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 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(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