TOTAL PHENOLIC AND TOTAL FLAVONOIDS CONTENT OF PITAYA PEELS BY

TOTAL PHENOLIC AND TOTAL FLAVONOIDS CONTENT OF PITAYA PEELS BY

WATER EXTRACTION

NG WEI CHET

A thesis submitted in fulfillment of the

requirements of the award of degree of

Bachelor of Chemical Engineering

Faculty of Chemical and Natural Resources Engineering

Universiti Malaysia Pahang

APRIL 2009

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I declare that this thesis is entitled “Total Phenolic and Total Flavonoids Content of

Pitaya Peels by Water Extraction” is the result of my own research except as cited in the

references. The thesis has not been accepted for any degree and is not concurrently

submitted in candidature of any other degree.

Signature : …………………………

Name : NG WEI CHET

Date : 8 APRIL 2009

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Special dedication to my family members, my supervisor, staffs of FKKSA laboratory and all my beloved friends.

For all your love and support. Thank you very much.

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ACKNOWLEDGEMENT

In preparing this thesis, I have gained knowledge and experiences from many

specialists. Therefore, I would take this chance to acknowledge their contributions.

First and foremost, a special thank to my supervisor, Dr Mimi Sakinah for her

willingness in overseeing the progress of my research work from the initial phase till the

completion of this thesis. Her understanding, encouraging and personal guidance have

provided a good basis for the present thesis.

Last but not least, I would like to express my sincere gratitude to my family

members who always care and support me throughout my study at Universiti Malaysia

Pahang, UMP.

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ABSTRACT

Residues from the processing of fruits and vegetables which are traditionally

considered an environmental problem is now widely recognized for obtaining high-

phenolic products. This paper was designed to study the total phenolic and total

flavonoids contents in the pitaya peels. The total phenolic content was determined by

using the Folin-Ciocalteu assay while the total flavnoids was measured using aluminum

chloride colorimetric assay by UV-visible spectrometer. The result showed that the

highest total phenolic content in pitaya peels was extracted at the optimum dose of

aluminum sulfate concentration of 25 mg/L (3.32 mg gallic acid equivalents (GAE)/ 25

g at 80oC, 3.21 mg GAE/25g at 60oC and 1.73 mg GAE/25g at 40oC). The greatest total

flavonoids content in pitaya peels was extracted (2.24 mg catechin equivalents (CE)/25 g

at 60oC, 1.79 mg CE/25g at 80oC and 1.60 mg CE/25g at 40oC) at the concentration of

aluminum sulfate of 30 mg/L. The results showed that the value of the total phenolic

content decreased when the concentration of aluminum sulfate of 25 mg/L due to the

fact that the high concentration of aluminum sulfate (more than 25 mg/L) could have

reacted with the phenolic compound of the pitaya peels to form another compound. The

total flavonoid was found to be extracted with the highest value at temperature of 60oC

and the lowest at 80oC. This shows that the flavonoids compound was somehow

destroyed at temperature of 80oC.

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ABSTRAK Sisa pemprosesaan buah-buahan dan sayur-sayuran yang selalunya menjadi

punca masalah alam sekitar kini semakin mendapat perhatian ramai kerana sisa tersebut

mengandungi jumlah kandungan fenolik yang memberangsangkan. Kajian ini bertujuan

untuk mengenal pasti jumlah kandungan fenolik dan jumlah kandungan flavonoids yang

terkandung dalam kulit buah naga. Jumlah kandungan fenolik ditentukan melalui kaedah

Folin-Ciocalteu, manakala jumlah kandungan flavonoids pula ditentukan melalui kaedah

kalorimetri aluminum klorida dengan bantuan alat UV-Vis spectrometer. Keputusan

kajian menunjukkan bahawa jumlah kandungan fenolik yang tertinggi dalam kulit buah

naga ialah 25 mg/L sebanyak (3.32 asid gallic bersamaan (GAE/ 25 g pada 80oC, 3.21

mg GAE/ 25g pada 60oC dan 1.73 mg GAE/ 25g pada 40oC) iaitu pada aluminum

sulfate yang optimum. Bagi jumlah kandungan flavonoids yang dapat dalam kulit buah

naga, adalah sebanyak (2.24 mg catechin bersamaan CE/25g pada 60oC, 1.79 mg

CE/25g pada 80oC dan 1.60 mg CE/ 25g pada 40oC) pada kepekatan aluminum sulfate

30 mg/L. Keputusan juga menunjukan kecerunan graf pada setiap suhu bagi jumlah

kandungan fenolik adalah menurun selepas 25 mg/L. Ini berkemungkinan adalah asid

yang berkepekatan tinggi (selepas kepekatan 25 mg/L) bertindakbalas dengan jumlah

kandungan fenolik komponen menjadikan ianya berubah kepada komponen yang

berlainan. Bagi jumlah kandungan flavonoids, penghasilannya semakin bertambah

apabila kepekatan aluminum sulfate dicampurkan. Walaubagaimanapun, jumlah

kandungan flavonoids hanya menunjukkan hasil yang banyak pada suhu 60oC dan bukan

pada suhu 80oC. Ini menunjukkan jumlah kandungan flavonoids telah diubahkan dari

segi struktur pada suhu 80oC dan mengakibatkan komponen itu menjadi bahan yang lain.

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TABLE OF CONTENTS

CHAPTER TITLE PAGE DECLARATION ii DEDICATION iii ACKNOWLEDGEMENT iv ABSTRACT v ABSTRAK vi TABLE OF CONTENTS vii LIST OF TABLES xiii LIST OF FIGURES xiv LIST OF ABBREVIATIONS xvii 1 INTRODUCTION 1.1 Background of Research 1 1.2 Problem Statement 2 1.3 Significance of Study 4 1.4 Objectives 6 1.5 Scope of Study 6

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2 LITERATURE REVIEW 2.1 Pitaya 8 2.1.1 Types of Pitaya 9 2.1.1.1 Yellow Pitaya 9 2.1.1.2 Red Pitaya 10 2.1.2 Plant Stem 11 2.1.3 Inflorescence 11 2.1.4 Fruit 11 2.1.5 Propagation 12 2.2 Polyphenols 12 2.3 Flavonoids 13 2.4 Natural Dyes 14 2.4.1 Art of Making Natural Dyes History 15 2.4.2 Types of Dyes 16 2.4.2.1 Lac Dye 16 2.4.2.2 Annatto 16 2.4.2.3 Harda 16 2.4.2.4 Himalayan Rhubard 17 2.4.2.5 Indigo Blue 17 2.4.2.6 Kamala Dye 17 2.4.2.7 Manju Phal 18 2.4.2.8 Gum Arabic 18 2.4.2.9 Trigonella Foenum Graecum 18

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2.4.2.10 Gold Dock 19 2.4.3 Uses of Dyes 19 2.4.3.1 Textile 19 2.4.3.2 Cosmestic 19 2.4.3.3 Edible Dyes 20 2.4.3.4 Food Items 20 2.4.3.5 Leather 20 2.5 Extraction 21 2.5.1 Method of Extraction 21 2.5.1.1 Aqueous Method 21 2.5.1.2 Alkaline Method 22 2.5.1.3 Acidic Method 22 2.5.1.4 Alcoholic Method 22 2.5.2 Extraction Technology 23 2.5.2.1 Solvent Extraction 23 2.5.2.2 Supercritical Fluid Extraction 24 2.5.2.3 Microwave Assisted Extraction 24 2.5.2.4 Continuous Steam Distillation Process 25 2.6 Advantages of Natural Dyes 25 2.7 Disadvantages of Natural Dyes 25 2.8 Aqueous Extraction 26 2.9 Total Phenolic Content (TPC) 27 2.10 Total Flavonoids (TF) 28

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2.11 Ultra Violet Visible Spectrometer 29 2.12 Centrifuge 31 2.12.1 Differential Centrifugation 32 2.12.2 Density Gradient Centrifugation 32 2.12.3 Rate Zonal Separation 34 2.12.4 Isopycnic Separation 35 3 METHODOLOGY 3.0 Introduction 36 3.1 Methodology Flow Chart 36 3.2 Preparation of Sample 39 3.2.1 Drying of Samples 41 3.2.2 Blending of Pitaya Peels into The 43 Powder Form 3.3 Extraction of Pitaya Peels 44 3.3.1 Extraction of Pitaya Peels at The 44

First Stage

3.3.2 Extraction of Pitaya Peels at 45

Second Stage

3.4 Filtration of The Pitaya Peels From The Extraction 46

3.5 Flocculation of Natural Dye From Extraction of 47

Pitaya Peels

3.5.1 Preparation of Stock Solution of Aluminum 47

Sulfate

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3.5.2 Dilution of Concentration of 47

Aluminum Sulfate

3.5.3 Stirring of Mixture of Aluminum Sulfate 48

Solution and Pitaya Peels Filtrate

3.6 Filtration of Floc 49

3.7 Analysis of Total Phenolic Content and 51 Total Flavonoids 3.7.1 Preparation of 80% Methanolic 51 Pitaya Peels Extracts 3.7.2 Centrifugation 52 3.7.3 Analytical Determination of 54 Total Phenolic Content 3.7.4 Analytical Determination of 55 Total Flavonoids

4 RESULTS AND DISCUSSIONS

4.0 Results 57 4.1 Total Phenolic Content and Total Flavonoids Content 58 Obtained at 40oC 4.1.1 Total Phenolic Content 58 4.1.2 Total Flavonoids Content 61 4.2 Total Phenolic Content and Total Flavonoids Content 63 Obtained at 60oC 4.2.1 Total Phenolic Content 63 4.2.2 Total Flavonoids 65 4.3 Total Phenolic Content and Total Flavonoids Content 67 Obtained at 80oC

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4.3.1 Total Phenolic Content 67 4.3.2 Total Flavonoids 69 4.4 Comparison of The Total Phenolic Content at Different 71 Temperatures 4.5 Comparison of The Total Flavonoids at Different 75 Temperatures 4.6 Volume of Filtrate 78

4.7 Weight of Floc 82

4.8 Color of Supernatant at Different Temperatures 85

5 CONCLUSIONS AND RECOMMENDATIONS

5.1 Conclusion 88

5.2 Recommendations 89

REFERENCES 90

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LIST OF TABLES

TABLE NO TITLE PAGE 2.1 Examples of Research Using Aqueous Extraction 26 4.1 Values of Absorbance and Total Phenolic Content at 40oC 60 4.2 Values of Absorbance and Total Flavonoids Content at 40oC 62 4.3 Values of Absorbance and Total Phenolic Content at 60oC 64 4.4 Values of Absorbance and Total Flavonoids Content at 60oC 66 4.5 Values of Absorbance and Total Phenolic Content at 80oC 68 4.6 Values of Absorbance and Total Flavonoids Content at 80oC 70 4.7 Comparison of Total Phenolic Content at Different Temperatures 74 4.8 Comparison of Total Flavonoids Content at Different 77 Temperatures 4.9 Volume of Filtrate Obtained at Different Temperatures 80 4.10 Weight of Floc at Different Temperature 83

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LIST OF FIGURES

FIGURE NO TITLE PAGE 2.1 Yellow Pitaya 9 2.2 Red Pitaya with White Flesh 10 2.3 Red Pitaya with Red Flesh 10 2.4 Molecular Structure of The Flavonoids Backbone 28 2.5 A Diagram of The Component of A Typical Spectrometer 30 2.6 Refrigerated Centrifuge at FKKSA, UMP 31 2.7 Differential Centrifugation 33 2.8 Rate-zonal (size) Separation 34 2.9 Isopycnic Separation 35 3.1 The Flow Chart of the Extraction of Pitaya Peels by Water 37 3.2 Procedure of 80% Methanolic Extraction 38 3.3 Procedure of Total Phenolic Content Assay 38 3.4 Procedure of Total Flavonoids Assay 39 3.5 Sample of Pitaya Peels in Large Pieces 40 3.6 Cutting of Pitaya Peels into Small Pieces 40 3.7 The Fresh Pitaya Peels on the Tray 41 3.8 Pitaya Peels in the Oven Before Drying 42 3.9 Pitaya Peels After Being Dried 42

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3.10 Pitaya Peels Being Blended into Powder Form 45 3.11 Pitaya Peels in Powder Form 44 3.12 Each Plastic Sealed Bag Containing 25 g of Dried Pitaya Peels 45 3.13 Extraction of Pitaya Peels after 6 Hours 46 3.14 Pitaya Peels Filtrate and Aluminum Sulfate Solution 48 Being Stirred 3.15 Flocs Being Formed in the Solution 49 3.16 The Floc Being Filtered By Fabric Cotton 50 3.17 Wet Floc After Being Filtered for 24 Hours 50 3.18 The Mixture Being Shaken at 37oC in The Shaking 51 Incubator 3.19 The Mixture After Being Shaken for 1 Hour 52 3.20 Mixture Containing in the Container Being Placed in 53 Centrifuge 3.21 Mixture Before Being Centrifuged 53 3.22 The Clear Supernatant and The Sediment After Being 54 Centrifuged 3.23 Mixture to be Determined for Its Total Phenolic Content 55 at 765 nm 3.24 Mixture to be Determined for Its Total Flavonoids 56 at 510nm 3.25 Adjusting the Cuvette Position in the UV-Vis 56 Spectrometer 4.1 Total Phenolic Content Versus Concentration of 60 Aluminum Sulfate at 40oC 4.2 Total Flavonoids Versus Concentration of Aluminum 62 Sulfate at 40oC

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4.3 Total Phenolic Content Versus Concentration of 64 Aluminum Sulfate at 60oC 4.4 Total Flavonoids Versus Concentration of Aluminum 66 Sulfate at 60oC 4.5 Total Phenolic Content Versus Concentration of 68 Aluminum Sulfate at 80oC 4.6 Total Flavonoids Content Versus Concentration of 70 Aluminum Sulfate at 80oC 4.7 Comparison of Total Phenolic Content at Different 74 Temperatures 4.8 Comparison of Total Flavonoids Content at Different 78 Temperatures 4.9 Filtrate Volume Versus Concentration of Aluminum 80 Sulfate at 40oC 4.10 Filtrate Volume Versus Concentration of Aluminum 81 Sulfate at 60oC 4.11 Filtrate Volume Versus Concentration of Aluminum 81 Sulfate at 80oC 4.12 Volume of Filtrate at Different Concentration of 82 Aluminum Sulfate 4.13 Weight of Floc Versus Concentration of Aluminum 84 Sulfate at 40oC 4.14 Weight of Floc Versus Concentration of Aluminum 84 Sulfate at 60oC 4.15 Weight of Floc Versus Concentration of Aluminum 85 Sulfate at 80oC 4.16 Supernatant Color at 40oC 86 4.17 Supernatant Color at 60oC 86 4.18 Supernatant Color at 80oC 87

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LIST OF ABBREVIATIONS/SYMBOLS

% - Percent

AlCl3 - Aluminum chloride

CE - Catechin equivalent

F-C - Folin-Ciocalteu

G - Gram

g/L - Gram per liter

GAE - Gallic acid equivalent

kg - kilogram

L - Liter

mg/L - Milligram per liter

Na2CO3 - Sodium carbonate

NaNO2 - Sodium nitrate

NaOH - Sodium hydroxide

nm - Nanometer oC - Degree celsius

TF - Total flavonoids

TPC - Total phenolic content

UV-Vis - Ultra Violet-Visible

Vs - Versus

µL - Micro liter

CHAPTER 1

INTRODUCTION

1.1 Background of Research

Phenolic compounds are secondary metabolites of plants. They are naturally

present in fruits and vegetables. These compounds are a part of the everyday diet and

also used as medicines or supplements. Research has shown that fruits and vegetables

contain other antioxidant nutrients, in addition to vitamins C and E, and carotenoids,

which significantly contribute to their total antioxidant capacity. The major part of those

antioxidant nutrients is polyphenolic compounds, which are components of fruits and

vegetables having strong antioxidant capacity. Flavonoids have a wide range of

biological activities, such as cell-proliferation-inhibiting, apoptosis-inducing, enzyme-

inhibiting, antibacterial, and antioxidant effects. Moreover, some findings indicate that

flavonoids possess various clinical properties, such as antiatherosclerotic, anti-

inflammatory, antitumour, antithrombogenic, antiosteoporotic, and antiviral effects (Su

et al., 2008).

Since ancient times, the ability of natural dyes to color had been known. In the

earliest written record, the use of natural dyes was found in China dated 2600 BC.

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Another proof to the use of natural dyes in ancient time is by the 4th century AD, dyes

such as woad, Brazilwood and etcaeteras where Brazil was named for the wood found

there. Natural dyes can be obtained from plants, animals and minerals. For example, the

turkey red was extracted from madder plant was so popular and used for dyeing until the

mid 1800s.

Pitaya ( Hylocereus undatus(Haw)) or dragon fruit which have been planted in

Viet Nam for almost 100 years (Mizrahi et al.,1997). Nowadays, coloring foodstuffs are

the most popular way to achieve desired hues because of their application does not

require certification (Stich et al., 1999; Stintzing et al., 2004). In addition, natural dyes

from the plants or fruits are believed to be safe as they are non-toxic, non-carcinogenic

and biodegradable nature. So, natural dyes are widely produced nowadays from natural

resources like from the plants or fruits which its colors can be extracted through various

types of method. It is then safe to be used after detoxification process.

1.2 Problem Statement

Polyphenol which it is widely used for the antioxidants are normally used in fats,

oils, soap and cosmetics to prevent oxidative rancidity. Other than the natural

antioxidants, artificial antioxidants such as butylated hydroxyanisole (BHA), butylated

hydroxytoluene (BHT) and propyl gallate (PG) are extensively used in food and some

commercial products. Recently, there has been a study finding out that those artificial

antioxidants may cause loss of nourishment and even produce toxic substances to harm

people’s health. Hence, phenolic antioxidants are strictly controlled for its use with the

definition of the amounts of these additives to be allowed added in foods. Thus, it is

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crucial to find out more natural phenolic to supply to the needs of phenolic from the

natural sources. (Yong et al., 1999)

The present trend throughout the world is shifting towards the use of eco-friendly

and biodegradable commodities, ranging from food industry to textile industry. Hence,

the demand for natural dyes is increasing day by day (Bhuyan et al., 2004).

Recent studies by the European Union found out that the colouring, Red 2G

which is being used in all types of food especially in enhancing the colour of burgers

and sausages that could endanger the health of humans like destroying the genetic

material in cells and causing cancer has been banned with immediate effect by The

Health Ministry of Malaysia while EU had banned this type of colouring on July 27,

2007 (The Star, 2007). The Sudan Red is widely used for colouring solvents, oils, waxes

and floor polishes which is illegally used to enhance and maintain the colour of the food

products has been prohibited for use in food products by Regulations Relating to Food

Colorants (R.1008) of the Foodstuffs, Cosmetics and Disinfectant Act 54 of 1972 in

South Africa. Research indicates that the Sudan Red brings carcinogenic effect to human

beings, and genotoxic effect which is capable of causing damage to DNA.

While in textile industry, it can mostly produce and use approximately 1.3

million tones of dyes, pigments and dye precursors, valued at around $ 23 billion, almost

all of which is manufactured synthetically. However, synthetic dyes have some

limitations. First, the production process requires hazardous chemicals, creating worker

safety concerns. Second, they may generate hazardous wastes and third, these dyes are

not environment friendly (Sengupta et al., 2003). So in the textiles industry nowadays,

there is a revival of natural dyes in textiles is growing interest because of the green

chemistry, improved eco-balances and thereby leading to the niche products for special

markets (Bechtold et al., 2006).

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Consumers nowadays are more aware of the poisonous of consuming artificial

dyes where it can lead to fatal and demanding for natural product or natural sources as

natural dyes lack of toxicity during production and having full biodegradation and

reduction of the environmental impact.

1.3 Significance of Study

Phenolic compounds are secondary metabolites in plants. Over the past 10 years,

there has been increasing interest in phenolic compounds and their role in human health

and nutrition. Some phenolic compounds present in natural products have higher

antioxidant activities than those of synthetic antioxidants. These polyphenolic

antioxidants can also be used to preserve foods because of their protective effects against

microorganisms.

Residues from the processing of fruits and vegetables, traditionally considered as

an environmental problem, are being increasingly recognized as sources for obtaining

high-phenolic products. The polyphenolics from waste materials deriving from agro-

industry production may be used as functional food ingredients and as natural

antioxidants to replace their synthetic equivalents that have experienced growing

rejection. (Zhou et al., 2008)

Natural dyes can offer not only a rich and varied source of dyestuff, but also the

possibility of an income through sustainable harvest of the dye plants. Another aspect to

be considered by an imaginary supplier of natural dyes has been identified during an

extensive study of possible future us of natural dyes, namely that the plant material

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which contains the natural dye needs the same level of standardization as modern

synthetic dyes already have achieved at present (Ganglberger et al., 2003; Rappl, 2005).

Furthermore there is abundance of waste contributed by certain industries like in

Food industry which produces cans of Pitaya leaving the Pitaya peels which can be fully

utilized to be extracted for its natural red dyes by using aqueous extraction method

.Hence producing of natural dyes are much more to be encouraged to fulfill the demand

of the market and reduce the waste disposal.

Production of red natural dye from Pitaya waste is natural and its red colourants

are now growing interest from both food manufacturers and consumers in the continuing

replacement of synthetic dyes (Duhard et al., 1997). As we know the plants have

generally produced less amount of colouring component on extraction which do not

sufficient to provide for the needs of market. Also, there is a restriction in using the

natural dyes in comparison with synthetic dyes because of the cost of the yield of natural

dyes (Ali et al, 2008). With the appearance of synthetic dyes the use of natural dyes for

textile dyeing almost disappeared. The wide range of colours available with good

fastness properties at moderate costs was the main reason for the replacement of natural

dyes by their synthetic counterparts.

Hence, it is imperative to optimize more colouring component with keeping the

environment safe through alkaline extraction from Pitaya waste excluding any hazardous

application of organic solvents.

The present work is, therefore, undertaken to extract more colouring components

with keeping the environment friendly extraction procedure excluding the extensive

application of organic solvents.

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1.4 Objectives

The current research is done to achieve the following objectives:

i) To determine the effect of temperature and the concentration of the

aluminum sulfate to the extraction of pitaya peels.

ii) To determine the amount of total phenolic and total flavonoids content exist

in the pitaya peels.

1.5 Scope of Study

The scopes of the study are:

i) Only Red Pitaya peels are used in the experiment.

ii) Aqueous extraction is involved in the experiment.

iii) Pitaya waste is heated in 250ml for 4 hours in the water with temperature

different at 40oC, 60oC and 80oC.

iv) Pitaya waste is heated in 125ml at second heating stage for 2 hours with

temperature different at 40oC.

v) The aqueous extraction involves using Aluminum Sulfate at different

concentration (mg/L) for 10, 15, 20, 25 and 30 mg/L.

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vi) 80% methanol is used in methanolic extraction.

vii) Total Phenolic Content (TPC) is determined by using Folin-Ciocalteu

method.

viii) Total flavonoid content (TF) is determined by using the Catechin method.

xi) Each sample of TPC (Total Phenolic Content) and TF (Total Flavonoids) is

determined by UV-visible spectrometer.

CHAPTER 2

LITERATURE REVIEW

2.1 Pitaya

Pitaya (Hylocerus undatus (Haw.)) or Dragon Fruit is native to the Central

America which has been documented in Aztec literature since the 13th century. It was

then commercially grown in Colombia, Nicaragua and Vietnam (LJ Health Promotions,

2003). This fruit was then brought in by the French. Pitaya have been planted in Viet

Nam for more than 100 years, following their introduction by the French (Mizrahi et al.,

1997). In the local, dragon fruit is named as Thanh Long or in English, it is ‘Green

Dragon’. The green color is its immature color before ripening. While the ‘dragon’ is

used to name the fruit as its appearance has ‘dragon-like’ scales or bracts on the surface.

Dragon fruit is considered to be the member of Cactaceae. The trailing cladodes, stems

modified to act as leaves, bear spectacular ovoid fruit year-round which are a bright red

colour when it is ripened or becomes mature. The fruit contains white, crimson or dark

red color, or some with pale-yellow flesh depending on the cultivar and the flesh is

interspersed with small black seeds (Hoa et al., 2006).

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2.1.1 Types of Pitaya

2.1.1.1 Yellow Pitaya

Yellow Pitaya (Selenicereus megalanthus) is a vine cactus that needs a trellis

system for support. In Israel net-houses are required to avoid photo-inhibition and

bleaching of its stems. Colombia was the first country to sell S. megalanthus in the

world market under the name of “yellow pitaya”. This plant can tolerate high

temperatures more than the other vine cacti, yields spiny fruits where the spines abscise

easily upon ripening (Figure 2.1). The fruits are smaller than the other vine cacti fruits

but the taste is superior, hence, the higher prices obtained in the markets in comparison

with the others. Most of the plantations in Colombia have been uprooted due to the

heavy infestation with fungi.

Figure 2.1 Yellow Pitaya (Wikimedia, 2008)

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2.1.1.2 The Red Pitaya

The red Pitaya (Hylocereus undatus) is known in Latin America but the Asia

name is dragon-fruit (Figure 2.2).There are red flesh clones and some are produced in

Nicaragua and are considered Hylocereus costaricensis. Pigments differ among the

clones and species. For example the Hylocereus sp. Clone 10487 has red color while the

H. polyrhizuz clones show glowing purple a unique color which has been chemically

identified as hylocerenin and iso-hylocerenin (Mizrahi et al., 2002). Besides, there is the

red Pitaya with the peels are in red colored shown in the Figure 2.3 .

Figure 2.2 Red Pitaya with White Flesh (Mizrahi et al., 2002)

Figure 2.3 Red Pitaya with Red Flesh (Mizrahi et al., 2002)

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2.1.2 Plant Stem

Pitayas are the fast growing, perennial, terrestrial, epiphytic, and vine-like cacti.

The appearance is triangular which are 3-sided, sometimes 4 or 5-sided. It is green,

fleshy and having many branched stems. For each of the stem segment, it has 2 flat,

wavy wings, with corneous margins and may have 1-3 small spines or be spineless. The

stem forms root which is adhered to the surface upon which they grow and the stem may

reach about 6.1m long.

2.1.3 Inflorescence

The flowers of the pitaya are hermaphroditic which means a plant having

stamens and pistils in the same flower (Oxford Dictionary, 2005). However, some

species and cultivars are self incompatible. The showy, edible, white flowers are very

large, fragrant, bell shaped and may be 36 cm long and 23 cm wide. The stamens and

lobed stigmas are cream colored.

2.1.4 Fruit

The pitaya fruit is fleshy berry, oblong and the thickness is about 11cm with red

or yellow peels with scales. The pulp maybe white, red or magenta depending on the

species. They are seeds in the pulp which are very small, numerous and black.

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2.1.5 Propagation

Pitaya can be propagated from seed, however, the fruit and stem characteristics

are variable, and the time from planting to fruit production may be up to around 7 years.

Asexual propagation is preferred, and currently, the stem cuttings method is widely

applied by most people. The entire stem segments of 12-38 cm are usually used. To

make the stem base, the stem has to be cut slanted then being treated with fungicide and

dry and heal for 7-8 days in dry, shady location before being planted. Another method

which is applying root hormone to the cuttings after curing but before it is done before

planting. This cutting method grows very fast and many produce fruit in about 6 to 9

months after planting. Longer cuttings usually reach the trellis supports faster than

shorter ones. Another method which is not commonly practiced is the Pitayas may be

grafted. Grafting has potential for selection of rootstocks adaptable to various soil types

and problems. Cutting takes about 4-6 months to develop a good root system in pots and

be ready for planting.

2.2 Polyphenols

Polyphenols are antioxidants in plants that is believed to have a substantial

amount of health benefits. Among the most well known of the polyphenols are the

flavonoids which are grouping of several thousand individual compounds. These

cmpounds are found together in many different foods, all contributing in a unique way to

an individual’s overall health. They are most commonly introduced to the body through

the consumption of fruits and vegetables.

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In addition of flavonoids, other classes of polyphenols include tannins (both

condensed tannins and hydrolysable tannins), and lignins. Each of these polyphenols can

be found from many different sources. For example, tannins are prevalent in wines, tea

and fruits. Nearly all the plants will carry at least some of these polyphenols.

Polyphenols work by eliminating free radicals in the body, which are known to

cause a number of health problems. It is thought polyphenols help prevent premature

aging and help prevent cancer, among other things. Some, such as tannins, may even

have antibiotic benefits as well.

The effect of tannins can be seen in a number of different applications. It is the

substance responsible for the browning, or tanning, of leather. In fact, that is how the

group of chemicals received their name. In fact, that is how the group of chemicals

received their name. Tannic acid can be seen in some lakes and rivers, especially those

in the tropical and subtropical areas populated with cypress trees. However, it should be

noted that some tannic acid may be harmful to the body.

2.3 Flavonoids

Flavonoids, which is also referred as bioflavonoids, are polyphenol antioxidants

found naturally in plants. They are secondary metabolites, meaning they are organic

compounds that have no direct involvement with the growth or development of plants.

Flavonoids are plant nutrients that when being consumed in the form of fruits and

vegetables are non-toxic as well as potentially beneficial to human body.

14

Flavonoids are widely disbursed throughout plants. It gives the flowers and fruits

of many plants their vibrant colors. They also play a role in protecting the plants from

microbe and insect attacks. More importantly, the consumption of foods containing

flavonoids has been linked to numerous health benefits. Though research shows

flavonoids alone provide minimal antioxidant benefit due to slow absorption by the

body, there is indication that they biologically trigger the production of natural enzymes

that fight disease.

Recent research indicates that flavonoids can be nutritionally helpful by

triggering enzymes that reduce the risk of certain cancers, heart disease, and age-related

degenerative diseases. Some research also indicates flavonoids may help prevent tooth

decay and reduce the occurrence of common ailments such as the flu. These potential

health benefits, many of which have been proven, have become of particular interest to

consumers and food manufacturers. Food that contain high amount of flavonoids include

blueberries, red beans, cranberries and blackberries. Many of other foods, including red

and yellow fruits and vegetables and some nuts, also contain flavonoids. Red wine and

certain tea also are rich in flavonoids as well.

2.4 Natural Dye

No chemical dye has the luster, that under-glow of rich colour, the delicious

aromatic smell, that soft light and shadow that gives so much pleasure to the eye. These

colours are alive (Thurston, 1972). Dyes can be derived from nature through herbs and

plants, flowers, seeds, barks and roots. Natural dyes give subtle, rich, warm colors that

unique. They have a mystery and life that fascinates and satisfies. Natural dyes can be

categorized as either substantive adjective.

15

2.4.1 Art of Making Natural Dyes History

The art of making natural dyes is one of the oldest known to man and date back

to the dawn of Civilization. In India, it was widely used for the colouring of fabrics and

other materials. Though the very earliest dyes were discovered by accident using berries

and fruits. With the experimentation and gradual development the natural dyes have

resulted into a highly refined art.

India’s expertise in natural dyes date back to ancient times. Using mordant to

hold fast the dyes or resists to selectivity prevent them from touching the cloth were

printed bales of whisper soft textiles. From 19th centuries, block printed resist dyes

textiles from Gujarat and Deecan adorned Europeans and their homes. The discovery of

synthetic dyes in the west in 19th century dealt a massive blow to Indian Textile

Industry. Some of the chemical dyes earlier found associated with hazards effecting

human life creating skin diseases and lungs problems. The environmentalist, therefore,

started searching the substitute of synthetic items which has led the use of more natural

dyes. In recent days the inherent advantages of natural dyes has resulted in the revival

and use of natural dyes.

16

2.4.2 Types of Natural Dyes

2.4.2.1 Lac Dye

It is extracted from lacifer lacca insect. It is used for dyeing of wool, silk and

cotton fibers. It gives reddish with tin mordant and purplish with copper mordant.

2.4.2.2 Annatto

It is prepared from the seed of annatto. It is used in the dyeing of silk and wool.

It gives orange and peach colour. Its botanical name is bixin.

2.4.2.3 Harda

It is prepared from fruits of Harda and it yields yellow and gray colours with

aluminum and ferrous mordant respectively. It can be used in coloration of wool and

silk.

17

2.4.2.4 Himalayan Rhubard

It is manufactured from Himalayan herb. The roots of this plants is used fro the

manufacture of dye stuff. It gives yellow and oranges. It can be used directly and with

Alum Mordant on wool or silk.

2.4.2.5 Indigo Blue

It is a fermented dyes of leaves of indigo ferra tinctoria. It gives blue colour. It

can dye cotton, wool and silk.

2.4.2.6 Kamala Dye

It is prepared from the deposits on flowers of Kamala tree. It gives yellow colour

on wool and silk. It can be used directly of with mordant as well.

18

2.4.2.7 Manju Phal

It is manufactured from the nut galls of Manju Phal tree. It is used for dyeing of

silk and wool, both directly or with mordant. It gives cream and grey colours with alum

and iron mordant.

2.4.2.8 Gum Arabic

It is manufactured from the bark of Indian Gum Arabic tree. It is used for dyeing

of cotton with mordant. It yields brown shade having very good fastness.

2.4.2.9 Trigonella Foenum Graecum

It is prepared from the fenugreek seeds. It is used in the dyeing of cotton fabrics.

It gives yellow shade with metallic mordents like copper sulphate and feerous sulphate.

19

2.4.2.10 Gold Dock

It is prepared from rumexmaritinus seeds. It yields brown colour on cotton with

alum, copper sulphate and ferrous sulphate mordants.

2.4.3 Uses of Dye

2.4.3.1 Textile

There is a big potential for the use of natural dyes in textile industry. These dyes

can be used for coloration of textile material at different stages such as on the yarn, on

the fabric and even can be applied on the apparels.

2.4.3.2 Cosmetics

The natural dyes from dolu and rind of pomegranate are used in coloration of

lipsticks and other cosmetics. They are manufactured by water extraction method. The

coloring matter can be extracted with Super Critical Fluid Extraction Method.

20

2.4.3.3 Edible Dyes

Most common dye, which can be used for coloration of the edible items, are

annatto seeds. The water soluble extract can be used for coloration of butter and oil

soluble extract can be used for colouration of ghee and ice cream. They are simple

extracts of annotto seeds, which give 55 to 60% yields when their extract is prepared.

Lac dye is also derivative of lac and is similar to cochineal and has been in use for

colouring food besides fabrics since ancient times.

2.4.3.4 Food Items

This is the sector where natural dyes can be consumed in appreciable amount.

This sector consumes 10 to 20 ton of natural dyes in a year. Moreover, the consumption

of natural dyes in beverage sector can be up to 20 to 40 ton per year.

2.4.3.5 Leather

Leather industry is already using natural tannin for tanning of leather sole.

However, this use is confined to cottage and small-scale leather units. The large

manufacturers are using Chrome Tanning. At present, none of the leather units are using

natural dyes for coloration of their product. If sufficient and offensive efforts are done,

the natural dyes can capture in leather sector in a big way.

21

2.5 Extraction of Natural Dyes

The natural dyes extraction is broadly divided into extraction method and

extraction technology.

2.5.1 Method of Extraction

The extraction methods of natural dye basically depend on medium in which the

dye is extracted. There are mainly four methods used in extraction of natural dyes where

they are aqueous method, alkaline method, acidic method and alcoholic method.

2.5.1.1 Aqueous Method

The known amount of dyestuff is boiled in 100 ml of soft water at 100oC. Then

the dye solution is filtered and finally the optical density is recorded.

22

2.5.1.2 Alkaline Method

First, 1% alkaline solution is prepared with addition of 1g of NaOH in 100ml of

water. Then the dye material is entered and boiled at 100oC. Finally the dye solution is

filtered and the optical density is recorded.

2.5.1.3 Acidic Method

Acidic solution of 1 % is prepared by adding 1 ml of HCl in 10 ml of soft water.

Then the material is entered and boiled at 100oC. Finally, the dye solution is filtered and

the optical density is recorded.

2.5.1.4 Alcoholic Method

Alcohol of 50ml is added to 50 ml of water. Then the dye material is entered and

boiled and finally the dye solution is filtered.

23

2.5.2 Extraction Technology

The extraction can be carried out in aqueous, acid or alkaline medium. At

present, small scale producers/ manufacturers are using extraction technology method.

Even the local dyers using more crude method for extraction using metallic flax and

crude process in refined way using blender condenser, distillation plant and drier and

crystallization unit with the capacity of 300 ton per year. The modern techniques of

extraction are carried out with the use of extraction plant, reverse osmosis process and

the latest is supercritical fluid extraction method. This method is very common in

developed countries.

2.5.2.1 Solvent Extraction

This technique was developed just before the dawn of twentieth century. Now it

has been commercialized in recent years. This technology has been improved to reduce

waste generation and eco-effectiveness of extraction technology. Ultrasonic extraction

followed by micro-wave extraction of solid finds extensive use mainly on organic

solvents extraction

24

2.5.2.2 Supercritical Fluid Extraction

This is a further advancement making significant step over the use of

conventional solvent extraction technology. It uses CO2 as extraction media. This

technique is used for the extraction of natural products in food, pharmaceuticals and

chemical industries too.

It makes it possible to work at moderate temperature without affecting the

organoleptic qualities and the active ingredients of the extracts obtained. Moreover, it

makes it possible to obtain 100% natural extracts, completely free from extraction

solvent residues. At the end of the extraction, an expansion phase which is achieved by

reducing pressure causes the CO2 to change from the supercritical state to the gaseous

state which enables it to be removed completely from the CO2 extract obtained.

2.5.2.3 Microwave Assisted Extraction Technology

This is a high-speed method used to selectively target compounds from various

raw materials. The technology uses a microwave applicator as the energy source during

solvent extraction. The advantage of this technology faster the processing, produce

better yield, improve quality, lower energy consumption, reduce solvent level and low

capital investments.

25

2.5.2.4 Continuous Steam Distillation Process

The Continuous Steam Distillation Process is a separation process using steam as

a media but instead of batch type, this process is continuous. The process consists of a

totally insulated pneumatic conveying system using super heated steams as a carrier gas.

2.6 Advantages of Natural Dyes

i) High diversity of rich and complex colours.

ii) Different colours go well together and rarely clash.

iii) Beauty of the results.

iv) Not dependent on non-renewable materials.

v) Allow the replication of ancient technique.

2.7 Disadvantages of Natural Dyes

i) Require large quantities in comparison to chemical dyes. However,

several natural dyes are sold as extracts and small amount of cochineal,

26

bazilwood and logwood dye in large amount of fiber.

ii) Longer time required for dyeing. However, if you are using the sun as

as an energy source, you can leave the dye vat unattended for a long time.

iii) May be more costly.

2.8 Aqueous Extraction

The aqueous extraction is where the solvent involved is only water used to

extract the component in the interested substances. It is preferable in most of the

research especially involving plants or fruits for medication or consuming purposes

simply because this method is 100% safe for human being to consume as no chemical

solvent is introduced in during the process. Table 2.1 shows the research that involving

fruits or vegetables extraction using water.

Table 2.1 Examples of research using aqueous extraction

No. Description Reference

1. Extraction of Mucilage from whole white mustard

seed.

(David et al., 2000)

2. Extraction of Jordanian plant species to determine

its antioxidant and total phenolic content.

(Tawaha et al.,2007)

3. Direct extraction of oil from sunflower seeds by

twin-screw extruder.

(Evon et al., 2007)

4. Extraction of Bryophyllum Pinnatum Leaf for

determination of neuropharmacological effects.

(Salahdeen et al., 2006)

27

5. Determination of aqueous extracts of galls of

Querus infectoria as antibacterial agents.

(Basri et al., 2004)

6. Extraction of medicinal plants . (Nair et al., 2006)

7. Rosemary extracts added in gelatins from tuna-skin

and bovine-hide gelatin.

(Gomez-Estaca et al., 2008)

8. Extraction of Labiateae, Vitaceae and Cyperaceae

for antimicrobial activity against bacteria.

(Parekh et al., 2006)

9. Production of anti-inflammatory activity. (Falodun et al., 2006)

10. Extraction of Harpagophytum procumbens Roots

for African traditional medicine for treatment.

(Mahomed et al., 2006)

11. Extraction of Nigerian savannah plants for

comparative in vitro trypanocidal activities.

( Atawodi, 2004)

2.9 Total Phenolic Content (TPC)

Phenolic compounds are secondary metabolites in plants. In past 10 years,

phenolic compounds has increased the interest of its role in human health and nutrition

(Tew et al., 2002). In some phenolic compounds, it contains natural products which have

higher antioxidant activities than those in synthetic antioxidants (Lu et al., 2000). These

compounds are capable of modulating the activity of many enzymes (Carlo et al., 1999)

suggesting their involvement in biochemical and physiological processes, not only in

plants, but also in animals and humans (Rusak et al., 2008).These polyphenolic

antioxidants is suitable to be used for preserving the foods as it has protective effects

against microorganisms (Cowan, 1999; Vattem et al., 2004).

28

2.10 Total Flavonoids (TF)

Flavonoids are polyphenolic compounds which are commonly known for its

antioxidant activity. They are widely distributed in plants for their functions including

producing blue or red pigmentation to the fruits or vegetable and protection for the fruits

being attacked by the insect. The characteristics of flavonoids are also widely found in

some other foods such as tea, peanut, coffee, almond, beer, chocolate and wine.

Research found out that the flavonoids have strong antioxidant which is

important for the use in biological function and health. Nowadays, the new finding for

the use of flavonoids is beneficial to the health which can conquer the cancer and heart

disease (Filippos et al., 2007). However, the flavonoids are highly metabolized which

can change their chemical structure and diminish their ability to function as an

antioxidant when it is in human body. It will treat the flavonoids are foreign compounds

and modify them for rapid excretion in urine and bile. By following the activity of the

flavonoids, this process helps remove the unwanted compound is inducing, called Phase

II enzymes that is able to eliminate mutagens and carcinogens. Hence, it is valuable for

cancer prevention material (Stauth, 2007).

Figure 2.4 Molecular structure of The Flavonoids Backbone

29

2.11 Ultra Violet Visible Spectrometer

The UV-vis spectrometer consists of a light source, a sample compartment, a

diode-array detector, and a data acquisition computer. The sample compartment is

between the light source and the detector. The spectrometer measures the amount of

ultraviolet and visible light transmitted by a sample placed in the sample compartment.

Typically liquid samples are used, contained in a transparent ‘cuvette’ or ‘cell’. A flow-

through cell for kinetics experiments is currently in the sample compartment but another

standard cuvette can easily be substituted for it. The sample compartment in

spectrometer is made for 1 cm cuvette.

The wavelength at which a chemical absorbs light is a function of its electronic

structure, so a UV/Vis spectrum can be used to identify some chemical species. For a

particular chemical, the amount of the light absorbed is related to the amount of the

chemical between the light source and the detector, so a UV/Vis spectrum cab be used to

quantify some chemical species. To use UV/Vis data for quantitation ‘Transmittance’,

(T, the percentage of light transmitted by the sample) must be converted to

‘Absorbance’.

‘Beer’s Law’ states that absorbance and concentration in solution are linearly

related to a given chemical, so long as the length of the path through the solution is

fixed. Actually, this linear dependence only hold below Absorbance of 2. When less

than 1% of the light is transmitted (A>2), the detector behaves non-linearly. A UV/Vis

spectrum is only useful for quantifying chemical species after ‘calibration’. A series of

solutions of known concentration must be analyzed and their absorbencies are recorded.

30

UV-visible Spectrometry function is relatively straightforward. A beam of light

from a visible and/ or UV light source (colored red) is separated into its component

wavelengths by a prism or diffraction grating. Each monochromatic (single wavelength)

beam in turn is split two equal intensity beams by a half-mirrored device. One beam, the

sample beam (colored magenta), passes through a small transparent container (cuvette)

containing a solution of the compound being studied in a transparent solvent. The other

beam, the reference (colored blue), passes through an identical cuvette containing only

the solvent. The intensities of these light beams are then measured by electronic

detectors and compared. The intensity of the reference beam, which should have no light

absorption, is defined as Io. The intensity of the sample beam is defined as I over a short

period of time, the spectrometer automatically scans all the component wavelengths in

the manner described. The ultraviolet (UV) region scanned is normally from 200 to

400nm, and the visible portion is fro 400 to 800 nm.

Figure 2.5 A diagram of The Components of A Typical Spectrometer

31

2.12 Centrifuge

Centrifugation is one of the most important and widely applied research

techniques in biochemistry, cellular and molecular biology, and in medicine. A

centrifuge is a piece of equipment driven by a motor that puts an object in rotation

around a fixed axis, applying force perpendicular to the axis. Current research and

clinical applications rely on isolation of cells, subcellular organelles, and

macromolecules, often in high yields.

A centrifuge uses centrifugal force (g-force) to isolate suspended particles from

their surrounding medium on either a batch or a continuous-flow basis. Applications for

centrifugation are many and may include sedimentation of cells and viruses, separation

of subcellular organelles, and isolation of macromolecules such as DNA, RNA, proteins,

or lipids.

Figure 2.6 Refrigerated Centrifuge at FKKSA, UMP

32

2.12.1 Differential Centrifugation

Separation is achieved primarily based on the size of the particles in differential

centrifugation. This type of separation is commonly used in simple pelleting and in

obtaining partially-pure preparation of subcellular organelles and macromolecules. For

the study of subcellular organelles, tissue or cells are first disrupted to release their

internal contents. This crude disrupted cell mixture is referred to as a homogenate.

During centrifugation of a cell homogenate, larger particles sediment faster than smaller

ones and this provides the basis for obtaining crude organelle fractions by differential

centrifugation. A cell homogenate can be centrifuged at a series of progressively higher

g-forces and times to generate pellets of partially-purified organelles.

When a cell homogenate is centrifuged, unbroken cells and heavy nuclei pellet to

the bottom of the tube. The supernatant can be further centrifuged to pellet subcellular

organelles of intermediate velocities such as mitochondria, Iysosomes, and microbodies.

Some of these sedimenting organelles can be obtained in partial purity and are typically

contaminated with other particles. Repeated washing of the pellets by resuspending in

isotonic solvents and re-pelleting may result in removal of contaminants that are smaller

size (Figure 2.7) obtaining partially-purified organelles by centrifugal separation

(density gradient separation).

33

Figure 2.7 Differential Centrifugation (Rickwood et al., 2001)

2.12.2 Density Gradient Centrifugation

Density gradient centrifugation is the preferred method to purify subcellular

organelles and macromolecules. It can be generated by placing layer after layer of

gradient such as sucrose in a tube with the heaviest layer at the bottom and the lightest at

the top in either a discontinuous or continuous mode. The cell fraction to be separated is

placed on top of the layer and centrifuged. Density gradient separation can be classified

into two categories where they are Rate-zonal separation and Isopycnic separation.

34

2.12.3 Rate Zonal Separation

Rate-zonal separation takes advantage of particle size and mass instead of

particle density for sedimentation. Figure 2.8 illustrates a rate-zonal separation process

and the criteria for successful rate-zonal separation. Examples of common applications

include separation of cellular organelles such as endosomes or separation of proteins,

such as antibodies. For instance, Antibody classes all have very similar densities, but

different masses. Thus, separation based on mass will separate the different classes,

whereas separation based on density will not be able to resolve these antibody classes.

Figure 2.8 Rate-zonal Separation (Rickwood et al., 2001)

35

2.12.4 Isopycnic Separation (Density)

In this type of separation, a particle of a particular density will sink during

centrifugation until a position is reached where the density of the surrounding solution is

exactly the same as the density of the particle. Once this quasi-equilibrium is reached,

the length of centrifugation does not have any influence on the migration of the particle.

A common example for this method is separation of nucleic acids in a CsCl gradient.

Figure 2.9 illustrates the isopycnic separation and criteria for successful separation. A

variety of gradient media can be used for isopycnic separation and their biological

(Rickwood, 2001)

Figure 2.9 Isopycnic Separation (Rickwood et al., 2001)

CHAPTER 3

METHODOLOGY

3.0 Introduction In chapter 3, it discusses about the methodology with a specific focus on the

running of extraction of Pitaya peel. This method of extraction of pitaya peels by using

water as its solvent simply because of the natural of the water that do not bring harmful

side effect to human being and environmental friendly. The method that has been used

for this study is based on the study of which the title is Process balance and product

quality in the production of natural indigo from Polygonum tinctorium Ait applying

low-technology methods by Bechtold et al. (2003).

3.1 Methodology of Flow Chart

Methodology flow chart is used as a guideline for the experiment. As illustrated

in Figure 3.1, the process begins with the first heating stage and end with analysis by

using Ultra Violet visible spectrometer.

37

Figure 3.1 The Flow Chart of the Extraction of Pitaya Peels by Water

25g of dried Pitaya peels was added into 350mL of water and extracted with temperature ranging from 40oC to 80oC for 4 hours in the oven

The mixture was added with 150mL of water at temperature of 40oC for 2 hours and being filtered to obtain the filtrate.

Aluminum Sulfate solution with concentration (mg/L) ranging from 10 to 30 mg/L was added to the filtrate.

The floc was filtered by cotton fabric.

10g of wet floc was added into 80% of methanol.

Analysis

Total Phenolic Content (TPC)

Total Flavonoids (TF)

The mixture was centrifuged at 6000 rpm for 30 minutes. Supernatant was used for analysis

Pitaya peels were dried in the oven at 60oC and then being milled into particle size.

38

Figure 3.2 Procedure of 80% Methanolic Extraction

Figure 3.3 Procedure of Total Phenolic Content Assay

50 uL of methanolic extract was added to 5 mL of water.

500 uL of 1M Folin reagent and 500 uL of 20% w/v Na2CO3 were added in the mixture.

The mixture was mixed for 1 hour at room temperature.

The samples were analyzed by UV-vis spectrometer at 765 nm.

10 g of wet floc was added into 10 mL of 80% methanol in the flask

The solution was shaken for 2 hours at 37oC

The solution was filled in the centrifuge container in Refrigerated Centrifuge for 30 min at 6000 rpm.

The supernatant was taken for analysis of TPC and TF.

39

Figure 3.4Procedure of Total Flavonoids Assay

3.2 Preparation of Sample

Fresh pitaya was brought from the farm located at Kuantan Road for 40kgs. All

the pitaya fruits that have been bought from the farm were then being peeled at the same

time to ensure the condition of the properties is same for every single piece of the peels

(Figure 3.5). After that, the peels were cut into pieces (about 2 x 2 cm in size) so that the

area exposed is larger instead of drying the whole big piece of the pitaya peels. The

process of cutting the pitaya peels is shown in the Figure 3.6

0.23 mL sample from 80% methanolic extract was added with 1.25 mL of water.

75 mL of 5% NaNO2 was added in the solution.

After 6 min at room temperature, 150mL of 10% AlCl3 was added in the previous solution.

After 5 min at room temperature, 0.5mL of 1M NaOH was added and mixed.

The samples were analyzed by UV-vis spectrometer at 510 nm.

40

Figure 3.5 Sample of Pitaya Peels in Large Pieces

Figure 3.6 Cutting of Pitaya Peels into Small Pieces

41

3.2.1 Drying of Pitaya Peels

Before the drying of pitaya peels in the oven, it was weighed by using the

balance provided in FKKSA lab. Then the tray was covered with aluminum foil to

ensure that the peels were not contaminated by any possible chemical substances. In the

Figure 3.7, it shows that the pitaya peels distributing evenly on the tray. Figure 3.8

shows pitaya peels being heated at temperature of 60oC for 24 hours in the oven

(HERAEUS/ MODEL UT 6). According to the previous study, temperature of 60oC is

suitable for the fruit of pitaya as it would not destroy its properties. After 24 hours, the

dried pitaya peels (Figure 3.9) were weighed to ensure its amount is enough for the use

in this experiment.

Figure 3.7 The Fresh Pitaya Peels on the Tray

42

Figure 3.8 Pitaya Peels in the Oven Before Drying

Figure 3.9 Pitaya Peels After Being Dried

43

3.2.2 Blending of Pitaya Peels into the Powder Form

Dried pitaya peels were then being blended by using the stainless steel blender

provided in the lab of FKKSA. Small amount of dried pitaya peels was added into the

blender so that all the dried pitaya peels would be blended into powder form. As shown

in the Figure 3.10, the pitaya peels were being blended into powder form. Figure 3.11

shows the dried pitaya peels in powder form.

Figure 3.10 Pitaya Peels Being Blended into Powder Form

44

Figure3.11 Pitaya Peels in Powder Form

3.3 Extraction of Pitaya Peels

3.3.1 Extraction of Pitaya Peels at the First Stage

In this experiment, extraction of pitaya waste by using water was introduced by

Bechtold et al. (2006). Based on this journal, the extraction was done at two stages. The

first one is the temperature varies from 40 to 80oC for 4 hours while the second stage is

at temperature of 40oC for 2 hours.

The dried pitaya peels were weighed and five grams of it was packed into each

plastic sealed bag as shown in the Figure 3.12. Each 5 g of dried pitaya peels was

introduced into the 1L conical flask. Then water of 350 ml was added into it. The

mixture was being stirred and put in the oven in FKKSA laboratory for 4 hours. For

convenience purpose, five conical flasks containing the 25 g of dried pitaya peels and

45

350ml of water were put in the oven at the same time for temperature of 40oC, 60oC and

80oC for 4 hours.

Figure 3.12 Each Plastic Sealed Bag Containing 25 g of Dried Pitaya Peels

3.3.2 Extraction of Pitaya Peels at Second Stage

After the first stage of extraction of pitaya peels was done for 4 hours and at the

temperature as stated. The mixture was then continued to be extracted at the temperature

of 40oC for 2 hours. Figure 3.13 shows that the extraction of pitaya peels in the conical

flasks after 2 hours at the temperature of 40oC.

46

Figure 3.13 Extraction of Pitaya Peels After 6 Hours

3.4 Filtration of The Pitaya Peels From The Extraction

After the extraction processes were completely done for the two stages heating of

pitaya peels in the conical flasks in the oven. The mixtures were then filtered by using

the fabric cotton to separate the pitaya peels and the filtrate. The filtrate obtained was

measured by using measuring cylinder and put aside to be ready for being added in the

different concentration of solution of aluminum sulfate.

47

3.5 Flocculation of Natural Dye From Extraction of Pitaya Peels

Flocculation is the process of binding small particles in the water together into

larger, heavier clumps which settle out relatively quickly. The larger particles are known

as floc. Properly formed floc will be settled out of the water quickly.

3.5.1 Preparation of Stock Solution of Aluminum Sulfate

For the preparation of stock solution of aluminum sulfate, 10g of alum was

weighed by using the analytical balance. It was then put in the 1L of volumetric flask

and 1L of water was added into it. The alum was dissolved by mixing it with the

magnetic stirrer.

3.5.2 Dilution of Concentration of Aluminum Sulfate

Concentration of aluminum sulfate is also one of the variables in this study. In

this experiment, concentration of aluminum sulfate is varies from 10mg/L, 15 mg/L,

20mg/L, 25mg/L and 30mg/L. In order to get the different concentration from the stock

solution, amount of the volume needed from the stock solution to obtain the desired

concentration of aluminum sulfate was calculated.

48

3.5.3 Stirring of Mixture of Aluminum Sulfate Solution and Pitaya Peels Filtrate

After the calculated concentration of aluminum sulfate from 10 to 30mg/L and

the filtrate of pitaya peels were mixed into the 5 beakers respectively, the stirring

process was done in the Basic Science Laboratory at FKKSA of Universiti Malaysia

Pahang. The mixtures were stirred at the speed at 20 rpm which is the best speed to be

used according to the previous study for 30 minutes. As shown in the Figure 3.14, the

mixtures being stirred at the speed of 20 rpm. The floc was formed slowly during the

stirring (Figure 3.15).

Figure 3.14 Pitaya Peels Filtrate and Aluminum Sulfate Solution Being Stirred

49

Figure 3.15 Flocs Being Formed in the Solution

3.6 Filtration of Floc

After 30 minutes stirring of the mixture at speed of 20 rpm, the floc formed was

needed to be filtered out by using fabric cotton as shown in the Figure 3.16. The floc

was used for the analysis of total phenolic content and total flavonoids. The filtration of

the floc process was left for 24 hours to ensure the filtrate was filtered out at maximum.

Figure 3.17 shows the floc obtained after 24 hours.

50

Figure 3.16 The Floc Being Filtered By Fabric Cotton

Figure 3.17 Wet Floc After Being Filtered for 24 Hours

51

3.7 Analysis of Total Phenolic Content and Total Flavonoids

3.7.1 Preparation of 80% Methanolic Pitaya Peels Extracts

For the method to prepare the 80% of methanol, 10mL of water was mixed with

8mL of methanol to obtain 80% of methanolic solution. Then 10g of wet floc extracted

from the pitaya peels extracts were weighed and 10 uL of 80% methanolic solution was

mixed with it in the 250 mL of conical flask. Aluminum foil was used as a lid to avoid

the vapour of the 80% methanolic escape in the air.

After all the conical flasks filled with 10g of floc and 10 uL of 80% methanolic

solution were sealed with aluminum foils, they were then being put in the shaking

incubator as shown in Figure 3.18 at 6000 rpm for one hour at 37oC. Figure 3.19 shows

the mixture in the conical flask after being shaken under the specific condition as stated

above.

Figure 3.18 The Mixture Being Shaken at 37oC in The Shaking Incubator

52

Figure 3.19 The Mixture After Being Shaken for 1 Hour

3.7.2 Centrifugation

After shaking for mixture of the floc from the pitaya peels extracts and the 80%

of methanolic solution for one hour at the temperature of 37oC, the mixture were then

brought to be centrifuged at the speed of 6000 rpm for 30 minutes. The mixture has to be

filled in the 10mL container with tightly closed of the lid. When placing the container in

the Centrifuge, balancing is important as imbalance of the placement of the containers in

the Centrifuge would affect the spinning of the machine. Figure 3.20 shows that the

containers being placed in the Centrifuge. Figure 3.21 shows that the mixture before

being centrifuged while Figure 3.22 shows the clear supernatant after being centrifuged.

The sediment can be seen clearer than before being centrifuged.

53

Figure 3.20 The Mixture Containing in the Container Being Placed in Centrifuge

Figure 3.21 The Mixture Before Being Centrifuged

54

Figure 3.22 The Clear Supernatant and The Sediment After Being Centrifuged

3.7.3 Analytical Determination of Total Phenolic Content

The total phenolic content (TPC) was determined according to the Folin-

Ciocalteu method. 50 uL of extract (80% methanolic pitaya peels extract) was fixed in

5mL of distilled deionized water. Folin-Ciocalteu reagent (500uL, 1M) and Na2CO3

(500 uL, 20% w/v) were added, and the mixture was mixed thoroughly and allowed to

stand for 60 min at room temperature before the absorbance was measured at 765 nm.

The final results were expressed as gallic acid equivalents (GAE) in milligrams per gram

of wet weight. The total Phenolic Content of Pitaya is determined according to the Folin-

Ciocalteu colorimetric method (Bao et al., 1999). Figure 3.23 shows the mixture waiting

for being analysed at 765 nm.

55

Figure 3.23 Mixture to be Determined for Its Total Phenolic Content at 765 nm

3.7.4 Analytical Determination of Total Flavonoids

Total flavonoid content was measured by the aluminum chloride colorimetric

assay. An aliquot (1ml) of 80% methonlic extracts was added to 10ml volumetric flask

containing 4ml of added H2O. The flask was added 0.3ml of 5% NaNO2. After 5 min,

0.3ml of the 10% AlCl3 was added. At 6 min, 2ml of 1M of NaOH was added and the

total volume was made up to 10ml add with H2O. The solution was mixed well and the

absorbance was measured against prepared reagent blank at 510nm (Figure 3.25) in the

UV-vis spectrometer. Total flavonoid content of pitaya peels were expressed as mg

catechin equivalents (CE)/25 g dry mass. Samples were analysed in duplicates. Figure

3.24 shows the mixture waiting to be analysed at 510 nm.

56

Figure 3.24 Mixture to be Determined for Its Total Flavonoids at 510nm

Figure 3.25 Adjusting the Cuvette Position in the UV-Vis Spectrometer

CHAPTER 4

RESULTS AND DISCUSSIONS

4.0 Result

The Folin-Ciocalteau assay is a fast and simple method to rapidly determine the

content of phenolics in samples. Phenolics or polyphenols are secondary plant

metabolites that are ubiquitously present in plants and plants products. Many of the

phenolics have been shown to contain high level of antioxidant activities. This

information has led to the determination of the total phenolic content of the sample

understudy. Furthermore, several studies have reported a significant correlation between

antioxidant activity present in some tropical vegetables with their total phenolic content,

suggesting that plants containing high phenolics can be a good source of antioxidants. In

addition, various studies have shown that phenolic antioxidants such as quercetin have

potential application as therapeutic drugs against free radical reactions (Rusak et al.,

2008).

58

4.1 Total Phenolic Content and Total Flavonoids Obtained at 40oC

The Folin-Ciocalteu method measures the reduction of the reagent by phenolic

compounds with the formation of a blue complex that can be measured at 750 nm

against gallic acid as a standard.

In this study, the objective of this experiment is to extract the natural color from

the pitaya waste by using water as the solvent in this extraction method. At the end of

this experiment, the total phenolic content and the total flavanoids that content in the

pitaya waste were determined by using UV-spectrometer.

4.1.1 Total Phenolic Content

In this work, water was used to extract the total phenolic and flavanoids content

from the pitaya peels. The temperature was varying from 40oC to 80oC. In order to

improve the flocculation and precipitation of the natural red color, different

concentration of the aluminum sulfate was also varied from 10mg/L to 30mg/L. Table

4.1 shows the absorbance of the red color by using the UV-spectrometer, and the total

phenolic content and total flavanoids content are expressed as gallic acid equivalents and

quercetin equivalents respectively.

From the data obtained which is shown in Table 4.1, the extract of the total

phenolic content in the pitaya peels was found to have increased as the concentration of

the aluminum sulfate increased. For this 40oC heating extraction of the pitaya peels,

59

10mg/L of the concentration of alum provided the lowest absorbance value which is

only 0.074 %T ABS at 710nm wavelength. This absorbance value can also be expressed

as 1.286 mg GAE/g. For the highest absorbance value that was obtained from this

extraction of pitaya waste is 0.174 %T ABS where it was contributed by the

concentration of alum at 25mg/L. Its gallic acid equivalent is 6.780 mg GAE/g. For the

concentration of alum sulfate solution at 25mg/L to 30mg/L, the value of the absorbance

has a slight decrease with the different of 0.769 %T ABS. Heating of the pitaya waste at

40oC could have said to provide average the total phenolic content ranging from 6.011 to

6.505 mg GAE/g.

From the concentration of 10mg/L of aluminum sulfate solution to 15 mg/L,

there is an increase of 61.86 % of the total phenolic content. From concentration of

15mg/L to 20 mg/L of aluminum sulfate, there is also an increase of the total phenolic

content of 27.12 % and also it does for concentration of 20 mg/L to 25 mg/L of

aluminum sulfate solution, the increase of the total phenolic content is 6.35 %. So it is

clear that the total phenolic content increases from 10mg/L to 25 mg/L of concentration

of aluminum sulfate solution with 55.51 %. But there is a sudden decrease with 80 % in

the total phenolic content from 25 mg/L to 30 mg/L of the aluminum sulfate solution.

60

Table 4.1 Value of absorbance and total phenolic content at 40oC

Temperature

40oC

Concentration of Aluminum Sulfate, mg/L

10

15

20

25

30

Absorbance,

%T ABS

0.059

0.072

0.080

0.082

0.068

Total

Phenolic

Content,

mg GAE/ga

0.451

1.181

1.621

1.731

0.932

a Total Phenolic Content is expressed as mg gallic acid equivalents (GAE) in 25 g of dry weight material.

Total Phenolic Content vs Concentration of Aluminum Sulfate

0

0.5

1

1.5

2

10 15 20 25 30


To

tal P

hen

olic

Co

nte

nt,

mg

GA

E/g

Figure 4.1 Total Phenolic Content Versus Concentration of Aluminum Sulfate at 40oC

61

4.1.2 Total Flavonoids

While for the total flavonoids content in this study, its content increased when

there is an increase in the concentration of the aluminum sulfate (Table 4.2). From the

graph (Figure 4.2), it shows the amount of total flavanoids increases gradually. For this

40oC heating extraction of the pitaya waste, 10 mg/L of the concentration of alum

provided the lowest absorbance value which is only 0.066 %T ABS at 510 nm

wavelength. This absorbance value can also be expressed in 1.213 mg CE/g. For the

highest absorbance value that was obtained from this extraction of pitaya waste is 0.085

%T ABS where it was contributed by the concentration of alum at 30 mg/L.

At the concentration of aluminum sulfate at 10 mg/L, the value of the total

flavonoids that can be obtained is 1.21 mg CE/g while at concentration of 15mg/L of the

alum sulfate solution, the value of the total flavonoids is 1.253 mg CE/g, so there is an

increase of the total flavonoids of 3.24 %. For the concentration at 15 mg/L to 20mg/L

of aluminum sulfate solution, the percentage of the increase in the total flavonoids is

3.15 While from 20 mg/L to 25 mg/L of concentration of aluminum sulfate, there is also

an increase in the total flavonoids of 8.68 %, and for the last one, from 25 mg/L to 30

mg/L concentration of aluminum sulfate, the increase is 11.62 %. It is very obvious to

see that there is an increase of the total flavonoids in 40oC extraction from 10 mg/L to 30

mg/L of the concentration of aluminum sulfate with the percentage of 72.11.

62

Table 4.2 Absorbance value and the total flavanoids at 40oC

Temperature

40oC


10

15

20

25

30

Absorbance,

%T ABS

0.066

0.068

0.07

0.076

0.085

Total

Flavanoids ,

mg QE/gb

1.213

1.253

1.294

1.417

1.603

b Total Flavanoid Content is expressed as catechin equivalents (CE) in 25 g of dry weight material.

Total Flavanoids vs Concentration of Aluminum Sulfate

0

0.5

1

1.5

2

10 15 20 25 30


To

tal

Fla

van

oid

s,

mg

CE

/ g

Figure 4.2 Total Flavonoids Versus Concentration of Aluminum Sulfate at 40oC

63

4.2 Total Phenolic and Total Flavonoids Content Obtained at 60oC

4.2.1 Total Phenolic Content

Pitaya waste that was being heated in the oven by using water at the first heating

stage at 60oC for 4 hours, it was then heated again at 40oC for 2 hours according to the

previous study. Only then it was filtered and aluminum sulfate was added with

concentration ranging from 10 mg/L to 30 mg/L. The result (Table 4.3) shows that the

total phenolic content of the pitaya waste increased gradually.

From the data collected in Table 4.3, the value of the absorbance for the

extraction of pitaya waste at 60oC has increased gradually from 10 mg/L to 30 mg/L of

concentration of Alum. But there is a decrease in the value of total phenolic content at

25 mg/L concentration of alum and then increase again at 30 mg/L concentration of

alum. It can be explained for this happen as the optimum dose for the extraction of this

experiment is at 20 mg/L of concentration of alum to be used.

The total phenolic content increased from 0.846 to 2.055 mg GAE/g from

concentration of aluminum sulfate solution at 40oC at 10 mg/L to 15 mg/L with 58.82 %.

While from 15 mg/L to 20 mg/L of the concentration of aluminum sulfate solution being

added, the value of the total phenolic content increased with amount of 1.15 mg GAE/g

where the increase in the percentage is 35.96. From 20 mg/L to 25 mg/L of the

concentration of aluminum sulfate solution, it shows that there is a sudden decrease of

the total phenolic content where 48.22 % has been decreased but it increased with 11.26

% after 25 mg/L of the concentration of aluminum sulfate solution with the value of total

phenolic content at 30 mg/L of concentration of aluminum sulfate is 2.44. So, from 10

64

mg/L to 20 mg/L of the concentration of aluminum sulfate, there is an increase of

73.63% in the total phenolic content.

Table 4.3 Values of absorbance and total phenolic content at 60oC

Temperature

60oC


10

15

20

25

30

Absorbance,

%T ABS

0.066

0.088

0.109

0.09

0.095

Total

Phenolic

Content ,

mg GAE/ga

0.846

2.055

3.209

2.165

2.439


65


0

0.5

1

1.5

2

2.5

3

3.5

10 15 20 25 30


To

tal

Ph

en

oli

c C

on

ten

t,

mg

GA

E/g



While for the study of total flavonoids content in this experiment, it is clearly

showing that the total flavonoids content increase its content gradually as the

concentration of the aluminum sulfate is increased. The lowest value of total flavonoids

content is 1.458 mg CE/g at the concentration of aluminum sulfate of 10 mg/L. The

highest value of the total flavonoid content is 2.238 mg CE/g at the concentration of 30

mg/L.

In the extraction of the pitaya peels, the concentration of aluminum sulfate

produced total flavonoids at 10 mg/L and 15 mg/L are 1.46 mg CE/g and 1.62 mg CE/g

respectively. The percentage of the increase of the total flavonoids is 10.22. From 15

66

mg/L to 20 mg/L concentration of aluminum sulfate, the total flavonoids increased from

1.62 CE/g to 1.83 CE/g, which has increased 11.44 %, while from 20 mg/L to 25 mg/L

of concentration of aluminum sulfate, the total flavonoids increased with amount of

0.276 mg CE/g equivalent to 13.09 %. Then from 25 mg/L to 30 mg/L of the aluminum

sulfate concentration solution, the total flavonoids increased to 2.24 mg CE/g. Overall

the total flavonoids in this extraction of pitaya peels at the temperature of 60oC has

increased gradually from 10 mg/L to 30 mg/L of the concentration of aluminum sulfate

solution.

Table 4.4 Values of absorbance value and total flavonoids content at 60oC

Temperature

60oC


10

15

20

25

30

Absorbance,

%T ABS

0.078

0.086

0.0.096

0.109

0.115

Total

Flavanoids ,

mg QE/gb

1.458

1.624

1.833

2.109

2.238


67


0

0.5

1

1.5

2

2.5

10 15 20 25 30


To

tal

Fla

va

no

ids

, m

g C

E/ 2

5g

Figure 4.4 Total Flavonoids Versus Concentration of Aluminum Sulfate at 60oC

4.3 Total Phenolic and Total Flavonoids Obtained at 80oC

4.3.1 Total Phenolic

In this experiment, the data in Table 4.5 shows the lowest value of the total

phenolic contents obtained is 1.286 mg GAE/g at the concentration of alum sulfate at 10

mg/L. While for the highest value of total phenolic content is 3.319 mg GAE/g at the

concentration of aluminum sulfate at 20 mg/L. So it can be concluded that the optimum

dosage for this experiment to extract the optimum total phenolic content is at 20 mg/L of

the concentration of aluminum sulfate.

68

From the graph in the Figure 4.5, it shows that initially the value of the total

phenolic content increased from from 10 mg/L until 20 mg/L of concentration of

aluminum sulfate solution for 61.26 % but the value of the total phenolic content

decreased start from 20 mg/L until 30 mg/L of the concentration of aluminum sulfate

solution for 36.42 %. From the concentration of aluminum sulfate solution at 10 mg/L to

15 mg/L, there is 23.03 % increase in the total phenolic content, also for the

concentration of aluminum sulfate solution at 15 mg/L to 20 mg/L, where there is 49.67

% increase in the total phenolic content. While for the concentration of aluminum sulfate

solution from 20 mg/L to 25 mg/L, it decreased for 39.17 % of the total phenolic content

also same for 25 mg/L to 30 mg/L of the concentration of aluminum sulfate where 13.02

% decrease in the value of total phenolic content.

Table 4.5 Values of absorbance value and total phenolic content at 80oC

Temperature

80oC


10

15

20

25

30

Absorbance,

%T ABS

0.074

0.081

0.111

0.094

0.089

Total

Phenolic

Content ,

mg GAE/ga

1.286

1.670

3.319

2.385

2.101


69


00.5

11.5

22.5

33.5

10 15 20 25 30


To

tal P

he

no

lic C

on

ten

t,

mg

GA

E/ 2

5g



While for the study of total flavonoids content in this experiment, the data in

Table 4.6 the lowest value obtained is 0.433 mg CE/g at the concentration of aluminum

sulfate of 10 mg/L. For the highest total flavanoids content is 1.833 mg CE/g at the

concentration of aluminum sulfate at 20 mg/L.

From the graph obtained in Figure 4.6, it shows that there is an increase in the

value of total flavonoids from 10 mg/L to 20 mg/L of the concentration of aluminum

sulfate solution, after that there is just a small decrease in the value of the total

flavonoids for 4.56 % from 20 mg/L to 30 mg/L concentration of aluminum sulfate

solution. At 10 mg/L of the concentration of aluminum sulfate solution, the value of the

70

total flavonoids is 1.33 mg CE/g and it increased to 1.39 mg CE/g at 15 mg/L

concentration of aluminum sulfate solution. Only 4.49% of the total flavonoids is

increased and it is also for the total flavonoids increased from the concentration of

aluminum sulfate solution from 15 mg/L to 20 mg/L, where 21.97 % of the total

flavonoids is increased. But the value of the total flavonoids decreased from 1.79 mg

CE/g to 1.69 mg CE/g, where only 6.08 % has been decreased, the value of the total

flavonoids then increased to 1.71 mg CE/g at the concentration of aluminum sulfate of

30 mg/L. So from the overall, there is an increase of the total flavonoids with 25.47%

from 10 mg/L to 20 mg/L of the concentration of aluminum sulfate solution. Meanwhile,

there is a decrease of total flavonoids with 4.57 % from 20 mg/L to 30 mg/L of the

concentration of aluminum sulfate solution.

Table 4.6 Values of absorbance and total flavonoids content at 80oC

Temperature

80oC


10

15

20

25

30

Absorbance,

%T ABS

0.026

0.075

0.096

0.089

0.090

Total

Flavanoids ,

mg CE/gb

0.433

1.396

1.833

1.686

1.707


71


0

0.5

1

1.5

2

10 15 20 25 30


To

tal

Fla

von

oid

s,

mg

CE

/ 25

g

Figure 4.6 Total Flavonoids Content Versus Concentration of Aluminum Sulfate at

80oC

4.4 Comparison of The Total Phenolic Content at Different Temperatures

From the graph in Figure 4.7, it shows clearly that the extraction at 40oC of

pitaya waste using water produced the lowest content of total phenolic among the 60oC

and 80oC. The reason for the extraction produced at the low content of total phenolic at

40oC is because at this temperature which might be considered as low, is not enough to

extract the optimum total phenolic contented in the pitaya waste.

With the comparison of the total phenolic content at the concentration of

aluminum sulfate at 10 mg/L, we can see from the graph showing that total phenolic

content produced at the highest value is at temperature of 80oC which is 1.286 mg

GAE/mg. The lowest value of total phenolic content is 0.451 mg GAE/g which is at the

72

temperature of 40oC. For heating at temperature of 60oC, the total phenolic content is

0.846 mg GAE/g.

For the comparison of total phenolic content at 15 mg/L, concentration of

aluminum sulfate, the highest value of total phenolic content is 2.055 mg GAE/g at 60oC

while for the lowest value of total phenolic content is 1.181 mg GAE/g at the

temperature of 40oC. For heating at the first stage which the temperature at 80oC, the

total phenolic content has the value of 1.670 mg GAE/g.

Concentration of aluminum sulfate at 20 mg/L actually can be said to be at the

optimum dose for this extraction of total phenolic content from the pitaya waste by using

water. From the graph (Figure 4.7), it shows very clear that it produced the highest value

of total phenolic content among the concentration of aluminum sulfate used ranging

from 10 to 30 mg/L. The highest value of the total phenolic content is 3.319 mg GAE/g

which is at the temperature of 80oC. The lowest value of the total phenolic content is

1.621 mg GAE/g at the temperature of 40oC. For heating the pitaya waste at temperature

of 60oC, its total phenolic content is 3.209 mg GAE/g.

At the concentration of 25 mg/L of aluminum sulfate, the highest value of total

phenolic content is 2.385 mg GAE/g which is at the temperature of 80oC while for the

lowest value of total phenolic content is 1.731 mg GAE/g at the temperature of 40oC.

For extraction of pitaya waste at temperature of 60oC, the value of the total phenolic

content is 2.165 mg GAE/g.

At 30 mg/L of concentration of aluminum sulfate, extraction of pitaya waste at

the temperature of 60oC has produced the highest value of total phenolic content which

is 2.439 mg GAE/mg. The lowest value of total phenolic content is 0.932 mg GAE/mg

73

which is at the temperature of 40oC. For temperature of 80oC, the value of the total

phenolic content is 2.101 mg GAE/g.

The different between the values of the highest total phenolic content at 80oC

which is 3.310 mg GAE/g and the lowest total phenolic content at 40oC which is 3.210

mg GAE/g is 47.7 %. While when the highest value of total phenolic content is

compared with the second highest total phenolic content where the value is 3.210 mg

GAE/g, the different is 3.1%.

It is very clear to see from the graph shown in Figure 4.7 that the different

temperatures have produced the highest total phenolic content at the concentration of

aluminum sulfate at 25 mg/L only. So it can be concluded that the optimum dose of

concentration of aluminum sulfate to be used for the extraction of pitaya peels is at 25

mg/L.

The graph also shows clearly that the values of the total phenolic content

decreased start after 25 mg/L of the concentration of aluminum sulfate for all the

temperatures. The reason for this phenomenon can be said that the high concentration of

aluminum sulfate (larger than 25 mg/L) would have been very acidic. The high acidity

of the aluminum sulfate reacted with the compound of total phenolic content which have

changed it to the another type of compound. Since the total phenolic content have been

changed to the another compound after the reaction of the high concentration of

aluminum sulfate, hence, the total phenolic content could not be detected during the

analysis for it content.

74

Table 4.7 Comparison of total phenolic content at different temperatures

Total

Phenolic

Content,

mg GAE/g


10

15

20

25

30

40 oC

0.451

1.181

1.621

1.731

0.932

60 oC

0.846

2.055

3.209

2.165

2.439

80 oC

1.286

1.670

3.319

2.385

2.101


00.5

11.5

22.5

33.5

10 15 20 25 30


To

tal

Ph

eno

lic

Co

nte

nt,

m

g G

AE

/ 25

g

40°C

60°C

80°C

Figure 4.7 Comparison of Total Phenolic Content at Different Temperatures

75

4.5 Comparison of The Total Flavonoids at Different Temperatures

Total flavonoids are compared with different concentration of aluminum sulfate

in the graph shown in Figure 4.8. From the graph in Figure 4.8, it is clearly showing that

the extraction at 80oC is in the middle of the two lines. While the blue color line which

represents the extraction at the temperature of 40oC is at the bottom which also means it

has the lowest content of total flavanoids at that specific temperature. While for the top

one the line of pink is seen to have the highest content of total flavonoids among all

three variables temperature. The highest content of total flavonoids is at the temperature

of 60oC. So for the pitaya waste to be extracted for its optimum content, temperature of

60oC is the most preferable compared with 40oC and 80oC.

Concentration of aluminum sulfate at 10 mg/L produced at almost the same

amount of total flavonoids content in the pitaya peels. The highest of the total flavonoids

content that was extracted is 1.457 mg CE/g at the temperature of 60oC. While for the

lowest content of total flavonoids in 10mg/L concentration of aluminum sulfate is 1.212

mg CE/g which is at the temperature of 40oC. For temperature at 80oC, the extraction of

the content of total flavonoids from pitaya peels is found to be at 1.457 mg CE/g.

For the concentration of aluminum sulfate at 15 mg/L, it shows the highest

extraction of total flavonoids is at the temperature of 60oC which the value is 1.624 mg

CE/g. Lowest content of total flavonoids being extracted in this concentration of

aluminum sulfate is 1.253 mg CE/g at the temperature of 40oC. Meanwhile, under the

temperature of 80oC, the extraction of total flavonoids was able to be obtained is found

to be 1.396 CE/g.

Extraction of pitaya waste at the concentration of 20 mg/L was studied in this

research as well. The highest total flavonoids content that could be obtained in this

76

concentration of aluminum sulfate was found to be at the temperature of 60oC which the

value of the total flavonoids content is 1.833 mg CE/g. The lowest total flavonoids

content that was obtained from this concentration of aluminum sulfate is at the

temperature of 40oC where the value of the content of total flavonoids is 1.294 mg CE/g.

For the extraction of pitaya waste at the temperature of 80oC, the value of total

flavonoids obtained is 1.789 mg CE/g.

Pitaya peels being extracted at 25 mg/L of the concentration of aluminum sulfate

was found to have the highest content of total flavonoids at the temperature of 60oC and

value is 2.109 mg CE/g. While value of the total flavonoids being extracted out from the

pitaya waste found to be at the lowest one is 1.417 mg CE/g at the temperature of 40oC.

For the extraction at the temperature of 80oC, the value of total flavanoids is 1.686 mg

CE/g.

Finally for the concentration of aluminum sulfate at 30 mg/L, the highest value

of total flavonoids is 2.238 mg CE/g at the temperature of 60oC. The lowest value of

total flavonoids in this extraction method at this concentration is 1.603 mg CE/g at the

temperature of 40oC. For the temperature of 80oC, the extraction of the total flavonoids

is found to be at the value of 1.707 mg CE/g.

The different between the values of the highest of the total flavonoids at 60oC

which is 2.23 mg CE/g and lowest of the total flavonoids content at 1.71 mg CE/g at

40oC of the concentration of aluminum sulfate at 30 mg/L is 28.2 %. While comparing

with the second highest value of total flavonoids content at 80oC is 23.3 %.

From the graph of the comparison of the three different temperatures producing

the total flavonoids content, it is clear to see that the values of the total flavonoids

77

content was getting much higher when the concentration of aluminum sulfate increased.

It can be said that the total flavonoids compound was not reacted with the aluminum

sulfate when the concentration was increased whilst it can be extracted more and the

total flavonoids content increased when the concentration of aluminum sulfate is higher.

The graph also shows that the value of the total flavonoids being extracted at the

temperature of 60oC but not 80oC because it can be explained that the total flavonoids

compound was destroyed by the temperature which is higher than 60oC. As a result, total

flavonoids could not be detected during the analysis for its content. Hence, the total

flavonoids content can be higher at the temperature of the range of 60oC to

80oC.

Table 4.8 Comparison of total flavonoids content at different temperatures

Total

Flavonoids

Content,

mg CE/g


10

15

20

25

30

40 oC

1.212

1.252

1.293

1.416

1.602

60 oC

1.457

1.623

1.833

2.109

2.238

80 oC

1.333

1.395

1.789

1.686

1.707

78

Total Flavonoids vs Concentration of Aluminum Sulfate

0

0.5

1

1.5

2

2.5

10 15 20 25 30


To

tal

Fla

von

oid

s,m

g C

E/

25g

40°C

60°C

80°C

Figure 4.8 Total Flavonoids Versus Concentration of Aluminum Sulfate at Different

Temperatures

4.6 Volume of Filtrate

Volume of filtrate was obtained from the filtration by using the cotton fabric.

The floc was used for the determination of total phenolic content and total flavonoids

analysis while the filtrate obtained is just to show the relation between the concentration

of the aluminum sulfate and the filtrate collected after one day filtration process. The

result is shown in Table 4.9.

For the extraction of pitaya waste at the temperature of 40oC, it shows that the

volume of filtrate increases when there is an increase in the concentration of the

aluminum sulfate. The filtrate volume was measured using measuring cylinder and the

reading is 195 mL at the concentration of 10 mg/L which is the highest volume that was

79

obtained. While for the lowest filtrate volume that was measured is 70 mL at the

concentration of aluminum sulfate of 30 mg/L.

Extraction of pitaya waste at temperature of 60oC can be seen to have the same

trend as in the temperature of 40oC, where the higher the concentration of aluminum

sulfate was added to the solution, the lesser the volume of filtrate could be obtained. So

in this extraction of pitaya waste at 60oC, the highest volume of filtrate that was

measured is 122 mL while for the lowest one, which is 62 mL at the temperature of

60oC.

While for the extraction at the temperature of 80oC, the trend of increase in the

volume of filtrate is same as in the previous variables, 40oC and 60oC. The highest

volume of filtrate that was obtained is in the concentration of aluminum sulfate of

10mg/L. For the volume which is at the lowest value is at 30mg/L, concentration of

aluminum sulfate.

Hence, it can be concluded that the increase in the concentration of the aluminum

sulfate would actually decrease the volume of the filtrate from the after filtration of the

floc by using the cotton fabric in this experiment. This phenomenon is because the

higher the concentration of the aluminum sulfate is able to form more floc in the

solution. Aluminum sulfate functions as a coagulant to coagulate the red dyes extraction

from after the heating of the pitaya waste with different temperature.

80

Table 4.9 Volume of filtrate obtained at different temperature

Volume of

Filtrate, ml


10 15 20 25 30

40oC 195 95 71 73 70

60oC 122 120 95 62 62

80oC 80 60 40 39 35

Volume of Filtrate vs Concentration of Aluminum Sulfate

0

50

100

150

200

250

10 15 20 25 30


Vo

lum

e o

f F

iltr

ate,

ml

Figure 4.9 Filtrate Volume Versus Concentration of Aluminum Sulfate at 40oC

81


0204060

80100120140

10 15 20 25 30


Vo

lum

e o

f F

iltr

ate,

ml



0

20

40

60

80

100

10 15 20 25 30


Vo

lum

e o

f F

iltr

ate,

ml


82

Figure 4.12 Volume of Filtrate at Different Concentration of Aluminum Sulfate

4.7 Weight of Floc

The extraction of the pitaya waste process was done by heating it in the oven, the

pitaya waste was filtered by using the plastic sieve. The filtrate is collected and then

concentration of aluminum sulfate ranging from 10mg/L, 15mg/L, 20mg/L, 25mg/L and

30 mg/L was added into five beakers respectively. The solution in the five beakers was

stirred at the speed of 20 rpm for 45 minutes. During the stirring process, the floc could

be seen increase in its size. After 45 minutes, the stirring process was stopped and the

filtration of this solution was done by using cotton fabric.

From the graph that is shown in Figure 4.13, it shows the weight of the floc

obtained at the extraction of pitaya peels at 40oC increased in its weight gradually from

83

67.94 g to 285.8 g. The percentage of weight floc being increased from 10 mg/L to 30

mg/L of the concentration of aluminum sulfate is 76.

For the extraction of pitaya peels at 60oC, it has the same trend like the one in the

extraction of pitaya peels at 40oC where from 10 mg/L to 30 mg/L of the concentration

of aluminum sulfate solution, the weight of the floc obtained increase as well for

33.41%.

At the temperature of 80oC of the extraction of pitaya peels, the trend of the

graph as shown in Figure 4.15 is the wet floc weight increases gradually from 10 mg/L

to 30 mg/L of the concentration of aluminum sulfate solution with 29.67 %.

Table 4.10 Weight of floc at different temperatures

Weight of

Floc, g


10

15

20

25

30

40oC 67.94 175.00 223.32 277.2 285.8

60oC 195.88 248.9 244.95 280.56 294.17

80oC 187.1 211.6 238.00 249.48 266.06

84

Weight of Wet Floc vs Concentration of Aluminum Sulfate

050

100150200250300350

10 15 20 25 30


Wei

gh

t o

f W

et F

loc,

g

Figure 4.13 Weight of Floc Versus Concentration of Aluminum Sulfate at 40oC

Weight of Wet Floc vs Concentration of Aluminum Sulfate

050

100150

200250300350

10 15 20 25 30


Wei

gh

t o

f W

et F

loc,

g


85

Weight of Wet Foc vs Concentration of Aluminum Sulfate

0

50

100

150

200

250

300

10 15 20 25 30


Wei

gh

t o

f W

et F

loc,

g


4.8 Color of Supernatant at Different Temperatures

From the observation during the experiment, the brightness of the color of the

supernatant changed from low to high of the concentration of the aluminum sulfate. As

shown in the figures below, it is clear to see the color getting pale when the

concentration of the aluminum sulfate is higher. It can be explained that the acidity of

the aluminum sulfate can actually result in reducing of the brightness of the pitaya peels

red color.

86

Figure 4.16 Supernatant Color at 40oC

Figure 4.17 Supernatant Color at 60oC

87

Figure 4.18 Supernatant Colors at 80oC

CHAPTER 5

CONCLUSION AND RECOMMENDATIONS

5.1 Conclusion

Experiment of the extraction of total phenolic and total flavonoids content

involving temperature ranging from 40oC to 80oC and also the different concentration of

aluminum sulfate from 10 mg/L to 30 mg/L. After this experiment had been done, this

study found out that different temperatures at different concentration of aluminum

sulfate do influence the value of the total phenolic and total flavonoids content. From the

observation during the experiment, the color of the supernatant was observed that the

colors of extract change due to the concentration of aluminum sulfate.

From this study, the highest value of total phenolic content is 3.31 mg GAE/ 25 g

at the temperature of 80oC. While for the highest value of total flavonoids content is 2.23

mg CE/ 25 g at the temperature of 60oC. From overall, temperature of 40oC produced the

less total phenolic and total flavonoids content among all the temperatures studied in this

experiment. Here, it can be concluded that temperature of 80oC could have produced

higher total phenolic content and the total flavonoids could have produced at

89

temperature at 60oC. So, it is clearly showing that temperature of 40oC is not enough to

extract the total phenolic and total flavonoids content.

5.2 Recommendations

This study can actually be improved by many aspects either in chemical used or

the equipments used to analyze the total phenolic and total flavonoids content to obtain

the high accuracy of the extract values from the pitaya peels.

The coagulant used in this study is aluminum sulfate. It is recommended that the

α-cyclodextrin is to be used as the flocculant to improve the flocculation of the extract of

pitaya peels. By using this flocculant, it might show the obvious different of the values

of total phenolic and total flavonoids content at different temperatures.

The presented data for total phenolic and total flavonoid content are a basis for

assessment of the preventive role of fruits and vegetables against free radicals effects.

There is a necessarily to study towards in-depth on the spectrum of multiple phenolics

and flavonoids representatives. Betacyanins should also be studied because it also

contributes to the total phenolics due to its phenols structure in the molecules.

The temperature to extract the pitaya peels can be up to 80oC to study that

whether the higher temperature could have extracted higher total phenolic content whilst

the temperature with small interval from 60oC to 80oC to obtain the exact temperature of

total flavonoids to be extracted at its highest temperature between the range.

90

REFERENCES

Ali, S., Hussain, T. and Nawaz, R., (2008). Optimatization of Alkaline Extraction of

Natural Dye From HennaLleaves and Its Dyeing on Cotton by Exhaust Method.

Journal of Cleaner Production.

Atawodi, S.E. (2004). Comparative in Iitro Trypanocidal Activities of Petroleum Ether,

Chloroform, Methanol and Aqueous Extracts of Some Nigerian Savannah Plants.

African Journal of Biotechnology, Vol.4 (2), pp. 177-182.

Basri, D.F. and Fan, S.H. (2004). The Potential of Aqueous And Acetone Extracts of

Galls of Quercus Infectoria As Antibacterial Agents. Department of Biomedical

Science, Universiti Kebangsaan Malaysia.

Bechtold, T., Mahmud-Ali, A. and Mussak, R. (2006). Natural Dyes For Textile Dyeing:

A Comparison of Methods to Assess The Quality of Canadian Golden Rod Plant

Material. Dyes and Pigments, 75; 287-293.

Bechtold, T., Turcanu, A., Ganglberger, E. and Geissler, S. (2003). Natural Dyes in

Modern Textile Dyehouses-How to Combine Experiences of Two Centuries to

Meet The Demands of The Future. J Clean Prod 2003; 11: 499-509.

Bhuyan, R., Saikia, C.N., and Das, K.K. (2004). Extraction And Identification of Colour

Components From The Bark of Mimusops Elengi And Terminalia Arjuna and

Evaluation of Their Dyeing Characteristics on Wool. Indian Journal of Fibre &

Textile Research; 29:470–6.

Carlo, D., G., Mascolo, N., Izzo, A. A. and Capasso, F. (1999). Flavonoids: Old and

New Aspects of A Class of Natural Therapeutic Drugs. Life Sciences, 65, 337-

353

91

Cowan, M. M. (1999). Plant Products as Antimicrobial Agents. Clinical Microbiology

Review, 12(4), 564-582.

David, T.B., and Levente, L.D. ( 2000 ). Rapid Aqueous Extraction of Mucilage from

Whole White Mustard Seed.

Druding, S.S. (2007, November 30). Dye History From 2600 BC to The 20th Century.

Dye Seminar at Covergence ’82.

Duhard, V., Garnier, J.C. and Megard, D. (1997). Comparison of The Stability of

Selected Anthocyanin Colorants in Drink Model Systems. Agro-Food-Industry

Hi-Tech, 8, 28–34.

Evon, Ph., Vandenbossche, V., Pontalier, P.Y. and Rigal, L. (2007). Direct Extraction

of Oil From Sunflower Seeds by Twin-screw Extruder According to An Aqueous

Extraction Process: Feasibility Study and Influence of Operation Conditions.

Industrial Crops and Products, 26(3), P.351-359.

Falodun, A., Okunrobo, L.O. and Uzoamaka, N. (2006). Phytochemical Screening and

Anti-inflammatory Evaluation of Methanolic and Aqueous Extracts of Euphorbia

Heterophylla Linn (Euphorbiaceae). African Journal of Biotechnology, Vol.5,

pp.529.

Filippos, V., Emmanouil, T., Carl, D., Guenter, V., Georg, K. and Nickolas, P. (October

2007). Biotechnology of Flavonoids and Other Phenylpropanoid-Derived

Natural Products. Part I: Chemical Diversity, Impacts on Plant Biology and

Human Health. Biotechnology Journal 2 (10).

Ganglberger, E., Geissler, S., Farb and Stoff, (2003). Sustainable Development

Durchneue Kooperationen und Prozesse, Project Report. Wien.

Bundesministerium fur Verkehr, Innovation und Technologie.

92

Gómez-Estaca, J., Bravo, L., Gómez-Guillén, M.C., Alemán, A., and Montero, P.

(2008). Antioxidant Properties of Tuna-skin and Bovine-hide Gelatin Films

Induces by The Addition of Oregano and Rosemary Extracts. Food Chemistry

112; 18-25.

Hoa, T.T, Clark, C.J., Waddell, B.C. and Woolf, A.B. (2006). Postharvest Quality of

Dragon Fruit (Hylocereus undatus) Following Disinfesting Hot Air Treatments.

Postharvest Biology and Technology 41; 62-69.

Li, H., Wang, X., Yong, L., Li, P. and Wang, H. (2008). Polyphenoic Compounds and

Antioxidant Properties of Selected China Wines. Food Chemistry, 112; 454-460.

LJ Health Promotions. Retrived November 11, 2008, from

http://www.dragonfruit.com.my/about.html

Lu, Y., and Foo, Y. (2000). Antioxidant and Radical Scavenging Activities of

Polyphenols From Apple Pomace. Food Chemistry, 68, 81-85.

Mahomed, I.M. and Ojewole, J.A.O. (2006). Oxytocin-Like Effect of Harpagophytum

Procumbens Dc [Pedaliaceae] Secondary Root Aqueous Extract On Rat Isolated

Uterus. Afr. J. Trad. CAM 3(1); 82-89.

Marinova, D., Ribarova, F. and Atanassova, M. (2005) Total Phenolics and Total

Flavonoids in Bulgarian Fruits and Vegetables. Journal of the University of

Chemical Technology and Metallurgy, page 255-260.

Mizrahi, Y., Nerd, A. and Nobel, P.S. (1997). Cacti As Crops. Hort. Rev. 18, 291–319.

Nair, R., Chanda, S.(2006). Activity of Some Medicinal Plants Against Certain

Pathogenic Bacterial Strains. Indian Journal of Pharmacol, Volume 38; 142-4.

93

Norziah, M. H., Ruri, A. S. and Fazilah, A. (2008). Utilization of Red Pitaya Fruit Peels

for Value Added Food Ingredients. International Conference on Environmental

Research and Technology (ICERT 2008).

Parekh, J., and Chanda, S.(2006). In-vitro Antimicrobial Activities of Extracts of

Launaea Procumbens Roxb. (Labiateae), Vitis vinifera L. (Vitaceae) and

Cyperus rotundus L. (Cyperaceae). African Journal of Biomedical Research,

Vol.9; 89-93.

Rappl, B. (2005). Trademark Farb and Stoff e Von der Idee zum marktfahigen

Handelsprodukt: Pflanzenfarben fur die Textilindustrie, project report. Wien:

Bundesministerium fur Verkehr, Innovation und Technologie.

Rickwood, D. and Graham, J.M. (2001). Biological Centrifugation. Springer Verlag.

Rusak, G., Komes, D., Likic, S., Horzic, D. and Kovac, M. (2008). Phenolic Content

and Antioxidative Capacity of Green and White Tea Extracts Depending on

Extraction Condition and The Solvent Used. Food Chemistry, 110; 852-858.

Salahdeen, H.M. and Yemitan, O.K.(2006). Neuropharmacological Effects of Aqueous

Leaf Extract of Bryophyllum Pinnatum in Mice. African Journal of Biomedical

Research, vol.9; 101-107.

Sengupta, S. and Singh, B. R. (2003). Natural “Green” Dyes for the Textile Industry.

The Massachusetts Toxics Use Reduction Institute, University of Massachusetts

Lowell, 2003.

Singleton, V.L. and Rossi, J.A. (1965). Colorimetryof Total Phenolics with

Phosphomolybdic-phosphotungstic Acid Reagents. American Journal of Enology

and Viticulture, 16, 144-158.

94

South Texas Unit of The Herb Society of America (1993). Dyeing. Retrieved November

10, 2008, from http://www.herbsociety-stu.org/Dyeing.htm#Resources

Stauth, D. (2007). Studies Force New View on Biology, Nutritional Action of

Flavonoids. Linus Pauling Institute. Retrieved from

http://www.eurekalert.org/pub_releases/2007-03/osu-sfn030507.php

Stich, E., Kloos, K. and Hoeck, H. (1999). Natur, Technik und Recht: miteinander, nicht

Gegeneinander. Zucker- und Subwaren Wirts-chaft, 52, 10–14.

Stintzing, F. C., and Carle, R. (2004). Functional Properties of Waterman, P. G. (1998).

Alkaloid chemosystematics. In G. A. Cordell anthocyanins and betalains in

plants, food, and in human (Ed.). The alkaloids – Chemistry and

pharmakology ,vol. 50, nutrition. Trends in Food Science and Technology, 15,

19–38.

Su, M. S., Shyu, Y. T. and Chien, P. J. (2008) Antioxidant Activities of Citrus Herbal

Product Extracts. Food Chemistry , page 892-896.

Suzuki, M., Watanabe, T., Miura, A., Harashima, E., Nakagawa, Y. and Tsuji, K.

(2002). An Extraction Solvent Optimum for Analyzing Polyphenol Contents by

Folin-Denis assay. Nippon Shokuhin Kagaku Kaishi, 49, 507-511.

Tapiero, H., Tew, K. D., Nguyen, B. G. and Mathé, G. (2002). Polyphenols: Do They

Play A Role in The Prevention of Human Pathologies. Biomedicine and

Pharmacotherapy, 56, 200-207.

Tawaha, K., Alali, F.Q., Gharaibeh, M., Mohammad, M. and El-Elimat, T. (2007).

Antioxidant Activity and Total Phenolic Content of Selected Jordanian Plant

Species. Food Chemistry 104, 1372-1378.

95

The Star. October 12, 2007. “ Dye Used in Burgers and Sausages Banned”. Retrieved from http://thestar.com.my/news/story.asp?file=/2007/10/12/nation/19148893&sec=na tion Vattem, D. A., Lin, Y.-T. and Shetty, L. K. (2004). Antimicrobial Activity Against Select

Food-borne Pathogens by Phenolic Antioxidants Enriched in Cranberry Pomace

by Solid-state Bioprocessing Using The Food Grade Fungus Rhizopus

Oligosporus. Process Biochemistry, 39, 1939-1946.

Wu, L. C., Hsu, H. W., Chen, Y.C., Chiu, C. C., Lin, Y.I. and Annie, H. (2005)

Antioxidant and anitproliferative activities of red pitaya. Food Chemistry page

319-327.

Yongnian, N., and Liu, C.(1999). Artificial Neural Networks and Multivariate

Calibration for Spectrophotometric Differential Kinetic Determinations of Food

Antioxidants. Food Chemistry .Volume 396, page 221-230.

Zhou, S.H., Fang, Z.X., Lu, Y., Chen, J.C., Liu, D.H. and Ye, X.Q. (2008). Phenolics

and Antioxidant Properties of Bayberry (Myrica rubra Sieb. Et Zucc.) Pomace.

Food Chemistry, 112; 394-39

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