Advances in the extraction and preservation of anthocyanin from vegetables a review

Several drawbacks of the methods were reported such as time consuming, insufficient extraction rate, degradation of anthocyanin due to high temperature use, hydrolysis of anthocyanin by

School of Bioscience Master Science of Crop Biotechnology

Research Project 3: Crop Biotechnology D24CB3

Dissertation

“Advances In The Extraction And Preservation Of Anthocyanin From Vegetables: A Review”

Abstract

Nowadays, natural colourants have high demand for use in food industry rather than synthetic colourants which might cause adverse human health effects Since the mid-1970s, anthocyanin extracted from fruits and vegetables have been found to be a great natural colorant Anthocyanin pigments are used in chewing gum, yogurts, candies, jams, beverages, fruit preparation and confectionery Depending on pH values, they account for colour of the plant leaves, flowers and fruits with red, pink, violet and blue Besides giving colour to plants, anthocyanins also have antioxidant and antihyperglycemic properties; hence, they are used as therapeutic source for many treatments of diabetes, coronary heart disease and cancer Many anthocyanin extraction methods such

as conventional acidified water (CAW), ultrasound, microwave pre-treatment, supercritical fluid extraction and pulsed electric field (PEF) have been proposed

by researchers and discussed in this review Several drawbacks of the methods were reported such as time consuming, insufficient extraction rate, degradation

of anthocyanin due to high temperature use, hydrolysis of anthocyanin by using acidified organic solvents; and no single extraction method could be applied for all plants From the literature, ultrasound and microwave-assisted extraction are two putative methods for extraction of anthocyanins from vegetables They have significant advantages such as cheap, easy to be manipulated, suitable for laboratory, domestic and large-scale industrial applications, less time-consuming, matrix independent, free sample particle size, less solvent used and long-term preservation Importantly, with those properties, they help enhance the yield of anthocyanin and also suitable for application of most vegetables from nature Furthermore, two putative methods could serve as a sound base for future large scale production of anthocyanin with high efficient and fast rate

by further investigations, modifiers and optimizations Apart from it, high pressure processing strengthen for the extracted anthocyanins with long-term preservation and industrial uses compared with the conventional preservation process, thermal processing Generally, anthocyanins deserve to be deeply investigated for future use as natural colorant in the future

Declaration

I hereby declare that this thesis is, except where otherwise stated, entirely my own work and that it have not been submitted as a dissertation for a master degree at any other university

-August 13, 2012

Nguyen Di Khanh

Acknowledgement

After months of hard works in completing this research project, it finally comes

to a day of expressing our gratitude to a number of people First of all, I would like to thank my supervisor, Dr Yin Sze Lim, lecturer in Nutrition research in School of Bioscience for guiding me and forinvaluable inputs to my research

I had some difficulties in doing this task, but she taught me patiently until I knew what to do She have tried and tried to teach me until I understand what I supposed to do with the project work Moreover, she helped me a lot with English consultation and grammar correction for my improved thesis write-up Internet, books, computers and all that as my source to complete this project, they also supported me and encouraged me to complete this task so that I will not procrastinate in doing it

I thank to my family and friends for their encouragement and patience when I was doing this project Without their support, I could not have done so much Last but not least, would also like to thank the University of Nottingham Malaysia Campus for giving a chance to conduct this project From this project, I would able to gain more knowledge for my future in the bioscience world

Table of Contents

Abstract i

Declaration ii

Acknowledgement iii

List of figures vii

List of tables ix

CHAPTER 1: INTRODUCTION 1

1.1 Research context 1

1.2 Main objectives 3

CHAPTER 2: BACKGROUND 4

2.1 Phenolic compounds in vegetables 4

2.1.1 Introduction 4

2.1.2 Chemical properties of phenolic compounds from vegetables 4

2.1.3 Flavonoids 5

2.1.4 Other classes of phenolic compounds 6

2.1.4.1 Phenolic acid 6

2.1.4.2 Tannins 7

2.1.4.3 Stilbenes 8

2.1.4.4 Lignans 9

2.1.5 Synthesis and metabolic processes of phenolic compounds 9

2.1.6 Phenolic compounds in vegetables and their health-promoting properties 11

2.2 Anthocyanins from vegetables 13

2.2.1 Introduction 13

2.2.2 Vegetables – a great sources of anthocyanins 13

2.2.3 Biosynthesis pathways of anthocyanins 13

2.2.4 Anthocyanins chemical properties and functions in nature 15

2.2.5 Anthocyanins stability 18

2.2.6 Anthocyanins biological properties 21

2.2.6.1 Antioxidant activities 21

2.2.6.2 Other biological properties 21

2.2.6.3 Anthocyanins and human health effects 22

2.2.7 Anthocyanins as natural colorants 23

2.2.8 Extraction and preservation processes of anthocyanins from vegetables 24

CHAPTER 3: METHODOLOGY 28

3.1 Searching method 28

3.2 Referencing 28

CHAPTER 4: RESULTS AND DISCUSSIONS 29

4.1 Methods for extraction of anthocyanins from vegetables 29

4.1.1 Conventional extraction method - Soxhlet technique 29

4.1.1.1 Introduction 29

4.1.1.2 Principles and mechanisms 29

4.1.1.3 Practical design 30

4.1.1.4 Advantages and disadvantages 32

4.1.1.5 Potential applications 32

4.1.2 Ultrasound-assisted extraction 33

4.1.2.1 Introduction 33

4.1.2.2 Principles and mechanisms 34

4.1.2.3 Practical design 35

4.1.2.4 Advantages and disadvantages 36

4.1.2.5 Potential applications 38

4.1.3 Microwave-assisted extraction 38

4.1.3.1 Introduction 38

4.1.3.2 Principles and mechanism 39

4.1.3.3 Practical design 40

4.1.3.4 Advantages and disadvantages 41

4.1.3.5 Recent applications 42

4.1.4 Supercritical fluid extraction 42

4.1.4.1 Introduction 42

4.1.4.2 Principles and mechanism 42

4.1.4.3 Practical design 43

4.1.4.4 Advantages and disadvantages 44

4.1.4.5 Potential applications 45

4.1.5 Accelerated solvent extraction 45

4.1.5.1 Introduction 45

4.1.5.2 Principles and mechanism 45

4.1.5.3 Advantages and disadvantages 46

4.1.5.4 Potential applications 47

4.1.6 Pulse electric field extraction 47

4.1.6.1 Introduction 47

4.1.6.2 Principles and mechanism 47

4.1.6.3 Practical design 48

4.1.6.4 Advantages and disadvantages 48

4.1.6.5 Recent applications 49

4.1.7 Comparisons among methods for extraction of anthocyanins from vegetables 49 4.2 Preservation processes for extracted anthocyanins 52

4.3.1 Introduction 52

4.2.2 Thermal processing 52

4.2.2.1 Introduction 52

4.2.2.2 Principles of thermal processing 53

4.2.2.3 Advantages and disadvantages 53

4.2.3 High pressure processing 54

4.2.3.1 Introduction 54

4.2.3.2 Principles and mechanism 54

4.2.3.3 Advantages and disadvantages 54

4.2.4 Comparison between the thermal and high pressure processing 54

4.2.5 Novel alternative techniques for preservation processes 55

CHAPTER 5: CONCLUSION 56

List of references 57

List of figures

Figure 1.1: Chemical structure of acylated anthocyanins 2

Figure 2.1: Chemical structure of subclasses of flavonoids 6

Figure 2.2: Two sub-groups of phenolic acids, hydroxybenzoic and hydrocinnamic acids 7

Figure 2.3: Chemical structure of two sub-groups of tannins - pro-anthocyanidins and gallotannins 7

Figure 2.4:Chemical structure of stibenes 8

Figure 2.5: Chemical structure of Lignans 9

Figure 2.6: Biosynthesis pathways of anthocyanins 14

Figure 2.7: General structure of anthocyanins 15

Figure 2.8: (a) General structure of the six common anthocyanidins;(b) Classification of anthocyanins with six common sub-groups 17

Figure 2.9: Thermal degradation of anthocyanins 19

Figure 2.10: Chemical structures of anthocyanins in corresponding to pH values and the degradation reaction 20

Figure 2.11: Mechanism of anthocyanins in preventing cancer 22

Figure 2.12: Steps involved in the whole extraction process of anthoyanins from vegetables 25

Figure 2.13: Two commonly used preserved processes: (a) thermal processing, (b) high pressure processing 26

Figure 4.1: Schematically experimental apparatus of Soxhlet extraction technique: (a) laboratory Soxhlet extractor, (b) schematic diagram of Soxhlet extraction apparatus 29

Figure 4.2: Two common ultrasound-assisted extraction apparatuses: (a) Ultrsound bath, (b) Ultrasound probe 33

Figure 4.3: Laboratory and schematic illustration diagram of 3 litter volume

ultrasound-assisted extractor 34

Figure 4.4: Industrial ultrasonic equipment with three different volumes 50L, 500L and 1000L 37

Figure 4.5: Schematic diagram of microwave-assisted extraction system 38

Figure 4.6: Schematic diagram of supercritical fluid extractor 42

Figure 4.7: Schematic diagram of Accelerated solvent extraction 45

Figure 4.8: Sketch of the pulsed electric field treatment chamber 47

Figure 4.9: Summery of the determination of anthocyanins including the extraction techniques have been used recently 49

List of tables

Table 2.1: Flavonoid group, their sub-groups, chemical characteristics together with some typical rich food sources 5 Table 2.2: Fruits and vegetable – great sources of phenolics 12 Table 2.3: Names and abbreviations of common varieties of anthocyanins 16 Table 2.4: Summary of functions of anthocyanins in some plants in nature 18 Table 2.5: Fruits and vegetables – common sources of anthocyanins with different indicated concentrations 24 Table 4.1: Advantages of ultrasound-assisted extraction technique compared with other techniques: MAE (microwave-assisted extraction), SFE (supercritical fluid extraction) and ASE (accelerated-solvent extraction) 36 Table 4.2: Summary of comparisons of the characteristics of the methods have

vegetables……….50

CHAPTER 1: INTRODUCTION

1.1 Research context

Nowadays, natural colorants have high demand for use in food industry rather than synthetic colorants which might cause adverse human health effects (Zarena and Sankar, 2012) The neurological and behavioural effects caused by the synthetic dyes used in food industry are adverse to human health (Lu et al., 2010) Therefore, anthocyanins, with their high potential in terms of high stability, high colorant power and low cost, have been considered as a great candidate for this requirement of new sources of pigments

Since the mid-1970s, anthocyanins have been extracted from fruits, vegetables, cereals and flowers of a great variety of plants Those have been comprehensively evaluated and accessed as potential sources for the extraction

of anthocyanins Depending on pH values, they account for colour of the plant leaves, flowers and fruits with red, pink, violet, blue and green (Lu et al., 2010; Naczk et al., 2011) The word anthocyanins came from Greek, in which anthos means flower and kyanos means blue Anthocyanins, belong to the flavonoid family, are a group of phenolic compound that can be soluble in water Chemically, they are glycosides of polymethoxy and polyhydroxy derivatives of flavylium or 2-phenylbenzopyrylium salts (Xu et al., 2010; Lu et al., 2010) Besides giving responsibility of colouration with bright colour to plants, anthocyanins also have antioxidant and antihyperglycemic properties (Arapitsas

et al., 2008; Lu et al., 2010; Zarena and Sankar, 2012); hence, they are investigated as therapeutic source for many treatments of diabetes, coronary heart disease and cancer, preventing the process of aging (Arapitsas et al., 2008) Their antioxidant properties give health promoting benefits and protect against various oxidants by diverse actions on various enzymes and metabolic processes (Zu et al., 2010; Lu et al., 2010; Zarena and Sankar, 2012)

Various health benefits associated with anthocyanins have been well studied such as antioxidant capacity, treatment of various blood circulation disorders based on capillary fragility, conservation of normal vascular porousness, augmentation of sight acuteness, radiation-protective agents, anti-neoplastic and chemo-protective agents, embarrassment of platelet combination, regulation for diabetes, vaso-protective and anti-inflammatory

properties (Zu et al., 2010) For instance, a total of twenty-four types of anthocyanins have been isolated and characterized in red cabbage Most of them have cyaniding in the form of mono or di-glycoside, and acylated form of anthocyanins (see figure 1.1) (Arapitsas et al., 2008; Zu et al., 2010)

Source: Xu et al., 2010 Figure 1.1: Chemical structure of acylated anthocyanins

The first problem associated with the use of anthocyanins in food systems is the method design for extraction process So far, many anthocyanin extraction methods such as conventional acidified water (Soxhlet extraction),

pre-treatment, accelerated fluid extraction and pulsed electric field (PEF) have been proposed by researchers (Vera and Mercadante, 2007; Welch et al., 2008; Arapitsas and Turner, 2008; Gachovska et al., 2010; Ignat et al., 2010; Lu et al., 2010; Xu et al., 2010; Jacob et al., 2011) However, several drawbacks of the methods were reported such as time consuming, insufficient rate, degradation of anthocyanin due to high temperature use, hydrolysis of anthocyanin by using acidified organic solvents; and no single extraction method could be applied for all plants For instance, in traditional techniques, the solvents such as ethanol, methanol, acetone or water which acidified with

hydrochloric acid or sulphure dioxide were commonly used Moreover, the products extracted need further purification steps (Zarena and Sankar, 2012) Extracted anthocyanins not only need further purification steps but also a suitable preservation process for long-term uses and applications in food industry Together with thermal processing, which is investigated and used for a long time ago, the high pressure processing is now taken advantaged for the preservation process of anthocyanins extracted from vegetables (Lu et al, 2010; Routray and Orsat, 2012; Idham et al., 2012)

Anoptimum putative extraction and preservation method that enhances the yield of anthocyanin suitable for application to most vegetables is extremely essential Hence, current study was carried out to review all of the methods that have been used for anthocyanins extraction and preservation with comparisons and contrasts to find out the best available method

1.2 Main objectives

i To investigate methods used for extraction of anthocyanin from vegetables

ii To elucidate advantages and disadvantages of the methods used for the extraction of anthocyanin

iii To investigate the preservation techniques of extracted anthocyanin

iv To propose a putative method applicable for extraction and preservation of anthocyanin for vegetables

2.1.2 Chemical properties of phenolic compounds from vegetables

Polyphenols, which are secondary metabolites produced by plants, are the highest antioxidant phenolic compounds antioxidants in human diets (Jay, 2008) One single compound is formed by an aromatic ring binding by some structural elements They are normally hydroxyl mioties (Jay, 2008)

Based on the number of phenol rings and those elements, polyphenols are classified into many sub-groups (Beecher, 2003; Jay, 2008; Laura et al., 2010) Flavonoids and non-flavonoids are two main groups of polyphenols First, flavonoid group consists of anthocynidins, proanthocyanidins, flavan-3-ols, flavonols, dihydroflavonol, flavones, flavanones and isoflavones They are chemically C6-C3-C6 struture (see table 2.1) Second, non-flavonoids group consist of simple phenols secoiridoids, lignans, chalcones, xanthones, benzophenones, coumarins, cinnamic acids, phenylacetic and acetophenones, hydrolysable tannins and benzoic acids (Jay, 2008) This study mainly focuses

on anthocyanins thereforethe flavonoid group will be more discussed in details

Table2.1: Flavonoid group, their sub-groups, chemical characteristics together with some typical rich food sources

Source: Jay, 2008

2.1.3 Flavonoids

Flavonoids are glycosides formed from several small number of flavonoid aglycones (Jay, 2008; Huang and Cai, 2010; Laura et al., 2010) The chemical structu

res of subclasses of flovanoids are introduced in figure 2.1 They are most water-soluble and can be found in the vacuoles of plant cells Within plants, these compounds function as pigments which have chemically defence ability against attacked microorganisms as well as some particular insects (Jay, 2008) Moreover, flavonoids are also involved in some other biological interactions in plants (Laura et al., 2010) Nevertheless, the active researches recently which are mainly focusing on the antioxidant activities of flavonoids are their possiblehuman healtheffects (Huang and Cai, 2010) It is stated that high-flavonoid consumption in human diets could contribute to mitigation in risks of some typical diseases and certain cancers (Liu, 2004; Huang and Cai, 2010; Laura et al., 2010) Particularly, fruits and vegetables are the great potential in such aspect

Source: Huang and Cai, 2010 Figure 2.1: Chemical structure of subclasses of flavonoids

2.1.4 Other classes of phenolic compounds

2.1.4.1 Phenolic acids

Phenolic acids account for about one-third of the dietary phenols (Ignat

et al., 2011) They can be found with free or bound forms within plants.Generally, the phenolic acids with bound forms are linked to some other components due to their acetal bonds and through ester and ether (Robbins, 2003; Zadernowski et al., 2009).Depend on certain extraction conditions and certain susceptibilities to degradation, phenolic acids will express in different forms (Ross et al., 2009; Ignat et al., 2011) There are two main sub-groups of phenolic acids (see figure 2.2), the hydroxycinnamic acids and the hydroxybenzoic acids (Ignat et al., 2011)

Source: Ignat et al., 2011 Figure 2.2: Two sub-groups of phenolic acids, hydroxybenzoic and hydrocinnamic acids

The hydroxycinnamic acids commonly have C6-C3 structure and consist

of some representatives such as sinapic, p-coumaric, ferulic and caffeic acids

On the other hand, the hydroxybenzoic acids with C6-C1 structure consist of syringic, vanillic, protocatechuic, p-hydroxybenzoic and gallic acids (Ignat et al., 2011)

2.1.4.2 Tannins

Tannins, which are high molecular compounds, constitute an important role in phenolic groups (Scalbert, 1991; Ignat et al., 2011) They consist of two sub-groups, pro-anthocyanidins (or condensed tannins) and gallotannins tannins (or hydrolysable tannins) (see figure 2.3)

Although the tannins show their potential in functioning as biological antioxidants, protein precipitating agents and metal ion chelators, their biological activities are difficult to be predicted within one particular biological system (Ignat et al., 2011) The reasons explain for that is because of their enormous structural variation as well as their varied biological roles Future work needs to study the relationships between structure and their activity inorder to predict the biological activities of tannins in any system (Scalbert, 1991; Ignat et al., 2011)

Source: Ignat et al., 2011.Figure 2.3: Chemical structure of two sub-groups of tannins - pro-anthocyanidins and gallotannins

2.1.4.3 Stilbenes

Stilbenes are present in human diet with very low quantities (Bavaresco, 2003) Resveratrol is the main representative of stilbenes, which is regularly in glycosylated forms (see figure 2.4) Resveratrol exists in both cis and trans isomeric forms (Bavaresco, 2003; Ignat et al., 2011) When the plants are infected by pathogens or are under some of stress conditions, they will produce stilbene compounds in response to those stresses More than 70 plant species including peanuts, berries and grapes have been detected to exhibit that kind of reaction (Bavaresco, 2003; Delmas et al., 2006; Ignat et al., 2011)

Source: Ignat et al., 2011.Figure 2.4: Chemical structure of stibenes

2.1.4.4 Lignans

Lignans are present in plants mainly as an aglycone (see figure 2.5) The glycoside derivatives of lignans in contrast, are mostly in minor forms Lignans are normally constituted from oxidative dimerization of two phenyl-propane units (Ignat et al., 2011) Many efforts have been made to specialized studies of lignans as well as their synthetic derivatives due to their potential applications in various pharmacological capabilities and cancer chemo-therapy (Saleem et al., 2005; Ignat et al., 2011)

Source: Ignat et al., 2011 Figure 2.5: Chemical structure of Lignans

2.1.5 Synthesis and metabolic processes of phenolic compounds

In plant metabolism, there are two different pathways named primary and secondary (Liu, 2004; Laura et al., 2010) Both of them can be found in all cells and in specialized cells, respectively The primary pathways manipulate a group of basic compounds; while in the secondary pathways, a wide variety of unique compounds will be produced of this kind of metabolic mechanism (Laura

et al., 2010; Ignat et al., 2011)

In the primary pathways, the metabolism of nucleic acids, proteins, lipids and carbohydrates are taken under the help of various reactions such as the biosynthesis of nucleic, protein and lipid, the pentose phosphate shunt, the tri-carboxylic acid cycles, and most importantly, glycolysis (Saleem et al., 2005) Differ from those metabolites, the products of secondary pathways which include coumarins, flavonoids, lignin, alkaloids, terpenes, phenylpropanoids and some other related compounds, are chemically produced by some specific

pathways such as the methylerythritol phosphate pathway or mevalonic acid pathway (Laura et al., 2010)

The synthesis of phenolic compounds in plant can proceed due to various pathways and it leads to the diversity of metabolic sub-groups (Ignat et al., 2011) The roles of each phenolic compound in plant also varied due to their diversity (Liu, 2004; Laura et al., 2010) For instance, some play roles in protecting plants against excessive water loss and/or harmful ultraviolet solar radiation, whereas some are responsible for mechanical supports (Ignat et al., 2011) In addition, some of these compounds fascinate seed dispersers as well

as pollinators for the plants (Laura et al., 2010) Some also can serve as signal molecules important for abiotic and biotic stress defense mechanisms (Liu, 2004) Moreover, some join in function of competition of the plant within the living conditions with others Products of secondary metabolites usually present

in plants with high amounts Second to cellulose, phenolic compounds contribute about more than 40% of organic matter assemblage to the biosphere (Liu, 2004; Laura et al., 2010)

The induced synthesis of phenolic compounds generally facilitate in growth and development of plants, especially vegetables (Laura et al., 2010; Ignat et al., 2011) However, the innate capacity, which helps the plants more adapted to natural environment by responding to various abiotic and biotic stresses, may induce metabolic responses It can result in reduction of product quality (Laura et al., 2010) For instance, jasmonic and salicylic acid are products of lipid and phenolic metabolism, which has resulted from the responsibility of the plants to a multitude of stresses Actually, when the plants respond to volatile stresses, some induced hormones such as jasmonic acid and ethylene can possess positive effect to the synthesis and accumulation of phenolic compounds up to high levels In addition to that aspect of signalling pathway, plant hormones such as ethylene and abscisic acid have also been induced (Ignat et al., 2011)

The excessive accumulation and/or over-production of constitutive synthesis of some particular phenolic compounds can cause reduction in product quality (Laura et al., 2010) Therefore, to improve the quality of food production using formulate techniques and management of cultural procedures, deeply insights in the synthesis and metabolism of phenolic compounds are extremely important The main target is reducing the phenolic metabolism and its effect in product quality (Giovannucci et al., 2003)

2.1.6 Phenolic compounds in vegetables and their

health-promoting properties

Vegetables are not only great source of essential nutrients and fibres, but also of various phytochemicals(see table 2.2) (Liu, 2004; Laura et al., 2010; Ignat et al., 2011) It has been shown that fruits and vegetables can give health-promoting benefits although the evidence and data proving it are contradictory among group of researchers and need more observations and studies (Giovannucci et al., 2003; Liu, 2004; Laura et al., 2010) For that reason, the effects of vegetable consumption in terms of treatments of various diseases as well as the mechanisms of each phytochemical component from vegetables are still needs more investigations and clinical intervention trials Fruits and vegetables are recently considered as functional food due to the fact that various phytochemicals extracted from them express in high antioxidant capacity (Syngletary et al., 2005; Laura et al., 2010) In addition to phenolic compounds, vegetable also provide other phytochemicals such as carotenoids and ascorbic acid Most of them have significant implications for human health (Liu, 2004; Laura et al., 2010)

Table 2.2: Fruits and vegetable – great sources of phenolics

Source: Ignat et al., 2011

By eating high-antioxidant level vegetables can help in increasing antioxidant concentration in blood and the whole body tissues, consequently protect cells and tissues from oxidative damage Evidences shown that, increasing dietary consumption of fruits and vegetables together with a proper way of control of infections as well as avoidance of smoking positively help to reduce some serious diseases and different types of cancers (Giovannucci et al., 2003; Liu, 2004; Syngletary et al., 2005; Laura et al., 2010) According to World Health Organization (WHO), a recommendation for public health strategy

is considered with about 400g/person in daily dietary consumption of fruits and vegetables with different colour ranging from white-green, green, orange-yellow, orange, red-purple, yellow-green and red for a better health care (Johnston et al., 2000; Liu, 2004; Laura et al., 2010)

2.2 Anthocyanins from vegetables

2.2.1 Introduction

Anthocyanins are the most common subgroup of flavonoids that can be found in fruits and vegetables (Abudullah et al., 2006) The name anthocyanins (from the Greek, anthos means a flower, and kyanos means dark blue) was first coined by Marquart (1835) and still be used up to the present day (He and Giusti, 2010) Those pigments are responsible for natural colour of plant species and their products, ranging from vivid red to blue (Boulton, 2001; Eiro and Heinonen, 2002; Abudullah et al., 2006; Abou-Arab et al., 2011)

Up-to-date, more than 635 anthocyanins have been identified in plants (Harborne and Williams, 2001) Anthocyanins are found in fruit, flowers, seeds, leaves, stems and root tissues of plants Flavanonols, flavanones, flavones, flavan-3-ols and flavonols are other sub-groups of flavonoids that differ in their oxidation state from the anthocyanins When dissolved, they are colourless or pale yellow (Harborne and Williams 2001; Kong et al., 2003)

2.2.2 Vegetables – a great sources of anthocyanins

Natural sources of anthocyanins vary greatly including coloured fruits, vegetables, nuts, and spices These are berries, grapes, dates, onion, eggplant, corn and importantly various vegetables such as red cabbage, purple sweet potato, broccoli, Roselle, and radish (Kong et al., 2003; Ignat and Popa, 2010) Among all kinds of vegetables in nature, red cabbage is one of the highest contained sources of anthocyanins (Arapitsas et al., 2008) The concentration and type(s) of anthocyanins presented in each coloured species vary substantially For instance, blueberries and grapes contain most of anthocyanidin derivatives while red cabbage has only cyanidin derivatives (Kong

et al., 2003)

2.2.3 Biosynthesis pathways of anthocyanins

There are many enzymes involved in biosynthesis pathway of anthocyanins which are also required for the synthesis of other flavonoids compounds (see figure 2.6) Many steps are involved in the pathway in which the product of the previous step of reaction will provide the substrate(s) for the

next leading step(s) (Rausher, 2008; Ignat and Popa, 2010).The pathway is started by the formation of chalcones from the reaction between malonyl CoA and 4-Coumaroyl CoA

Source: Rausher, 2008.Figure 2.6: Biosynthesis pathways of anthocyanins Thefirst step is catalysed by enzyme chalcone synthase (CHS) Various kinds of enzymes, which are listed in Italic, uppercase (chalcone (CHS), chalcone-3-hydroxylase (CHI), flavonoid 3’5’hydroxylase (F3H), leucoanthocyanidins reductase (LCR), flavone synthase (FS), flavonol synthase (FLS), isoflavone synthase (IFS), aureusidin synthase (AUS), dihydro (DH), leuco (L)) while specific compounds produced after single step by step of reactions are listed in lowercase (chalcone, naringenin, DH-kaempferol, DH-quecitiin, …) and classes of compounds are presented in bold

ANTHOCYANIDINS,…).Three coloured branches of the pathway are leaded to three different final classes of anthocyanins with three different hydroxyl groups

1, 2 and 3 Branches which are not coloured are be ended by other types of flavonoids (Rausher, 2008)

Anthocyanins present in anthocyanoplasts which are intensively pigmented organelles that can be found in anthocyanin-producing cells in more than 70 species (Rausher, 2008).The anthocyanoplasts are formed in the vacuole or in the cytoplasm during pigment synthesis and then they are dispersed to produce a pigmented vacuole Within plants, anthocyanins are exclusively accumulated and located differently depended on specific parts To

be more specifically, in grapes and/or berries, anthocyanins are located in epidermal cell layers, while in some flowers, they are found in epidermal cell and rarely in mesophyll cells (Robert and Catherine, 1980; Rausher, 2008)

sub-2.2.4 Anthocyanins chemical properties and functions in nature

The basic structures of anthocyanins are anthocyanidins which consist of

an aromatic ring [A], a heterocyclic ring [C] and another aromatic ring [B] (Castaneda-Ovando, et al., 2009) The [C] ring contains oxygen molecule, which

is bonded to the [B] ring by carbon-carbon bond (see figure 2.7) When the anthocyanidins are in glycoside form, they are called anthocyanins

Source: Castaneda-Ovando, et al., 2009 Figure 2.7: General structure of anthocyanins

Up-to-date, there is a huge variety of anthocyanins found in nature Based on the differences in the number of hydroxylated groups, the nature and number of aromatic or aliphatic acids attached to sugars in the molecule, the nature and number of sugars that attached to the molecule as well as the position of this attachment, individual anthocyanins can be distinguished Among more than 635 different anthocyanins and 23 anthocyanidins (see table 4.1), only six of them can be found in higher or vascular plants including delphinidin (Dp),pelargonidin (Pg), cyanidin (Cy),peonidin (Pn), petunidin (Pt)

and malvidin (Mv) (Harborne and Williams 2001; Kong et al., 2003; Ovando et al., 2009)

Castaneda-Table 2.3: Names and abbreviations of common varieties of anthocyanins

Source: Castaneda-Ovando et al., 2009

In particular, the glycosidesof Pg, Dp and Cy, three non-methylated anthocyanidins, are the most common found in nature with 80% in pigmented leaves, 69% in fruits and about 50% in flowers In fruits and vegetables, the distribution of six common anthocyanidins (see figure 2.8) is revealed with different percentages as followed: Mv (7%), Pt (7%), Dp (12%), Pn (12%), Pg (12%) and Cy (50%) (Castaneda-Ovando et al.,2009)

(a)

(b) Source: Castaneda-Ovando et al.,2009 Figure 2.8: (a) General structure of the six common anthocyanidins (Cabrita

et al., 2010); (b) Classification of anthocyanins with six common sub-groups Based on several reviews in anthocyanins recently, it is revealed that anthocyanins have an enormous variety in nature being found as a very interesting but also very complex group of flavonoids Apart from the ability of counting for colour of plants and plant products, some of anthocyanins might function usefully in chemotaxonomic investigations, cold tolerance, photo-inhibition tolerance, phytoalexin anti-microbial antioxidants and/or in pollinationprocess of different fruits and vegetables (see table 2.4) (García-Benítez, Cabello, & Revilla, 2003; Kong et al., 2003; Castaneda-Ovando et al.,2009)

Table 2.4: Summary of functions of anthocyanins in some plants in nature

NOTE: “?” represented for not investigated function

Source: Kong et al., 2003

2.2.5 Anthocyanins stability

Anthocyanins stability which is important for a long-term industrial use,

is easy to affected by various factors including the presence of metallic ions, proteins, flavonoids and enzymes; solvents; light; oxygen; chemical structure; pH; concentration; storage temperature (Castaneda-Ovando et al., 2009) The chemical stabilisation is now become one of the main attentions in recent anthocyanins studies Being good alternatives to artificial colorants in future based on their great potential applications and beneficial effects, the stabilisation of isolated anthocyanins need to be studied well in order to avoid unwanted degradation (Reiersen et al., 2003; Kong et al., 2003; Castaneda-Ovando et al., 2009)

Solvents used and their concentrations in the extraction processessuch

as ethanol, water, acetonitrile: water, dioxane, 2-butanone possess have been revealed to affect colour of final extracted anthocyanins products Ito and his co-workers (2002) investigated that the colour of synthetic flavylium salts (FVs)

in those mentioned nature is changed due to FVs and solvent concentrations Particularly, when FVs are in protic solvent, they exhibit red colour and they

differ in case of using aprotic solvents with yellow colour ones.It was proposed that the yellow and red species resemble dimer and monomer respectfully in order to explain for that fact of changing colour In addition, water concentration also plays a fundamental role in this circumstance when it is added to the acetonitrile: water mixtures with higher volume and leading to the converting of monomer into a green dimer.Water also facilitates the dimerization of flavylium salts due to the fact that water molecules may help to neutralizing the electrostatic repulsions of flavylium salts which are required for the dimerization (Ito et al., 2002; Castaneda-Ovando et al., 2009)

Depending on the pH value of the solution, the chemical forms of anthocyanins can be varied significantly (Kennedy and Waterhouse, 2000; Fleschhut et al., 2006; Castaneda-Ovando et al., 2009) Ranging from red, purple, blue, green and/or colourless, the pH value is from 1 to 6 correspondingly (see figure 2.10) In that, figure [A] with pH 1 corresponds to red and purple colours, figure [B], [C] and [D] at pH value between 2 and 4 correspond to blue colour Figure [E] and [F] represent for two colourless species carbinol pseudobase and chalcone, respectfully At pH values higher than 7, degradation reaction of anthocyanins (see figure 2.9) will occur depended on their substituent groups (Kennedy and Waterhouse, 2000; Cabrita, Fossen and Andersen, 2000)

Source: Cabrita, Fossen and Andersen, 2000

Figure 2.9: Thermal degradation of anthocyanins

The degradation of anthocyanins results with phenolic acid and aldehyde (figure 2.10, degradation reaction) Normally, when the temperature is higher than 700C, it will rapidly cause the degradation as well as discoloration of anthocyanins (Cabrita, Fossen and Andersen, 2000).The stability of anthocyanins is affected by the ring [B] within their structure The additional methoxyl or hydroxyl groups which influent the stability of the aglycon when the anthocyanins arein neutral media Among various kinds of anthocyanidins, Pg have been stated as the most stable one due to the presence of the hydroxyl group in its structure (Fleschhut et al., 2006; Castaneda-Ovando et al., 2009) Within the anthocyanin structure, the alkaline region is the most instable and easy to be affected by pH values revealing by the investigations about anthocyanins stability and the effect of pH value on the colour exhibition (Cabrita, Fossen and Andersen, 2000; Fleschhut et al., 2006; Castaneda-Ovando et al., 2009)

Source: Castaneda-Ovando et al., 2009

Figure 2.10: Chemical structures of anthocyanins in corresponding to pH values and the degradation reaction

2.2.6 Anthocyanins biological properties

2.2.6.1 Antioxidant activities

Anthocyanins and anthocyanidins are easy to oxidise therefore they have high antioxidant properties as compared to other compounds (Castaneda-Ovando et al., 2009) The compounds contain 1,4-hydroquinone or catechol groups are very easy to oxidise due to ability to stabilizing of the phenoxyl radical with oxygen Those compounds do not extract any hydrogen molecules

to other substances They will react with another semiquinone to generate phenol group and a quinone by using two radicals (Castaneda-Ovando et al., 2009) Generally, the antioxidant mechanisms can be explained using protein biding, metal chelation and hydrogen donation (Kong et al., 2003) In fact, the anthocyanins can donate phenolic hydrogen atoms or a free electron to capture free radicals Anthocyanins not only show higher antioxidant activities than vitamins C and E, but also might contribute to the fruits and vegetables some other anti-carcinogenic, mutagenesis activities against chronic and degenerative diseases (Wang and Lin, 2000; Castaneda-Ovando et al., 2009)

In nature, besides the phenolic compounds, some other phytochemical substances also exhibit antioxidant activity including ascorbic acid, carotenoids, tocopherols and some nitrogenous compounds (Nichenametla et al., 2006) Several studies have reported that there is a linear correlation between the anthocyanins content in some fruits and vegetables and the values of the antioxidant capacity (Wang and Lin, 2000; Nichenametla et al., 2006; Castaneda-Ovando et al., 2009)

2.2.6.2 Other biological properties

Apart from the antioxidant capacity, anthocyanins also possess some other pharmaceutical properties from which they are used by humans in therapeutic treatments Those include anti-oedema, anti-inflammatory, cell-regeneration, antimicrobial, chemo-preventive, vasoactive (Kong et al., 2003; Xavier et al., 2008) Based on those pharmaceutical application potentials, anthocyanins have been investigated whether they might have effects on cancer development(see figure 2.11) (Hou, 2003; Kong et al., 2003; Xavier et al.,

2008, Zu et al., 2010) Several studies have been conducted to test the effects

of anthocyanins in some illnesses or diseases such as tissue inflammation, capillary permeability and fragility, tumor, cardiovascular disease, liver damage (Pawlowicz et al., 2000; Jankowski et al., 2000; Kong et al., 2003; Castaneda-Ovando et al., 2009)

Source: Hou, 2003 Figure 2.11: Mechanism of anthocyanins in preventing cancer

According to Meiers et al., (2001), the two anthocyanidins cyanidin (Cy) and delphinidin (Dp) showed the ability to inhibit the tumour cells or epidermal growth-factor receptor (EGFR) in human In that experiment, malvidin (Mv), another kind of anthocyanidin normally found in grapes, showed less effect than

Cy and Dp Another anthocyanin activity study was conducted by Wang and Mazza in 2002 which test the potentials of anthocyanins in antiulcer activity and capacity to protect from ultra-violet (UV) radiation (Sharma, 2001; Wang and Mazza, 2002) They also tested for the inhibitory effects of other phenolic compounds on nitric oxide (NO) production From theirfinding, it is stated that

NO is associated with some chronic inflammatory diseases (Kong et al., 2003)

2.2.6.3 Anthocyanins and human health effects

As compared to other flavonoids, anthocyanins have high dietary consumption due to the fact that they are wide distributed in plant materials (He

et al., 2010) Evidences from various cell-line studies and some human clinical trials suggested that anthocyanins have great potential in health-promoting benefits due to their antioxidant properties (Hou, 2003; He et al., 2010)

Anthocyanins chemically can possess diabetes alleviation properties, obesity control, cardiovascular disease prevention, anti-carcinogenic activity and anti-inflammatory In human digestive system, anthocyanins are mainly absorbed by the epithelial tissues of stomach and small intestine Anthocyanins possibly possess health benefits as dietary antioxidants (Kong et al., 2003; Castaneda-Ovando et al., 2009)

Due to the roles of anthocyanins and some other phytochemicals as phyto-protective agents and pollination attractants, nowadays plant physiologists and botanists have investigated on anthocyanins in various taxonomic studies (Castaneda-Ovando et al., 2009) Moreover, food scientists and horticulturists also conducted many studies on these compounds due to their noticeable importance as natural colorant in industrial food system Most of the sources come from fruits and vegetables The utility of anthocyanins as natural food colorants have been studied with many subsequent projects and research (Gachovska et al., 2010; Lu et al., 2010; Xu et al., 2010; Geetha et al., 2011; Ignat et al., 2011; Idham et al., 2012)

For a better use of anthocyanins in preventing diseases and cancers in human, there is much to be learned about their mechanisms, how they are metabolized, the relationships between their structure and activity as well as the bio-availability Apart from it, the side effects of these compounds in certain dosage levels also need to be identified in future research Consequently, the advantages of anthocyanins in both antioxidant health benefits and natural colorant for industrial uses in food system will be more sufficient

2.2.7 Anthocyanins as natural colorants

Consumers have been increasing concerns health-care worldwide The uses of anthocyanins, natural pigments, as food colorants to replace synthetic food dyes have been increased recently (Sanchez-Moreno et al., 2006) Anthocyanins from fruits and vegetables are generally considered as a great source with safe and health promoting properties Apart from it, betalains from beets, carotenoids from carrots have also been used as natural colours in domestic and industrial food (Gachovska et al., 2010)

In addition to fruits, some vegetables such as red cabbage, radishes, onion (see table 2.5) have been received more and more interests due to their high anthocyanin content and the susceptibility for the extraction process(Xavier

et al., 2008; Xu et al., 2010) There have been not only many extraction methods have been conducted in various kinds of vegetables, but also for the isolation, characterisation, quantification and separation of anthocyanins (Sanchez-Moreno et al., 2006; Stalikas, 2007; Arapitsas and Turner, 2008; Xavier et al., 2008; Patras et al., 2009; Vatai et al., 2009; Gachovska et al., 2010; Lu et al., 2010; Xu et al., 2010; Geetha et al., 2011; Ignat et al., 2011; Jacob et al., 2011; Idham et al., 2012)

Table 2.5: Fruits and vegetables – common sources of anthocyanins with different indicated concentrations

particles of plant materials, the researchers will choose a sufficient method for the extraction of anthocyanins Meaning, different kind of vegetables will lead to different methods selected for the extraction process (Fan et al., 2010; Macdonald et al., 2010) It is also concerned about the selection of solvents for

a high yield, high recovery, time-consuming and cheap result of extraction process Generally, there are three main steps involved in the whole extraction procedure, the pre-treatment step, extraction step, and the isolation and purification step (see figure 2.12) (Routray and Orsat, 2012) The processes involved in the pre-treatment step such as drying, milling, grinning, homogenisation and maceration facilitate for the whole extraction process with high yield, long-term storage and ease for further processes (preservation, usage and application for industrial food) They mix up and increase the contact surface between sample and solvent used, therefore facilitate the rupture and breakdown of cell walls Anthocyanins, for that reason, are easy coming out into the solid and easier to be obtained after extraction procedure (Macdonald et al., 2010; Fan et al., 2010; Khandare et al., 2011)

Source: Routray and Orsat, 2012 Figure 2.12: Steps involved in the whole extraction process of anthoyanins from vegetables

Along with pre-treatment step and selection of solvent used, nowadays, many novel extraction techniques including microwave-assisted extraction, supercritical, fluid extraction, accelerated fluid extraction, sonication assisted extraction, which are newly investigated and used as alternative for the conventional extraction method (such as Soxhlet extraction) The uses of

ultrasound, microwave and other assistance help disrupt plant cellular structures, increase the solvent penetration efficacy (Lu et al, 2010; Routray and Orsat, 2012) In such cases, high yield of extracted anthocyanins are obtained efficiently In addition, they help reduce large amount of solvent used like in conventional extraction method, which is one seriously environmental issue For the methods which use non-selective or toxic solvents (hexane), isolation and purification steps are essential in order to obtain the final extracted anthocyanins with highly purified and non-toxic to human and environment as well (Macdonald et al., 2010; Khandare et al., 2011)

Anthocyanins after extracted, for long-term usages and domestic and industrial food applications as natural colorant, needs to proceed undergo the preservation process There are two commonly preservation processes for anthocyanins The extracted anthocyanins need to be stored in darkness as a hydro-alcoholic solution at 40C or as a freeze–dried powder at 250C for further usage by using either thermal or high pressure processing (see figure 2.13)

Source: Idham et al., 2012 Figure 2.13: Two commonly used preserved processes: (a) thermal processing, (b) high pressure processing

Depending on pH values (ranging from 3, 5, 7 or 9), total antioxidant pigments as well as the antioxidant power will be affected (Idham et al., 2012) Therefore, the preservation process needs to be considered with sufficient technique for long-term storage of anthocyanins

After searching, all the information related to anthocyanins extraction and preservation processes was extracted, compared between different publicationsthat have discussed about the same parameters and objectives in order to figure out the reliable peer review

3.2 Referencing

In this thesis, standard “Harvard Referencing” which is extensively accepted in scholarly circles has been used to avoid plagiarism, a serious academic offence In text references are important not only for the evaluators easy to make further reading and checking, but also for juniors and/or readers can find the initial sources During the process to cite reference insert citations and produce bibliographies, some effective reference management software (RefWorks, Endnote) This is a good way compared to other different systems and applications to cite and create bibliography with correct, time-consuming and complex duty

CHAPTER 4: RESULTS AND DISCUSSIONS

4.1 Methods for extraction of anthocyanins from vegetables

There are a number of methods for extraction of anthocyanins have been proposed and used by researchers including Soxhlet extraction, ultrasound-assisted extraction, microwave-assisted extraction, accelerated solvent extraction, supercritical fluid extraction, pulsed electric field extraction In this chapter, these methods will be discussed along with principles, mechanisms, practical design and advantages and disadvantages of each method

4.1.1 Conventional extraction method - Soxhlet technique

4.1.1.1 Introduction

Most of the conventional techniques for solvent extraction of natural compounds from plant materials which include hydrostillation, maceration and Soxhlet, differ by choice of solvent and assistance of heat and/or agitation (Wang and Weller, 2006; Wang et al., 2011) Among those classical techniques, Soxhlet extraction was mostly used as a standard one and support as the main reference for other novel techniques available at present Originally, Soxhlet technique was used to determine of fat in milk (Luque de Castro and Garco Aa-Ayuso, 1998) It is the most general and well-established technique used for leaching or solid-liquid extraction methods (Wang and Weller, 2006) It is less efficient than other novel techniques such as ultrasound-assisted extraction and/or microwave-assisted extraction, hence there is a very limited number of reports for the applications of Soxhlet technique for extraction of anthocyanins from vegetables (Pinelo et al., 2004; Naczk and Shahidi, 2006; Wang and Weller, 2006; Wang et al., 2011)

4.1.1.2 Principles and mechanisms

In analytical processes, sample pre-treatment is considered as one of the most time consuming steps Soxhlet extraction (see figure 4.1) is the oldest way

of such kind of solid sample pre-treatment (Wang and Weller, 2006; Pinelo et al., 2004) It has been used for a long time and requires large amount of solvents

In Soxhlet extraction, solid plant materials are placed within a holder During the process, samples are filled with condensed solvent from a distillation flask (see figure 4.1) It is operated until the liquid reaches the overflow level Subsequently, the solute within the thimble-holder is aspirated

thimble-by a siphon and it is also unloaded back into the distillation flask The extracted liquid is then carried into the bulk liquid

Source: Wang and Weller, 2006 Figure 4.1: Schematically experimental apparatus of Soxhlet extraction technique: (a) laboratory Soxhlet extractor, (b) schematic diagram of Soxhlet extraction apparatus

In the solvent flask, distillation is used to separate the achieved solute which

is placed separately in flask while the fresh solvent is right back into the sample solid bed The process is reiterated flow after flow until a target extraction solute is accomplished (Luque de Castro and Garco Aa-Ayuso, 1998; Wang and Weller, 2006)

4.1.1.3 Practical design

Soxhlet extraction method needs a suitable solvent in order to obtain the final targeted phenolic compounds Depended on the extraction conditions, different solvents are selected and the results are also different in yields From

the literature, hexane is the most widely used solvent for Soxhlet extraction It

is a very good solvent because it is soluble and easy to recover or reused Nevertheless, according to Environmental Protection Agency, US, hexane, especially n-hexane which is the common commercial hexane is the most hazardous air pollutant (Mamidipally and Liu, 2004) Therefore, due to laboratory safety, human health cares as well as environmental concerns, researchers have been selected some other less toxic solvents for the operation such as water, hydrocarbons, d-limonene, ethanol and isopropanol (Pinelo et al., 2004; Pinelo et al., 2005; Wang and Weller, 2006)

Taking one example, Mamidipally and Liu (2004) conducted one experiment

to test and compare the efficacy of different solvent used on extracted oil yield from rice bran In that, they figured out that the extracted oil amount is significantly higher when the d-limonene solvent was used rather than hexane Another circumstance for water, the achieved oil content and free fatty acid were also observed with lower contents than the ones extracted using hexane (Hanmoungjai and Niranjan, 2000) Moreover, colour imparting components were obtained with lower performance From that, alternative solvents seem to

be resulted with lower yields as well as recovery capability (Wang and Weller, 2006) The reason for that is molecular affinity is decreased between solute and solvent used (Pinelo et al., 2004)

Cost of alternative solvents that have been used for Soxhlet extraction is generally higher than hexane Therefore, co-solvent(s) sometimes is added together with solvents as a better cost deal In addition, some of the co-solvent helps increase the polarity of the liquid phase within extraction process (Wang and Weller, 2004; Wang et al., 2011) According to Li and Weiss (2004), when co-solvent(s) (i.e isopropanol) is mixed with hexane, final extracted compounds and kinetics of extraction are increased significantly

There are many factors influent the Soxhlet extraction process First, matrix characteristics and particle size of samples strongly affect the final extracted solid compounds, especially anthocyanins They are considered as internal diffusion during the extraction process (Luque-Garcia and Luque de Castro, 2004) For instance, when two experiments with different particle size of sample are set up, the larger the particle size sample the longer the extraction process

to obtain the same result of extraction efficacy Next, temperature also affect to the extraction process because boiling temperature directly affect to the evaporation process which helps the solvent used easy to recover Temperature