clinical and epidemiological evidences and underlying mechanisms Journal of Medicinal Plants Research (4):473484 Erel, O 2004 A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation Clinical Biochemistry 37 (4):277-285 80 Fang, Y.-Z., S Yang, and G Wu 2002 Free radicals, antioxidants, and nutrition Nutrition 18 (10):872-879 Fernández-León, M., A Fernández-León, M Lozano, M Ayuso, M Amodio, G Colelli, and D González-Gómez 2013a Retention of quality and functional values of broccoli „Parthenon‟stored in modified atmosphere packaging Food Control 31 (2):302-313 Fernández-León, M., A Fernández-León, M Lozano, M Ayuso, and D González-Gómez 2012 Identification, quantification and comparison of the principal bioactive compounds and external quality parameters of two broccoli cultivars Journal of Functional Foods (2):465-473 Fernández-León 2013b Different postharvest strategies to preserve broccoli quality during storage and shelf life: Controlled atmosphere and 1MCP Food Chemistry 138 (1):564-573 Gawlik-Dziki, U., M Jeżyna, M Świeca, D Dziki, B Baraniak, and J Czyż 2012 Effect of bioaccessibility of phenolic compounds on in vitro anticancer activity of broccoli sprouts Food Research International 49 (1):469-476 Girard-Lalancette, K., A Pichette, and J Legault 2009 Sensitive cell-based assay using DCFH oxidation for the determination of pro-and antioxidant properties of compounds and mixtures: Analysis of fruit and vegetable juices Food Chemistry 115 (2):720-726 Gliszczyńska-Świgło, A., E Ciska, K Pawlak-Lemańska, J Chmielewski, T Borkowski, and B Tyrakowska 2006 Changes in the content of health-promoting compounds and antioxidant activity of broccoli after domestic processing Food Additives and Contaminants 23 (11):10881098 Gray, A 1982 Taxonomy and evolution of broccoli (Brassica oleracea var italica) Economic Botany 36 (4):397-410 Guo, R., G Yuan, and Q Wang 2011 Sucrose enhances the accumulation of anthocyanins and glucosinolates in broccoli sprouts Food Chemistry 129 (3):1080-1087 81 Haina, Y., Y Shanjing, Y Yuru, X Gongnian, and Y Qi 2010 Antioxidant activity of isothiocyanate extracts from broccoli Chinese Journal of Chemical Engineering 18 (2):312-321 Halliwell, B 1994 Free radicals, antioxidants, and human disease: curiosity, cause, or consequence? The Lancet 344 (8924):721-724 Halliwell, B 2007 Biochemistry of oxidative stress Biochemical Society Transactions 35 (5):1147-1150 Halliwell, B., and J M Gutteridge 1995 The definition and measurement of antioxidants in biological systems Free Radical Biology and Medicine 18 (1):125-126 Han, D., and K H Row 2011 Separation and purification of sulforaphane from broccoli by solid phase extraction International Journal of Molecular Sciences 12 (3):1854-1861 Hanlon, N., N Coldham, A Gielbert, M J Sauer, and C Ioannides 2009 Repeated intake of broccoli does not lead to higher plasma levels of sulforaphane in human volunteers Cancer Letter 284 (1):15-20 Hasperué, J H., A R Chaves, and G A Martínez 2011 End of day harvest delays postharvest senescence of broccoli florets Postharvest Biology and Technology 59 (1):64-70 Heres-Pulido, M E., I Duas-García, L Castañeda-Partida, L F SantosCruz, V Vega-Contreras, R Rebollar-Vega, J C Gómez-Luna, and Á Durán-Díaz 2010 Genotoxicity studies of organically grown broccoli (Brassica oleracea var italica) and its interactions with urethane, methyl methanesulfonate and 4-nitroquinoline-1-oxide genotoxicity in the wing spot test of Drosophila melanogaster Food and Chemical Toxicology 48 (1):120-128 Hsu, B., I M Coupar, and K Ng 2006 Antioxidant activity of hot water extract from the fruit of the Doum palm, Hyphaene thebaica Food Chemistry 98 (2):317-328 Huang, D., B Ou, and R L Prior 2005 The chemistry behind antioxidant capacity assays J Agric Food Chem 53 (6):1841-1856 82 Jagota, S., and H Dani 1982 A new colorimetric technique for the estimation of vitamin C using Folin phenol reagent Analytical Biochemistry 127 (1):178-182 Jahan, I A., M Mostafa, I Nimmi, M H Hossain, M Ahsan, and J U Chowdhury 2010 Chemical and antioxidant properties of broccoli growing in Bangladesh Dhaka University Journal of Pharmaceutical Sciences (1):31-37 Jaiswal, A K., N Abu‐Ghannam, and S Gupta 2012 A comparative study on the polyphenolic content, antibacterial activity and antioxidant capacity of different solvent extracts of Brassica oleracea vegetables International Journal of Food Science & Technology 47 (2):223-231 Jaiswal, A K., G Rajauria, N Abu-Ghannam, and S Gupta 2011 Phenolic composition, antioxidant capacity and antibacterial activity of selected Irish Brassica vegetables Food Chemistry 146 (1):648-663 Jasmina, Č., P Adisa, M Milka, and B Kasim 2012 Antioxidative and antitumor properties of in vitro-cultivated broccoli (Brassica oleracea var italica) Pharmaceutical Biology 50 (2):175-181 Jeffery, E., A Brown, A Kurilich, A Keck, N Matusheski, B Klein, and J Juvik 2003 Variation in content of bioactive components in broccoli Journal of Food Composition and Analysis 16 (3):323-330 Jeffery, E H., and M Araya 2009 Physiological effects of broccoli consumption Phytochemistry Reviews (1):283-298 Kaur, C., and H C Kapoor 2002 Anti‐oxidant activity and total phenolic content of some Asian vegetables International Journal of Food Science & Technology 37 (2):153-161 Kaur, C., K Kumar, D Anil, and H Kapoor 2007 Variations in antioxidant activity in broccoli (Brassica oleracea L.) cultivars Journal of Food Biochemistry 31 (5):621-638 Keck, A.-S., and J W Finley 2006 Aqueous extracts of selenium-fertilized broccoli increase selenoprotein activity and inhibit DNA single-strand 83 breaks, but decrease the activity of quinone reductase in Hepa 1c1c7 cells Food and Chemical Toxicology 44 (5):695-703 Koh, E., K Wimalasiri, A Chassy, and A Mitchell 2009 Content of ascorbic acid, quercetin, kaempferol and total phenolics in commercial broccoli Journal of Food Composition and Analysis 22 (7):637-643 Kurilich, A C., E H Jeffery, J A Juvik, M A Wallig, and B P Klein 2002 Antioxidant capacity of different broccoli (Brassica oleracea) genotypes using the oxygen radical absorbance capacity (ORAC) assay Journal of Agricultural and Food Chemistry 50 (18):5053-5057 Kusznierewicz, B., J Lewandowska, A Kruszyna, A Piasek, and A Bartoszek 2010 The antioxidative properties of white cabbage (Brassica oleracea var capitata f alba) fresh and submitted to culinary processing Journal of Food Biochemistry 34 (s1):262-285 Kwon, D., S Yoon, O Carter, G S Bailey, and R H Dashwood 2006 Antioxidant and antigenotoxic activities of Angelica keiskei, Oenanthe javanica and Brassica oleracea in the Salmonella mutagenicity assay and in HCT116 human colon cancer cells BioFactors (Oxford, England) 26 (4):231 Lam, T K., I Ruczinski, K J Helzlsouer, Y Y Shugart, L E Caulfield, and A J Alberg 2010 Cruciferous vegetable intake and lung cancer risk: a nested case-control study matched on cigarette smoking Cancer Epidemiology Biomarkers & Prevention 19 (10):2534-2540 Latté, K P., K.-E Appel, and A Lampen 2011 Health benefits and possible risks of broccoli–an overview Food and Chemical Toxicology 49 (12):3287-3309 Lee, K.-M., J Lim, S Nam, M Y Yoon, Y.-K Kwon, B Y Jung, Y Park, S Park, and S S Yoon 2011 Inhibitory effects of broccoli extract on Escherichia coli O157: H7 quorum sensing and in vivo virulence FEMS Microbiology Letters 321 (1):67-74 84 Lin, C.-H., and C.-Y Chang 2005 Textural change and antioxidant properties of broccoli under different cooking treatments Food Chemistry 90 (1):9-15 Lin, K.-H., Y.-Y Yang, C.-M Yang, M.-Y Huang, H.-F Lo, K.-C Liu, H.S Lin, and P.-Y Chao 2013 Antioxidant activity of herbaceous plant extracts protect against hydrogen peroxide-induced DNA damage in human lymphocytes BMC Research Notes (1):490 López-Alarcón, C., and A Denicola 2013 Evaluating the antioxidant capacity of natural products: a review on chemical and cellular-based assays Analytica Chimica Acta 763:1-10 Mahn, A., M Zamorano, H Barrientos, and A Reyes 2012 Optimization of a process to obtain selenium-enriched freeze-dried broccoli with high antioxidant properties LWT-Food Science and Technology 47 (2):267273 Martínez-Hernández, G B., F Artés-Hernández, P A Gómez, and F Artés 2013 Quality changes after vacuum-based and conventional industrial cooking of kailan-hybrid broccoli throughout retail cold storage LWTFood Science and Technology 50 (2):707-714 Martínez-Hernández, G B., P A Gómez, I Pradas, F Artés, and F ArtésHernández 2011 Moderate UV-C pretreatment as a quality enhancement tool in fresh-cut broccoli Postharvest Biology and Technology 62 (3):327-337 Martínez-Sánchez, A., A Gil-Izquierdo, M I Gil, and F Ferreres 2008 A comparative study of flavonoid compounds, vitamin C, and antioxidant properties of baby leaf Brassicaceae species Journal of Agricultural and Food Chemistry 56 (7):2330-2340 McDougall, G., I Martinussen, O Junttila, S Verrall, and D Stewart 2011 Assessing the influence of genotype and temperature on polyphenol composition in cloudberry (Rubus chamaemorus L.) using a novel mass spectrometric method Journal of Agricultural and Food Chemistry 59 (20):10860-10868 85 McDougall, G J., N N Kulkarni, and D Stewart 2009 Berry polyphenols inhibit pancreatic lipase activity in vitro Food Chemistry 115 (1):193199 Mirmiran, P., Z Bahadoran, F Hosseinpanah, A Keyzad, and F Azizi 2012 Effects of broccoli sprout with high sulforaphane concentration on inflammatory markers in type diabetic patients: A randomized double-blind placebo-controlled clinical trial Journal of Functional Foods (4):837-841 Moon, J.-K., J.-R Kim, Y.-J Ahn, and T Shibamoto 2010 Analysis and anti-Helicobacter activity of sulforaphane and related compounds present in broccoli (Brassica oleracea L.) sprouts Journal of Agricultural and Food Chemistry 58 (11):6672-6677 Moreno, D A., M Carvajal, C López-Berenguer, and C García-Viguera 2006 Chemical and biological characterisation of nutraceutical compounds of broccoli Journal of Agricultural and Food Chemistry 41 (5):1508-1522 Nichenametla, S N., T G Taruscio, D L Barney, and J H Exon 2006 A review of the effects and mechanisms of polyphenolics in cancer Critical Reviews in Food Science and Nutrition 46 (2):161-183 Oh, M M., and C Rajashekar 2009 Antioxidant content of edible sprouts: effects of environmental shocks J Sci Food Agric 89 (13):2221-2227 Pająk, P., R Socha, D Gałkowska, J Rożnowski, and T Fortuna 2014 Phenolic profile and antioxidant activity in selected seeds and sprouts Food Chemistry 143:300-306 Patras, A., N P Brunton, G Downey, A Rawson, K Warriner, and G Gernigon 2011a Application of principal component and hierarchical cluster analysis to classify fruits and vegetables commonly consumed in Ireland based on in vitro antioxidant activity Journal of Food Composition and Analysis 24 (2):250-256 Patras, A., B Tiwari, and N Brunton 2011b Influence of blanching and low temperature preservation strategies on antioxidant activity and 86 phytochemical content of carrots, green beans and broccoli LWT-Food Science and Technology 44 (1):299-306 Pérez-Balibrea, S., D A Moreno, and C García-Viguera 2011 Genotypic effects on the phytochemical quality of seeds and sprouts from commercial broccoli cultivars Food Chemistry 125 (2):348-354 Pham-Huy, L A., H He, and C Pham-Huy 2008 Free radicals, antioxidants in disease and health International Journal of Biomedical Science (2):89-103 Podsędek, A 2007 Natural antioxidants and antioxidant capacity of Brassica vegetables: A review LWT-Food Science and Technology 40 (1):1-11 Porter, Y 2012 Antioxidant properties of green broccoli and purple-sprouting broccoli under different cooking conditions Bioscience Horizons 5: 4484 Ramos, S., M Alía, L Bravo, and L Goya 2005 Comparative effects of food-derived polyphenols on the viability and apoptosis of a human hepatoma cell line (HepG2) Journal of Agricultural and Food Chemistry 53 (4):1271-1280 Raseetha, S., I Oey, D Burritt, S Heenan, and N Hamid 2013 Evolution of antioxidant enzymes activity and volatile release during storage of processed broccoli (Brassica oleracea L italica) LWT-Food Science and Technology 54 (1):216-223 Re, R., N Pellegrini, A Proteggente, A Pannala, M Yang, and C RiceEvans 1999 Antioxidant activity applying an improved ABTS radical cation decolorization assay Free Radical Biology and Medicine 26 (9):1231-1237 Riedl, M A., A Saxon, and D Diaz-Sanchez 2009 Oral sulforaphane increases Phase II antioxidant enzymes in the human upper airway Clinical Immunology 130 (3):244-251 Rodríguez-Cantú, L N., J A Gutiérrez-Uribe, J Arriola-Vucovich, R I Díaz-De La Garza, J W Fahey, and S O Serna-Saldivar 2011 Broccoli (Brassica oleracea var italica) sprouts and extracts rich in 87 glucosinolates and isothiocyanates affect cholesterol metabolism and genes involved in lipid homeostasis in hamsters Journal of Agricultural and Food Chemistry 59 (4):1095-1103 Rodríguez‐Hernández, M d C., D A Moreno, M Carvajal, C García‐ Viguera, and M d C Martínez‐Ballesta 2012 Natural antioxidants in purple sprouting broccoli under Mediterranean climate Journal Food Science 77 (10):C1058-C1063 Roy, M K., L R Juneja, S Isobe, and T Tsushida 2009 Steam processed broccoli (Brassica oleracea) has higher antioxidant activity in chemical and cellular assay systems Food Chemistry 114 (1):263-269 Rudolf, E., and M Červinka 2011 Sulforaphane induces cytotoxicity and lysosome-and mitochondria-dependent cell death in colon cancer cells with deleted p53 Toxicology in Vitro 25 (7):1302-1309 Seifried, H E., D E Anderson, E I Fisher, and J A Milner 2007 A review of the interaction among dietary antioxidants and reactive oxygen species The Journal of Nutritional Biochemistry 18 (9):567-579 Shahidi, F., and Y Zhong 2015 Measurement of antioxidant activity Journal of Functional Foods Sharma, C., L Sadrieh, A Priyani, M Ahmed, A H Hassan, and A Hussain 2011 Anti-carcinogenic effects of sulforaphane in association with its apoptosis-inducing and anti-inflammatory properties in human cervical cancer cells Cancer Epidemiol 35 (3):272-278 Sharma, O P., and T K Bhat 2009 DPPH antioxidant assay revisited Food Chemistry 113 (4):1202-1205 Sies, H 1997 Physiological society symposium: Impaired endothelial and smooth muscle cell function in oxidative stress Experimental Physiology 82:291-295 Sikora, E., E Cieślik, T Leszczyńska, A Filipiak-Florkiewicz, and P M Pisulewski 2008 The antioxidant activity of selected cruciferous vegetables subjected to aquathermal processing Food Chemistry 107 (1):55-59 88 Singh, J., A Upadhyay, K Prasad, A Bahadur, and M Rai 2007 Variability of carotenes, vitamin C, E and phenolics in Brassica vegetables Journal of Food Composition and Analysis 20 (2):106-112 Singhal, M., A Paul, and H P Singh 2014 Synthesis and reducing power assay of methyl semicarbazone derivatives Journal of Saudi Chemical Society 18 (2):121-127 Soengas, P., M Cartea, M Francisco, T Sotelo, and P Velasco 2012 New insights into antioxidant activity of Brassica crops Food Chemistry 134 (2):725-733 Soengas Pilar, M D P., T Sotelo Pérez, P Velasco Pazos, and M E Cartea González 2011 Antioxidant properties of Brassica vegetables Functional Plant Science and Biotechnology (2):43-55 Song, W., C M Derito, M K Liu, X He, M Dong, and R H Liu 2010 Cellular antioxidant activity of common vegetables Journal of Agricultural and Food Chemistry 58 (11):6621-6629 Sun, T., J R Powers, and J Tang 2007 Evaluation of the antioxidant activity of asparagus, broccoli and their juices Food Chemistry 105 (1):101-106 Thaipong, K., U Boonprakob, K Crosby, L Cisneros-Zevallos, and D H Byrne 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 Tomofuji, T., D Ekuni, T Azuma, K Irie, Y Endo, T Yamamoto, A Ishikado, T Sato, K Harada, and H Suido 2012 Supplementation of broccoli or Bifidobacterium longum–fermented broccoli suppresses serum lipid peroxidation and osteoclast differentiation on alveolar bone surface in rats fed a high-cholesterol diet Nutrition Research 32 (4):301-307 Traka, M., A V Gasper, A Melchini, J R Bacon, P W Needs, V Frost, A Chantry, A M Jones, C A Ortori, and D A Barrett 2008 Broccoli 89 consumption interacts with GSTM1 to perturb oncogenic signalling pathways in the prostate PLoS One (7):e2568 Vale, A P., H Cidade, M Pinto, and M B P Oliveira 2014 Effect of sprouting and light cycle on antioxidant activity of Brassica oleracea varieties Food Chemistry 165:379-387 Valko, M., D Leibfritz, J Moncol, M T Cronin, M Mazur, and J Telser 2007 Free radicals and antioxidants in normal physiological functions and human disease The International Journal of Biochemistry & Cell Biology 39 (1):44-84 Wu, X., L Gu, J Holden, D B Haytowitz, S E Gebhardt, G Beecher, and R L Prior 2004 Development of a database for total antioxidant capacity in foods: a preliminary study Journal of Food Composition and Analysis 17 (3):407-422 Zhang, D., and Y Hamauzu 2004 Phenolics, ascorbic acid, carotenoids and antioxidant activity of broccoli and their changes during conventional and microwave cooking Food Chemistry 88 (4):503-509 90 Biosketch of Author Name: Le Thanh Ninh (黎青寧） Nationality: Vietnamese Date of birth: 28th March, 1990 Email address: ltninh90@gmail.com Current address: International Master‟s Degree Program in Food Science, National Pingtung University of Science and Technology, Taiwan Address: The 05 Group, Quang Trung Ward, Thai Nguyen City, Thai Nguyen Province, Vietnam Education background: 2005-2008: Luong Ngoc Quyen High School, Thai Nguyen, Vietnam 2008-2012: Thai Nguyen University of Agriculture and Forestry, Vietnam 2013-2015: National Pingtung University of Science and Technology, Taiwan 91 ... Student ID: G10280008 Title of Thesis: Evaluation of antioxidant activity of various parts of broccoli Total Pages: 91 Name of Institute: National Pingtung University of Science and Technology... main cause of heart, cardiovascular and chronic diseases, and various forms of cancer Therefore, the main objective of the study is to investigate antioxidant activity of broccoli Broccoli was... potential of broccoli 25 Table List of the important assays to screen antioxidant activity 38 Table List of the important techniques used for antioxidants analysis 40 VIII List of Figures