Tài liệu 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 7 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 1. Introduction ................................................................................................. 1 2. Literature Review........................................................................................ 3 2.1. Broccoli................................................................................................. 3 2.1.1. Origin and taxonomy ...................................................................... 3 2.1.2. Health promoting compounds ........................................................ 6 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 3. 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 4. 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 5. Conclusion ................................................................................................ 76 6. References ................................................................................................. 77 Biosketch of Author ........................................................................................ 91 VII List of Tables Table 1. Ascorbic acid (AA) content of Brassica vegetables ......................... 14 Table 2. Applications in the analysis of vitamins C in broccoli samples. ...... 15 Table 3. Applications in the analysis of phenolic compounds in broccoli samples. ............................................................................................. 17 Table 4. Applications in the analysis of carotenoids in broccoli samples ...... 20 Table 5. Summary of works evaluating the antioxidant potential of broccoli ............................................................................................. 25 Table 6. List of the important assays to screen antioxidant activity ............... 38 Table 7. List of the important techniques used for antioxidants analysis ....... 40 VIII List of Figures Figure 1. Morphology of sprouting broccoli ..................................................... 5 Figure 2. The published works in the last years related to determination of broccoli compounds ...................................................................... 7 Figure 3. The extraction techniques used to analyze health promoting compounds of broccoli in the last five years. .................................... 8 Figure 4. The health promoting compounds analyzed of broccoli in the last five years ............................................................................................ 9 Figure 5. The characterization techniques used to analyze health promoting compounds of broccoli in the last five years ................................... 10 Figure 6. Summary of the different biological activities attributed to the health promoting compounds of broccoli in the last five years ...... 22 Figure 7. ORAC values of selected vegetables ............................................... 26 Figure 8. Structures of glucosinolate precursor (A) glucoraphanin and isothiocyanate hydrolysis product (B) sulforaphane ....................... 27 Figure 9. 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 Figure 17. Total flavonoid content of broccoli fresh weight in solvent extractions of sprout in different sprouting times (a) and different parts (b)............................................................................56 Figure 18. Total flavonoid content of broccoli dry weight in solvent extractions of sprout in different sprouting times (a) and different parts (b)............................................................................57 Figure 19. Vitamin C content of broccoli fresh weight in solvent extractions of sprout in different sprouting times (a) and different parts (b)............................................................................59 Figure 20. Vitamin C content of broccoli dry weight in solvent extractions of sprout in different sprouting times (a) and different parts (b)............................................................................60 Figure 21. DPPH scavenging activity of broccoli fresh weight in solvent extractions of sprout in different sprouting times (a) and different parts (b)............................................................................62 Figure 22. DPPH scavenging activity of broccoli dry weight in solvent extractions of sprout in different sprouting times (a) and different parts (b)............................................................................63 Figure 23. ABTS scavenging activity of broccoli fresh weight in solvent extractions of sprout in different sprouting times (a) and different parts (b)............................................................................65 Figure 24. ABTS scavenging activity of broccoli dry weight in solvent extractions of sprout in different sprouting times (a) and different parts (b)............................................................................66 Figure 25. Reducing power of broccoli fresh weight in solvent extractions of sprout in different sprouting times (a) and different parts (b)............................................................................68 X Figure 26. Reducing power of broccoli dry weight in solvent extractions of sprout in different sprouting times (a) and different parts (b)............................................................................69 Figure 27. Different concentrations of dried different broccoli part’s extracts on A549 cell viability by MTT method (a) and IC 50 value (b)................................................................................71 Figure 28. Different concentrations of dried different broccoli part’s extracts on HepG2 cell viability by MTT method (a) and IC 50 value (b)................................................................................73 Figure 29. Different concentrations of dried different broccoli part’s extracts on FL83B cell viability by MTT method (a) and IC 50 value (b)................................................................................75 XI 1. Introduction In recent years, the role of natural dietary antioxidants in disease prevention has been the focus of many investigations. Antioxidants inhibit free radical reactions, and may therefore protect cells against oxidative damage (Pham-Huy et al. 2008). Oxidative stress has long been described as a critical factor in the pathogenesis of various diseases, including cancer, chronic diseases and cardiovascular disease (Bjelakovic et al. 2012). The ability of an excess reactive oxygen species to damage macromolecules like DNA, protein, and lipids is often referred to the initiation and progression of carcinogenesis and abnormal vascular cell proliferation (Boivin et al. 2009; Carocho and Ferreira 2013). Several studies indicated that fruits and vegetables are good sources of many health promoting compounds and potentially protective phytochemicals including glucosinolates and various kinds of natural antioxidants such as phenolics, carotenoids, selenium, or vitamins (Kurilich et al. 2002; Wu et al. 2004). Among the most widely nutritional vegetables, broccoli (Brassica oleracea L.), which is thought to have originally come from the eastern Mediterranean area and to have been introduced to Europe long time ago (Branca 2008), is nowadays consumed worldwide and is highly valued by large groups of the population due to its flavor, but also due to its health promoting benefits with anticancer or antioxidant properties, which have been mainly attributed to glucosinolates as well as phenolic compounds, vitamins and minerals respectively (Latté et al. 2011). Consequently, incorporating some of these broccoli health promoting compounds directly or adding to pharmaceutical products (nutraceutical) or other foods (functional foods) once they have been isolated and extracted from this vegetable, is a safe and effective way to guard against many of today‟s most common diseases (Boivin et al. 2009; Bjelakovic et al. 2012). Due to the potential effects from high antioxidant properties of broccoli, it is important to be able to measure antioxidant activity using biologically relevant assays (Song et al. 2010). Over the past five years, the 1 rising interest in the extraction, isolation, characterization and determination of the biological activity of these beneficial broccoli compounds has been demonstrated by large number of published research papers dealing with this issue (Ares et al. 2013; Soengas Pilar et al. 2011). The previous researchers in different regions of the world have indicated that broccoli possessed high antioxidant capacity refers to different broccoli parts, diverse extraction treatments or various kinds of biological assays (Jahan et al. 2010). However, so far, no study has been carried out on antioxidant properties and chemical constituents of broccoli from Taiwan. Particularly, to date, only few studies determined the antioxidant capacity of broccoli leave, seed and sprout separately. In addition, currently, the critical relevance of the dietary intake of plant-origin nutrients and bioactive phytochemicals beneficial for health and well-being has become a paradigm in the design of modern diets and plans of pharmaceutical products or functional foods (Dominguez‐Perles et al. 2011; Moreno et al. 2006). Therefore, the main objective of the study is to investigate total antioxidant activity of broccoli floret, leave, seed and sprout. The results might have a significant impact on consumers‟ food selections and provide scientific information for in-depth research about commercial products. 2 2. Literature Review 2.1. Broccoli 2.1.1. Origin and taxonomy Broccoli (Brassica oleracea L.) is a traditional European crop that have become widespread in Asia in recent decades whereas its presence in Europe has been quite stable (Branca 2008). Broccoli is an edible green plant in the cabbage family whose large, flowering head is eaten as a popular vegetable. The word broccoli comes from the Italian plural of broccolo, which means "the flowering crest of a cabbage" (Gray 1982). Broccoli has large flower heads, usually green in color, arranged in a tree-like structure branching out from an edible stalk. The flower head is surrounded by leaves (Buck 1956). Broccoli resembles cauliflower, which is a different cultivar group of the same species (Branca 2008). In recent years, the interest in broccoli cultivation has grown because of the genetic improvement programs in several countries, mainly in Asia, Europe and the USA, and due to the new opportunities offered by the food industry in exploiting traditional and new phenotypes in transformation processes (Soengas et al. 2012; Latté et al. 2011). In addition, the healthy compounds contained in the produce of several brassicas which allows them to be defined as functional foods, are important for increasing the consumption of broccoli (Koh et al. 2009). The diversity present for broccoli in germplasm, which could be exploited to provide new horticultural items, is important for breeding programs aimed at meeting new consumer requirements (Kurilich et al. 2002). The origin of group to which broccoli belongs is the Brassicaceae and specifically to the Brassica genus (Buck 1956). The Brassicaceae is a large family comprised of approximately 3,000 described species apportioned among 350-380 genera (Kurilich et al. 2002). The many varieties show considerable diversity in form, with different parts of the plant being consumed as vegetables (Soengas Pilar et al. 2011). Most authorities today consider broccoli belong to the species Brassica oleracea L. and there are two 3 major varieties of broccoli, B. oleracea L. var. botrytis or cauliflower broccoli and B. oleracea L. var. italica Plenk or sprouting broccoli (commonly called broccoli, broccoli florets or broccoli heads) (Gray 1982). In fact, this species is strictly related to other species of the genus and indirectly shares a gene pool with them (Branca 2008). The sprouting broccolis are thought to originally come from the eastern Mediterranean area although it is not known when they first appeared (Buck 1956). The earliest description of sprouting broccoli was by Dalechamp in the 16th century. After that Miller's Gardeners' Dictionary of 1724 refers to "sprout colliflower" or "Italian asparagus". In their descriptions, the sprouting nature of the plant was indicated (Branca 2008). From the eastern Mediterranean, broccolis were introduced into Europe, especially to Italy, in the medieval times (Buck 1956). A lot of cultivars have been cultivated in Italy and later on in other parts of Europe as well as in America (Latté et al. 2011; Heres-Pulido et al. 2010). 4 Figure 1. Morphology of sprouting broccoli (Dominguez‐Perles et al. 2011) 5 2.1.2. Health promoting compounds The discovery of bioactive components in foods is exciting, suggesting the possibility of improved public health through diet (Jeffery et al. 2003). The critical relevance of the dietary intake of plant-origin nutrients and bioactive phytochemicals beneficial for health and well-being has become a paradigm in the design of modern diets and plans (Dominguez‐Perles et al. 2011). The Brassicaceae plants are amongst the most consumed vegetables in the world. These plant products have also been associated with beneficial health effects based on the presence of bioactive compounds, with antioxidant capacity, such as vitamins C and E, carotenoids, phenolics, flavonoids (Aires et al. 2011; Singh et al. 2007). Like other species of the Brassica family, broccoli contains a rich source of health promoting phytochemicals such as vitamins, glucosinolates, phenolic compounds, and dietary essential minerals (Gliszczyńska-Świgło et al. 2006; Jeffery et al. 2003). Because of the interest in its bioactive composition and nutritive value for health promotion, consumption of broccoli is increasing steadily worldwide over the years (Moreno et al. 2006). Over the past several years, the rising studies in the extraction, characterization and determination of the biological activity of these beneficial broccoli compounds have been demonstrated by the large number of published research papers dealing with this issue (Figure 2.). It is also interesting to mention that many extraction, separation and determination techniques have been employed to obtain and characterize these compounds from broccoli (Figure 3). As can be seen in Figure 4, several compounds have been investigated during the period of time such as glucosinolates and related compounds (33% of the publications), phenolic compounds (28%), vitamins (14%) or other essential elements like Se, minerals for human nutrition (Ca and Mg), main (Na, K, Cl and P) or trace (Fe, Zn, Cu, Mn, I, F, Se, Cr, Mo, Co, and Ni) elements and sugars (Ares et al. 2013; Dominguez‐Perles et al. 2011; Borowski et al. 2008). 6 Figure 2. The published works in the last years related to determination of broccoli compounds (data up to January 2013) (Ares et al. 2013) 7 Figure 3. The extraction techniques used to analyze health promoting compounds of broccoli in the last five years (Ares et al. 2013). 8