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

I

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

II

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

III

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)

IV

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 (40-

50% 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

V

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

VI

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

VII

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

VIII

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

IX

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