Evaluation of antioxidant activity of various parts of broccoli (LV thạc sĩ)

Evaluation of antioxidant activity of various parts of broccoli (LV thạc sĩ)Evaluation of antioxidant activity of various parts of broccoli (LV thạc sĩ)Evaluation of antioxidant activity of various parts of broccoli (LV thạc sĩ)Evaluation of antioxidant activity of various parts of broccoli (LV thạc sĩ)Evaluation of antioxidant activity of various parts of broccoli (LV thạc sĩ)Evaluation of antioxidant activity of various parts of broccoli (LV thạc sĩ)Evaluation of antioxidant activity of various parts of broccoli (LV thạc sĩ)Evaluation of antioxidant activity of various parts of broccoli (LV thạc sĩ)Evaluation of antioxidant activity of various parts of broccoli (LV thạc sĩ)Evaluation of antioxidant activity of various parts of broccoli (LV thạc sĩ)Evaluation of antioxidant activity of various parts of broccoli (LV thạc sĩ)Evaluation of antioxidant activity of various parts of broccoli (LV thạc sĩ)Evaluation of antioxidant activity of various parts of broccoli (LV thạc sĩ)Evaluation of antioxidant activity of various parts of broccoli (LV thạc sĩ)

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

研究生 (Student): 黎青寧 (Le Thanh Ninh)

物其 IC50 值約在 100 - 400µg/mL 間。這項研究可能對消費者的食品選

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

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)

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

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

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

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

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

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

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

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

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 benefitswith 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

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 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)

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)

Figure 1 Morphology of sprouting broccoli

(Dominguez‐Perles et al 2011)

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)

Figure 2 The published works in the last years related to determination of

broccoli compounds (data up to January 2013) (Ares et al 2013)

Figure 3 The extraction techniques used to analyze health promoting

compounds of broccoli in the last five years (Ares et al 2013)

Figure 4 The health promoting compounds analyzed of

broccoli in the last five years (Ares et al 2013)

Figure 5 The characterization techniques used to analyze health promoting

compounds of broccoli in the last five years (Ares et al 2013)

In some other cases, solid phase extraction (SPE) has been employed, especially when analyzing glucosinolates (GLSs), sulforaphane (SF) and vitamins, as it is an effective procedure to obtain cleaner and purer extracts, and at the same time it is possible to concentrate the sample Hydrolysis has been mainly employed to analyze proteins, GLSs and SF, while an acid digestion was recommended to determine proteins and essential elements

(Moreno et al 2006) Meanwhile, liquid chromatography (LC) coupled to

several detectors has mainly been used as characterization technique due to the physico-chemical characteristics of the broccoli health promoting compounds, and because it is possible to analyze the content in broccoli of

single compounds (Haina et al 2010; Ares et al 2013) (Figure 5)

Mass Spectrometry (MS) detectors have been employed in most cases

to confirm the presence of specific compounds when the standards were not available, rather than to quantify those compounds as it was not required a great sensitivity because they are usually found at high concentrations

(Fernández-León et al 2012; Haina et al., 2010; Arnáiz et al., 2012; Gliszczyńska-Świgło et al., 2006) In addition, spectrophotometric methods

(UV–vis) have been preferred to determine the total content or to study the profile of a group of phytochemicals, as they are simpler and cheaper than

other characterization techniques (Jeffery et al 2003; Martínez-Hernández et

al 2011) Moreover, gas chromatography (GC) has been chosen to analyze

lipids, proteins, SF and some essential elements, although it should be

pointed out that its use has been more limited than LC (Ares et al., 2013; Moreno et al., 2006; Dominguez‐Perles et al., 2011; Aires et al., 2011; Haina

et al 2010) In relation to other characterization techniques, it should be

2.1.3 Antioxidant properties

Broccoli is becoming increasingly popular as a potential healthy

vegetable and is a significant source of nutritional antioxidants (Sun et al

2007) Dietary antioxidants, such as water-soluble vitamin C and phenolic compounds, as well as lipid-soluble vitamin E and carotenoids, present in Broccoli contribute both to the first and second defense lines against

oxidative stress (Borowski et al 2008; Roy et al., 2009) The major antioxidants of vegetables are vitamins C and E, carotenoids, and phenolic compounds, especially flavonoids (Porter 2012) These antioxidants scavenge radicals and break the chain propagation (the second defense line) Vitamin E and carotenoids also contribute to the first defense line against oxidative stress, because they quench singlet oxygen Flavonoids as well as vitamin C

showed a protective activity to atocopherol in human LDL (Borowski et al 2008; Soengas Pilar et al 2011)

2.1.3.1 Water-soluble antioxidants

a Vitamin C

Broccoli contains high levels of vitamins, which are organic compounds essential in trace amounts for the normal growth and maintenance of life (Zhang and Hamauzu 2004) These compounds have diverse biochemical roles as regulators of mineral metabolism or of cell and tissue growth and differentiation, antioxidant activity and some are

precursors of enzyme cofactors (Kaur et al 2007) Reducing equivalent for

biochemical reactions is one of the most important physiological functions of ascorbic acid (vitamin C), and in some vegetables it is responsible for 35 -95% of antioxidant capacity Vitamin C and its oxidation product - dehydroascorbic acid have many biological activities in human body (Podsędek 2007) More than 85% of vitamin C in human diets is supplied by

fruits and vegetables (Soengas Pilar et al 2011) Biological function of

ascorbic acid can be defined as an enzyme cofactor, a radical scavenger, and

as a donor/acceptor in electron transport at the plasma membrane (Podsędek

2007; Fernández-León et al 2012) As can be seen in table 1, broccoli has much higher level of vitamins C than several other Brassica vegetables

Several sample treatments based on solvent extraction have been proposed in order to isolate and obtain ascorbic acid (AA) from broccoli As a result, a solvent mixture (C6H8O7/EDTA/NaF/methanol/water) or MPA were selected to perform this task (Table 2) Titrimetric and fluorimetric methods also were used to analyze vitamins They are simple popular However they

are not chemically specific for AA (Ares et al 2013; Fernández-León et al 2012; Jahan et al 2010)

Table 1 Ascorbic acid (AA) content of Brassica vegetables

(mg/100g edible portion)

(Fernández-León et al 2012)

Table 2 Applications in the analysis of vitamin C

in broccoli samples

(Podsędek 2007)

b Phenolic compounds

More than 4000 compounds divided into 12 subclasses belong to this group of natural compounds, and several of them can be found in broccoli

(Cartea et al 2010) Phenolics range from simple, low molecular weight,

single aromatic-ringed compounds to large and complex tannins and derived

polyphenols (Soengas Pilar et al 2011) The number and arrangement of their

carbon atoms are classified in flavonoids (flavonols, flavones, flavan-3-ols, anthocyanidins, flavanones, isoflavones and others) and non-flavonoids (phenolic acids, hydroxycinnamates, stilbenes and others), and are commonly

found conjugated to sugars and organic acids (Podsędek 2007; Cartea et al

2010) The main activity reported for phenolic compounds has been as antioxidants, although they are also associated with other health promoting effects such as anti-carcinogenic, antiinflammatory, anti-ageing, and anti-

thrombotic activity (Soengas Pilar et al 2011)

Several sample treatments have been published in order to extract these compounds from broccoli, the majority being solvent extractions or

microwave assisted extraction (MAE) (Ares et al 2013) Methanol a water

mixtures have been predominantly used to extract those compounds from broccoli, while methanol, ethanol and water have been also widely employed

(Mahn et al 2012; Soengas et al 2012; Soengas Pilar et al 2011) Moreover,

spectrophotometric techniques have been successfully used to determine the total phenolic content in broccoli However, in some cases, single phenolic compounds were specifically analyzed by using other techniques like LC, GC

and CE (Soengas et al 2012; Raseetha et al 2013; Haina et al 2010)

One of the classic methods used to analyze total phenolic content is the Folin Ciocalteu reagent (FCR), in which the measured colour change is associated with the reduction of a molybdate–tungstate reagent induced by the phenols in the sample This reagent does not only react with phenols, but with any reducing substance of the broccoli sample All types of broccoli parts are

analyzed in these studies (florets, heads, sprouts, etc.) (Table 3) (Banerjee et

al 2012; Ares et al 2013; Kaur et al 2007)

Table 3 Applications in the analysis of phenolic compounds

in broccoli samples

(Podsędek 2007)

Flavonoids are a family of phenolic compounds that have been

investigated in broccoli (Cartea et al 2010) Quercetin and kaempferol are the

predominant flavonoids in broccoli In general, their levels depend on several factors like environmental pressures, cultivar, postharvest transport or genotype (Podsędek, 2007) Several research papers have been published in order to determine their total content in broccoli samples As with total phenolic content, solvent extraction was mainly used to isolate flavonoids, although an additional step of mixing the broccoli extract with sodium nitrite,

aluminium chloride and sodium hydroxide was required (Table 3) (Soengas et

al 2012) (Ares et al 2013; Kaur et al 2007; Sun et al 2007)

Broccoli is a source of hydroxycinnamoyl derivatives The predominant hydroxycinnamoyl acids were identified as 1-sinapoyl-2-feruloylgentiobiose; 1,2-diferuloylgentiobiose; 1,2,20-trisinapoylgentiobiose, and neochlorogenic acid In addition, 1,20-disinapoyl-2-feruloylgentiobiose and 1-sinapoyl-2,2-diferuloyl gentiobiose and chlorogenic acids were found in broccoli Total amounts of feruloylsinapoyl esters of gentiobiose and caffeic acid derivatives

in 14 cultivars of broccoli varied from 0 - 8.25 mg/100 g, and from 0 - 3.82

mg/100 g, respectively (Cartea et al 2010; Podsędek 2007)

2.1.3.2 Lipid-soluble antioxidants

a Carotenoids

Carotenoids (carotens and xanthophylls) are yellow, orange and red pigments present in many fruits and vegetables (Zhang and Hamauzu 2004) Several of them are precursors of vitamin A (i.e β-carotene, £-carotene, and β-cryptoxanthin), and due to conjugated double bonds they are both radical scavengers and quenchers of singlet oxygen Lower serum β-carotene levels have been linked to higher rates of cancer and cardiovascular diseases, as well

as to increased risk of myocardial infarction among smokers (Soengas Pilar et

al 2011) (Singh et al 2007)

Carotenoids, which possess great importance by their nutritional and physiological activities, cannot be synthesized by animals Therefore they should be acquired through the diet Total or individual (lycopene or β-

carotene) carotenoid contents have been isolated in different broccoli parts

such as heads, florets and sprouts (Guo et al., 2011; Hasperué et al., 2011; Leja et al., 2001; Rodríguez‐Hernández et al., 2012) Solvent extraction with

different solvents or composition has been used to isolate these compounds, and in most cases acetone was predominantly used (alone or in mixtures with other solvents)(Table 4) (Singh et al., 2007; Ares et al., 2013)

In addition to lutein and trans-β-carotene, cis-β-carotene was founded

in broccoli Moreover, Brassica vegetables also contain cryptoxanthin,

neoxanthin and violaxanthin, but cryptoxanthin only in broccoli (0.024 mg/100 g) (Rodríguez‐Hernández et al 2012)

Table 4 Applications in the analysis of carotenoids

in broccoli samples

(Podsędek 2007)

b Vitamin E

In addition to carotenoids, vitamin E also belongs to a group of soluble antioxidants The biological activity of vitamin E exhibit tocopherols and tocotrienols, especially α-tocopherol (Podsędek 2007) The predominant reaction responsible for tocopherol antioxidant activity is hydrogen atom

lipid-donation, where a tocopheroxyl radical is formed (Soengas Pilar et al., 2011)

Vitamin E shows protective effects against the coronary heart disease due to

inhibition of LDL oxidation (Singh et al 2007)

In our diet, in addition to fats, oils and cereal grains, vegetables constitute the major source of vitamin E in our diet However there are only

few data of tocopherol content in vegetables (Singh et al 2007) In Brassica

vegetables, the best source of vitamin E is broccoli The descending order of

total tocopherols and tocotrienols in Brassica vegetables is as follows:

broccoli (0.82 mg/100 g); Brussels sprouts (0.40 mg/100 g); Cauliflower (0.35 mg/100 g); Chinese cabbage (0.24 mg/100 g); Red cabbage (0.05

mg/100 g); White cabbage (0.04 mg/100 g) (Soengas Pilar et al 2011)

(Podsędek 2007)

2.1.4 Biological activities and health

Broccoli is an excellent dietary source of phytochemicals, including lipids, phenolic compounds, proteins, amino acids, vitamins, glucosinolates and their breakdown products, and certain minerals and essential elements

(Singh et al 2007) The potential health promoting roles of these compounds

have been extensively studied in the last years Beneficial effects such as antioxidant, anticancer, antimicrobial, amongst several others (Figure 6),

Figure 6 Summary of the different biological activities

attributed to the health promoting compounds of

broccoli in the last five years (Ares et al 2013)

2.1.4.1 Antioxidant activities

Broccoli is renowned for its vast range of non-enzymatic bioactive compounds, being rich in both nutritional antioxidants like vitamins, and non-nutritional antioxidants as are carotenoids, and phenolic compounds, particularly flavonoids (Zhang and Hamauzu 2004) The antioxidant properties of glucosinolates and related compounds have been also reported Broccoli stands out as the most concentrated source of a premiere antioxidant nutrient – vitamin C This central antioxidant vitamin can provide longer-term support of oxygen metabolism in the body if it is accompanied by flavonoids

that allow it to recycle (Singh et al 2007; Latté et al 2011) Broccoli

provides many such flavonoids in significant amounts, including the

flavonoids kaempferol and quercitin (Cartea et al 2010) Also concentrated in

broccoli are the carotenoids lutein, zeaxanthin, and β-carotene All three of

these carotenoids function as key antioxidants (Singh et al 2007) In the case

of lutein and β-carotene, broccoli has been shown not only to provide significant amounts of these antioxidants but to significantly increase their blood levels when consumed in the amount of three cups (Jeffery and Araya 2009) Other antioxidants provided by broccoli in beneficial amounts include vitamin E and the minerals manganese and zinc (Podsędek 2007)

As an antioxidant group, the vitamins, minerals, flavonoids and carotenoids contained in broccoli work to lower risk of oxidative stress in the

body (Latté et al 2011) The ability of these nutrients to support oxygen

metabolism and avoid excess formation of overly reactive, oxygen-containing molecules makes them equally helpful in lowering risk of chronic

inflammation and risk of cancer (Soengas Pilar et al 2011) If cancer

2013; Jahan et al 2010; Anwar et al 2013); ferric reducing antioxidant power

(FRAP) (Dominguez-Perles et al 2011; Patras et al 2011a; Banerjee et al

2012; Martínez-Hernández et al 2013); oxygen radical absorbance capacity

(ORAC) (Lin and Chang 2005; Zhang and Hamauzu 2004;

Martínez-Hernández et al 2013); 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic

acid) (ABTS) radical method (Fernández-León et al 2013; Sun et al 2007);

Trolox equivalent antioxidant capacity (TEAC) (Roy et al 2009)or Reducing

power (Lin and Chang 2005; Jahan et al 2010; Anwar et al 2013) Finally, it

should also be mentioned that the majority of these research studies were

performed in vitro (Chaudhary et al 2014; Roy et al 2009)

In last few years, several researches investigated and compared the

antioxidant activity from numerousvegetables (Kaur and Kapoor 2002; Song

et al 2010; Sikora et al 2008; Soengas Pilar et al 2011) or various foods

(Wu et al 2004) The studies indicated that broccoli had the high total

antioxidant capacity when compared to other common vegetables and fruits

It was only lower than that of nut group (Wu et al 2004) For instance,

according to the study of Wei Song (Wei Song 2010), beets, broccoli, and red

pepper had the highORAC values of 27 selected vegetables (Figure 7)

Table 5 Summary of works evaluating the antioxidant

potential of broccoli

(Soengas Pilar et al 2011)

Figure 7 ORAC values of selected vegetables (Song et al 2010)

2.1.4.2 Anticancer activities

In the last few years, cancer prevention by natural products has received considerable attention The potentially protective role of cruciferous vegetables, including broccoli, and active components present in these vegetables (Beecher 1994), such as glucosinolates (GLSs) and related products (isothiocyanates (ITCs), especially sulforaphane (SF)) (Figure 8),

has been extensively studied in experimental in vitro and in vivo carcinogenesis models (Chaudhary et al 2012)

Figure 8 Structures of glucosinolate precursor (A) glucoraphanin

and isothiocyanate hydrolysis product (B) sulforaphane

(Clarke et al 2008)

Recent studies have demonstrated that in humans SF is rapidly absorbed following consumption of liquidized broccoli, but repeated intake of the vegetable does not lead to higher plasma levels, and subsequently to an

relation to prostate and breast cancer (Clarke et al 2008; Elbarbry and Elrody

2011; Rudolf and Červinka 2011) For all these reasons, it is not surprising that most of the publications devoted to investigating the anticancer activity

of broccoli and derived products were related to SF (Latté et al 2011; Han and Row 2011; Abdulah et al 2009; Sharma et al 2011; Traka et al 2008)

In one of these studies, it has been demonstrated that there is a synergy between SF inbroccoli sprouts and gemcitabine which may enhance the index

of prevention and treatment ofcervical cancer (Traka et al 2008)

Phenolic compounds have also been investigated for their potential

anticancer activity (Cartea et al 2010; Beecher 1994; Gawlik-Dziki et al 2012; Boivin et al 2009) For instance, an analysis was undertaken of the effect of the bio-accessibility of phenolic compounds on the in vitro anticancer activity of broccoli sprouts (Gawlik-Dziki et al 2012) After

performing RPLC-PAD-ESI-MS analyses, it was found that the compounds were mainly phenolics and glucosinolates

In addition, it should be mentioned that one study has been published suggesting that the intake of cruciferous vegetables is inversely associated

with lung cancer risk (Lam et al 2010), while a different study argued that is

not sufficient to eat broccoli frequently to prevent the occurrence of cancer

(Heres-Pulido et al 2010) Consequently, it can be concluded that the

consumption of broccoli provides the organism with several phytochemicals which have been shown to possess anticancer activity

2.1.4.3 Other biological activities

Several recent researches have been published focusing on several other biological activities, such as antimicrobial and antibacterial, or the potentially beneficial effects of broccoli for patients with diabetes, cholesterol, cardiovascular diseases and asthma The antibacterial and antimicrobial activities of broccoli extracts have been the subject of study in

several publications (Jaiswal et al 2011; Moon et al 2010; Alvarez et al 2013; Lee et al 2011) In these researches, a comparative study has been

conducted of the antibacterial activities of broccoli (florets and stalks) extracts