THE EFFECT OF HEAT PROCESSING ON TRITERPENE GLYCOSIDES AND

ANTIOXIDANT ACTIVITY OF HERBAL PEGAGA (Centella asiatica L. Urban)

DRINK

SANIAH BTE KORMIN

A thesis submitted in fulfilment of the

requirements for the award of the degree of

Master of Engineering (Bioprocess)

Faculty of Chemical and Natural Resources Engineering

Universiti Teknologi Malaysia

JUNE 2005

ii

“I declare that this thesis entitled The Effect of Heat Processing on Triterpene

Glycosides and Antioxidant Activity of Herbal Pegaga (Centella asiatica L.Urban)

Drink 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 of author : SANIAH BTE KORMIN

Date : …………………………………

iii

ACKNOWLEDGEMENT

First and foremost, thanks to God Almighty for the guidance and help in giving

me the strength to complete this thesis. I would also like to take this opportunity to

express my utmost gratitude to my supervisor, Prof. Dr. Mohd Roji Sarmidi for his

valuable guidance and advice throughout this thesis study.

Appreciation is also to Pn. Faridah Husin, Research officer, Food Technology

Center, MARDI Serdang, for her kindness in supporting this study. I would like to

express my sincere appreciation to research assistants in MARDI Johor Bahru for their

help during the various laboratory tasks. A word of thanks also goes to all personnel and

technicians in Chemical Engineering Pilot Plant, UTM due to their full support in my

research experiments especially to En. Abdul Rahim Abd. Rahman and En Muhammad

Subri Abd. Rahman.

Finally, I am also forever indebted to my lovely husband, Mohd Azli Sairan for

his continuous encouragement and many sacrifices.

iv

ABSTRACT

The health benefit of herbal pegaga drink, which is associated with triterpene

glycosides content and antioxidant activity attract a lot of interest from the public and

food and herbal industries. The works carried in this research investigated the effect of

heat processing at 65 C/15 minutes, 80 C/5minutes and pasteurization at 80 C/5minutes

followed by canning and boiling at 100 C/10 minutes on these phytochemicals and

compared to untreated herbal pegaga drink or fresh sample. The results revealed that the

untreated pegaga drink exhibited much higher (P<0.05) antioxidant activity than the

heat-treated samples. The Ferric Reducing Ability of Plasm (FRAP) values was 860

µmol/litre for the untreated sample and in the range of 404 - 740 µmol/litre for heat-

treated sample. The untreated drink inhibited about 72% of linoleic acid peroxidation

and the percentage inhibition of heat-treated samples were in the ranged of 26-56%. The

FRAP and Ferric Thiocyanate (FTC) assays were strongly correlated (r=0.93) towards

the assessment of antioxidant activity in pegaga drink samples. The concentration of

ascorbic acid and total polyphenol after heat treatment were 0.7 mg/100ml to 1.76

mg/100ml and 730.27 mg/100ml to 903.23 mg/100ml, respectively. Phenolic compound

was found as the major contributor to the antioxidant activity in pegaga drink. Analysis

of the triterpene glycosides content was performed using an isocratic High Peformance

Liquid Chromatography system (HPLC). Heat processing resulted in a several fold

decreased of total triterpene glycosides. The amount in untreated drink was 10.8 to

17.3% higher than those in heat-treated pegaga drinks. The present study indicated that

the herbal pegaga drinks samples still retain appreciable amount of madecassoside,

madecassic acid, asiaticoside, asiatic acid and polyphenol compounds. These

phytochemicals are good sources of antioxidant.

v

ABSTRAK

Faedah kesihatan bagi minuman herba pegaga yang dikaitkan dengan kehadiran

triterpena glikosida dan aktiviti pengantioksidan telah menarik minat yang tinggi

daripada orang awam dan pengusaha industri herba serta makanan. Kajian ini

dijalankan bagi menyiasat kesan proses pemanasan pada suhu 65 C/15 minit, 80 C/5

minit dan pempasturan pada 80 C/5 minit diikuti dengan pengetinan dan pendidihan

pada 100 C/10 minit ke atas perubahan fitokimia tersebut dan dibandingkan dengan

minuman tanpa rawatan atau sampel segar. Keputusan menunjukkan minuman pegaga

tanpa rawatan menghasilkan aktiviti pengantioksidan yang lebih tinggi (P<0.05)

berbanding sampel yang dipanaskan. Nilai ‘Ferric Reducing Ability of Plasma’ (FRAP)

adalah 860 µmol/liter bagi sampel tanpa rawatan dan dalam julat 404 - 740 µmol/liter

untuk sampel yang dipanaskan. Minuman tanpa rawatan merencat 72% pengoksidaan

asid linoleik dan peratus perencatan bagi sampel yang dipanaskan adalah di antara 26-

56%. Kaedah FRAP dan ‘Ferric Thiocyanate’ (FTC) berkorelasi tinggi (r=0.93) melalui

penilaian aktiviti pengantioksidan di dalam sampel minuman pegaga. Kepekatan asid

askorbik dan jumlah polifenol selepas pemanasan adalah 0.7 mg/100ml hingga 1.76

mg/100ml dan 730.27 mg/100ml hingga 903.23 mg/100ml setiap satunya. Sebatian

fenolik merupakan penyumbang utama kepada aktiviti pengantioksidan. Analisa bagi

kandungan triterpena glikosida dibuat menggunakan sistem isokratik Kromatografi

Cecair Berprestasi Tinggi (HPLC). Proses pemanasan turut menyebabkan penurunan

beberapa kali ganda amaun triterpena glikosida. Amaun di dalam minuman tanpa

rawatan panas adalah 10.8 hingga 17.3% lebih tinggi daripada minuman pegaga yang

dipanaskan. Kajian ini menunjukkan bahawa minuman herba pegaga masih

mengekalkan amaun madekasosida, asid madekasik , asiatikosida, asid asiatik dan

polifenol pada paras yang wajar diterima. Fitokimia ini adalah sumber pengantioksidan

yang baik.

vi

TABLE OF CONTENTS

CHAPTER TITLE PAGE

DECLARATION ii

ACKNOWLEDGEMENT iii

ABSTRACT iv

ABSTRAK v

TABLE OF CONTENTS x

LIST OF PLATE xi

LIST OF TABLES xii

LIST OF FIGURES xiii

LIST OF SYMBOLS xv

LIST OF APPENDICES xviii

1 INTRODUCTION 1

1.1 Objective 9

1.2 Scopes 9

2 LITERATURE RIVIEW 11

2.1 Medicinal Plants in Malaysia 11

2.2 Herbal Products in Food Industries 12

vii

2.3 Plant Material (Centella asiatica) 12

2.3.1 Plant Description 12

2.3.2 Medicinal Applications 13

2.3.3 Bioactive Constituents in Pegaga 14

2.4 Nutrient Composition 15

2.4.1 Proximate Composition and Nutritive Values 16

of Pegaga

2.5 Triterpene Glysoside (Asiaticoside, Madecassoside, 18

Asiatic acid, Madecassic acid)

2.5.1 Chemical Structure of Triterpene Glycosides 18

2.5.2 Health-Promoting Effect of Triterpene 20

Glycosides

2.5.3 Antioxidative Activity of Triterpene 20

Glycosides

2.5.4 Methods for Assessing Triterpene Glycosides 21

2.5.4.1 Extraction 21

2.5.4.2 HPLC Analysis 22

2.6 Ascorbic acid 22

2.6.1 The Contribution of Ascorbic acid in 24

Antioxidant Activity

2.7 Polyphenol 25

2.7.1 Phenolic Compounds in Pegaga 26

2.7.2 The Contribution of Phenolic compounds 26

in Antioxidant Activity

2.8 Antioxidant activity 28

2.8.1 Antioxidant activity in Herbs 30

2.8.2 Antioxidant Activity of Pegaga 30

2.8.3 The Role of Synergistic or Secondary 31

Antioxidants

2.8.3.1 Effect of citric acid 32

2.8.3.2 Effect of sulphites 32

viii

2.8.4 Effect of enzymatic oxidation on 33

antioxidant activity

2.8.5 Effect of concentration and sugar content 34

2.8.6 The Mechanism of Antioxidant Activity 35

2.8.7 Assesment of Antioxidant Activity 36

2.8.7.1 Ferric Reducing Ability of Plasma 37

(FRAP)

2.8.7.2 Ferric Thiocyanate (FTC) 37

2.9 Heat processing of Food and Beverages 38

2.9.1 The retention of nutrient and phytochemical 42

during processing of foods

2.9.2 Effect of food processing on nutrient 44

composition

2.9.3 Effect of heat processing on natural 45

antioxidant

2.9.4 Effect of heat processing on antioxidant 48

activity

2.9.4.1 Development of pro-oxidant 49

during heat processing

2.9.4.2 Development of heat-induced 50

antioxidant

2.10 Effect of heat processing on triterpene glycosides 52

3 MATERIAL AND METHODS 54

3.1 Introduction 54

3.2 Material and Sample Preparation 56

3.2.1 Juice Extraction 56

3.2.2 Preparation of Pegaga Drink 56

3.2.3 Commercial Pegaga Drink Sample 59

3.3 Experimentals and Analytical Methods 59

ix

3.3.1 Physico-chemical Characteristics 59

3.3.1.1 Colour Index 59

3.3.1.2 Total Soluble Solid and pH 60

3.3.1.3 Total Acidity 60

3.3.2 Proximate and Micronutrient Analysis 59

3.3.2.1 Moisture 60

3.3.2.2 Ash 60

3.3.2.3 Protein 61

3.3.2.4 Fat 62

3.3.2.5 Fibre 62

3.3.2.6 Carbohydrate and Energy 63

3.3.2.7 Microelement 63

3.3.3 Ascorbic Acid Assay 64

3.3.4 Total Polyphenol Assay 65

3.3.5 Antioxidant Assay 66

3.3.5.1 Ferric Reducing Ability of Plasm 66

(FRAP) Assay

3.3.5.2 Ferric Thiocyanate Method (FTC) 66

3.3.6 Study on Factors Influence to the Antioxidant 67

Activity of Pegaga Drink

3.3.7 Determination of Triterpene Glycosides 68

3.4 Statistical Analysis 69

4 RESULTS AND DISCUSSION 70

4.1 Introduction 70

4.2 Physico-chemical Characteristics of Pegaga Drink 71

4.3 Nutrient Composition 74

4.4 Total Polyphenol 77

4.5 Ascorbic Acid Content 81

4.6 Antioxidant Activity 83

x

4.6.1 Antioxidant Activity in Linoleic Acid 83

System (FTC Assay)

4.6.2 Antioxidant Activity by Ferric Reducing 86

Ability of Plasma (FRAP Assay)

4.6.3 Correlation of FTC Assay and FRAP Assay 90

4.7 Antioxidant Activity of Phenolic Compounds and 92

Ascorbic Acid

4.8 The Factors Influence on Antioxidant Activity 97

4.8.1 Effect of Citric Acid on Antioxidant Activity 98

4.8.2 Effect of Total Soluble Solid on 101

Antioxidant Activity

4.8.3 Effect of Sodium Metabisulphite 103

4.9 Triterpene Glycosides 106

4.9.1 Isocratic HPLC Assay 107

4.9.2 Quantitative Determination of Triterpene 114

Glycosides in Pegaga Drink

4.10 Antioxidant Activity of Asiaticoside 121

5 CONCLUSION AND RECOMMENDATION 122

5.1 Introduction 122

5.2 Conclusion 122

5.2 Recommendations and further works 124

REFERENCES 126

Appendices 147 – 155

xi

LIST OF PLATE

PLATE TITLE PAGE

1 Pegaga (Centella asiatica) 13

xii

LIST OF TABLES

TABLE TITLE PAGE

2.1 Nutritional composition of pegaga 17

2.2 Classification of food antioxidant 29

4.1 Physico-chemical characteristic of pegaga drink 72

4.2 The nutritional value and trace element of pegaga drink 76

4.3 Correlation (r) of antioxidant activity with total polyphenol 96

and ascorbic acid content of the pegaga drink

4.4 Results of HPLC analysis 114

4.5 Results for triterpene glycosides assay 116

xiii

LIST OF FIGURES

FIGURE TITLE PAGE

2.1 Structure of triterpene glycosides 19

2.2 The group of saponin glycosides 19

2.3 Structure of ascorbic acid 23

2.4 Influence of pH of heating medium on heat resistence 41

of spores

2.5 Changes in overall antioxidant activity due to 52

development of different stages Millard reaction

at different temperatures

3.1 Flowchart of the preparation of pegaga drink 57

3.2 Experimental layout 58

4.1 Total phenolic compounds (as ferrulic and gallic acid 80

equivalent) of different sample of pegaga drink

4.2 Ascorbic acid content of different sample of 82

pegaga drink

4.3 % inhibition of peroxidation as mean (n=3) in pegaga 85

drink and standard sample

4.4 FRAP activity as mean (n=3) in different thermal processing 88

of pegaga drink

4.5 Correlation of FRAP and FTC measurement of antioxidant 91

activity in pegaga drink

4.6 Regression of FRAP assay against FTC measurement of 92

antioxidant activity of pegaga drink, BHT, vitamin E and

vitamin C

4.7 Correlation coefficient of antioxidant activity (FRAP assay) 94

xiv

and total polyphenol content

4.8 The effect of citric acid on the antioxidant activity 99

(FTC assay) of pegaga drink.

4.9 The effect of citric acid on antioxidant activity (FRAP assay) 100

of pegaga drink.

4.10 The effect of total soluble solid on the antioxidant activity 102

(FRAP assay) of pegaga drink

4.11 The effect of total soluble solid on the antioxidant activity 103

of pegaga drink

4.12 The effect of sodium metabisulphite on the antioxidant 104

activity (FRAP assay) of pegaga drink

4.13 Correlation coefficient of antioxidant activity and 105

concentration of sodium metabisulphite

4.14 The effect of sodium metabisulphite on inhibition of linoleic 106

acid peroxidation of pegaga drink

4.15 HPLC-Chromatogram for standard madecassoside 108

4.16 HPLC-Chromatogram for standard asiaticoside 108

4.17 Calibration curve for madecassoside 109

4.18 Calibration curve for asiaticoside 110

4.19 HPLC-Chromatogram for madecassic acid 111

4.20 HPLC-Chromatogram for asiatic acid 112

4.21 Calibration curve for madecassis acid 112

4.22 Calibration curve for asiatic acid 113

4.23 Triterpenoid fraction (%) of pegaga extract from drink 120

samples

xvi

O2 - Superoxide radical

H2O2 - Hydrogen peroxide

OH. - Hydroxyl radical

LDL - Low debsity lipoprotein

CHO - Carbohydrate

HTST - High temperature short time

RP - Reverse phase

PPO - Polyphenol oxidase

DPPH - Radical scavenging activity

SS - Superoxide free radical scavenging activity

TBHQ - tert-butylhydroquinone

FDA - Food Drug and Administration

TBARS - Thiobarbituric acid reactive species

ORAC - Oxygen radical absorbance capacity

BCBT - -carotene bleaching test

ABTS - 2.2’, azino-bis(3-ethyl-benz-thiozoline-6-sulfonic acid)

CMC - Carboxy methylcellulose

TSS - Total soluble solid

TA - Total acidity

HCL - Hydrochloric acid

GAE - Gallic acid equivalent

TPTZ - Trypyridyl-s-triazine

UV - Ultraviolet-visible

HCL - Hydrochloric acid

Fe2SO4.7H2O - Ferum sulfate

NaOH - Sodium hydroxide

K2S04 - Pottasium sulfate

EDTA - Ethylenediamine tetra-acetic acid

DMRT - Duncan’s multiple range test

SAS - Statististical Analysis System

CIE - Commision Internationale de L’Eclairage

xv

LIST OF SYMBOLS

Rt - Retention time

L - Linearity

r2 - Correlation coefficient

L* - Colour index for lightness

a* - Colour index for redness

b* - Colour index for yellowness

ppm - part per million

rpm - rotation per minute

HPLC - High Performance Liquid Chromatography

GAE - Gallic acid equivalent (mg/100ml)

TSS - Total soluble solid

TA - Total acidity

Brix - Unit for total soluble solid

NEB - Non-enzymatic browning

RDA - Recommended Daily Allowance

TLC - Thin Layer Chromatography

FTC - Ferric Thiocyanate

FRAP - Ferric Reducing Ability of Plasma

TBA - Thiobarbituric acid

BHT - Butylated hydroxytoulene

BHA - Butylated hydroxy anisole

MRPs - Maillard Reaction Products

ESR - Electron Spin Resonance Spectroscopy

SO2 - Sodium dioxide

SD - Standard deviation

ROS - Reactive oxygen species

xvii

EGCg - epigallocatechin gallate

RE - Total vitamin A activity

B1 - Vitamin B1 (Thiamine)

B2 - Vitamin B2 (Riboflavin)

E.P - Edible portion

Vitamin C - Ascorbic acid

Ca - Calcium

Fe - Iron

Na - Sodium

K - Pottasium

xviii

LIST OF APPENDICES

APPENDIX TITLE PAGE

A1 HPLC-Chromatogram of methanol extract of triterpene acid 147

(Fresh sample and Sample A)

A2 HPLC-Chromatogram of methanol extract of triterpene acid 148

(Sample B and Sample C)

A3 HPLC-Chromatogram of methanol extract of triterpene acid 149

(Commercial sample CM1 and CM2)

B1 HPLC-Chromatogram of methanol extract of glycosides 150

(Fresh sample and Sample A)

B2 HPLC-Chromatogram of methanol extract of glycosides 151

(Sample B and Sample C)

B3 HPLC-Chromatogram of methanol extract of glycosides 152

(Commercial sample CM1 and CM2)

C HPLC-Chromatogram of water extract of triterpene acid and 153

Glycosides for fresh sample

D Calibration curve of standard FeSO4.7H20 154

E Standard calibration curve of gallic acid (GAE) 155

CHAPTER 1

INTRODUCTION

In recent year, the production and consumption of fruit and vegetable juice has

been increasing. The increased in demand is mainly because of their health benefit

(Wong, et al., 2001). Lately, attention has been given to pegaga-based products

(Faridah, 1998; Brinkhaus, et al., 2000).

Pegaga (Centella asiatica Linn.) is widely consumed as herb in different parts of

the world. Pegaga is generally used in health food and cosmetic products. This herb is

associated with wound healing agents (Vogel, et al., 1990). In Malaysia, it is commonly

consume as vegetable or ‘ulam’ and juice among the Malays and as a cooling drink by

the Chinese (Tiek, 1997; Zakaria and Mohd, 1994; Turton, 1993). The interest on herbal

beverages such as pegaga drink is because of its pharmacological activity. The

pharmacological activity is attributed to its phytochemical constituents such as

asiaticoside and antioxidant property.

Currently, several pegaga based herbal products have been developed and

marketed by Small and Medium Industries (SMI). They are marketed as herbal drink,

cosmetic products and herbal preparation in the form of capsule, tablet and powdered

products. Pegaga have also been developed into herbal confectionary.

2

The health benefit of pegaga is thought to be due to several saponin constituents

including triterpene acids (asiatic acid and madecassic acid) and their respective

glycosides (asiaticoside and madecassoside). Total triterpenoids; asiatic acid,

madecassic acid, asiaticoside and madecassoside have been shown to significantly

influence the synthesis of collagen, improve wound healing and ficronectin in human

skin fibroblasts culture (Vogel et al, 1990; Brinkhaus, et al., 2000). Pegaga extract that

contains 30 mg of triterpenic acids shows a good wound healing property (Faridah,

1998). Pegaga extract also has anti-ulcer effects especially with reference to its asiatic

acid and asiaticoside content (Cheng and Koo, 2000; Somchit, et al, 2002; Chatterjee, et

al., 1992). Asiaticoside is reported to possess strong antioxidant properties (Shukla, et

al., 1999b), act as antimicrobial (WHO, 1998) and anti-inflammatory (Chen, et al.,

1999).

Most of the phytochemical from plant extract have been identified to exhibit

antioxidant activity. A number of plant constituents have been recognized to have

positive effect against the oxygen reactive compounds in biological system (Hemeda and

Klein, 1990). There are several evidents indicated that antioxidants in diet provide

benefit for health and well-being. The reactive oxygen species (ROS), such as

superoxide radical (O2), hydrogen peroxide (H2O2) and the hydroxyl radical (OH.),

cause functional damage to man, carcinogenesis, aging and circulatory disturbances

(Tagi, 1987). The consumption of fruits and vegetables containing antioxidants has

been reported to provide protection against a wide range of degenerative diseases

including ageing, cancer, diabetes and cardiovascular diseases (Ames, 1983; Vimala and

Mohd Ilham Adenan, 1999; Caragay, 1992). Plants components contain antioxidative

properties to counteract ROS (Lu and Foo, 1995).

Antioxidants are compounds that inhibit or delay the oxidation damage in foods

and process products. It is well established that lipid peroxidation reaction is caused by

the formation of free radicals in cell and tissues. Oxidation reactions are also a concern

in food industry. They initiate and promote product deteriorations, thereby limiting the

3

shelf life of fresh and processed foods (Jadhav, et al., 1996). Antioxidants play an

important role as inhibitors of lipid peroxidation in food products snd in living cell

against oxidative damage (Vimala and Adenan, 1999; Lindsay, 1985).

Synthetic antioxiants such as butylated hydroxytoluene (BHT) and butylated

hydroxyanisole (BHA), and natural antioxidants such as tocopherol and ascorbic acid,

are widely used in food industries due to their protecting ability against oxidation-

reduction reactions (Roberto, et al., 2000). It is known that BHT and BHA retard lipid

oxidation, however, due to increasing consumer awareness of health aspect, their used is

slowly replaced by alternative antioxidants, which are without toxic effect. Recently,

there is growing interest in the used of natural antioxidant in food products. Natural

antioxidants are perceived as safe, less toxic and beneficial for human health, however it

is very expensive and not widely commercialized. Sources of natural antioxidants are

spices and herbs, and such materials have been used throughout history for flavouring

and preservative agent (Kikuzaki and Nakatani, 1993).

High concentrations of phytochemical in plant extracts are associated with strong

antioxidant activity. Ascorbic acid and phenolic compounds including vitamins,

pigments and flavonoids have been identified to be responsible for antioxidant properties

in most plants, for example anthocyanin in Roselle extract (Tsai, et al., 2002),

hydroycinnamic acid in blood orange juice (Arena, et al., 2001) and catechins in tea

extract (Kikuzaki and Nakatani, 1993). Polyphenols belong to a heterogeneous class of

compounds with great variety of effects. These compounds are reported to quench

oxygen-derived free radicals by donating a hydrogen atom or an electron to the free

radical (Yuting, et al., 1990). The antioxidant effect of polyhenols has been reported in

many in vitro studies including human low-density lipoprotein (LDL) and liposomes

(Teissedre, et al., 1996). The relationship between antioxidant activity with ascorbic

acid content and phenolic compounds has recently been discussed in many research

works (Gil-Izquierdo, et al., 2002; Arena, et al., 2001; Gil-Izquierdo, et al., 2001;

Dawes and Keene, 1999). The flovonols quercetin was identified as the antioxidant

4

property in Polygonum hydropiper, a medicinal herb (Haraguchi, et al., 1992) and onion

(Makris and Rossiter, 2001). The antioxidant activity of orange juice, pineapple juice

and many fruit juices are found to be associated with the concentration of ascorbic acid

(Gardner, et al., 2000). On the other hand, ascorbic acid is widely used as an antioxidant

in many food products, including processed fruits, vegetables, meat, fish, soft drinks and

beverages (Madhavi, et al., 1996b).

Nutritionally, pegaga contains appreciable level of asiaticoside (1-8%), -

carotene (2649 g), ascorbic acid (48.5 mg) and total phenolic (23000mg/100g)

(Brinkhaus, et al., 2000; Tee, et al., 1997; Fezah, et al., 2000). These compounds play

an important role on promoting human health through their antioxidant activity

(Velioglu, et al., 1998; Gil-Izquierdo et al., 2001; Jeniffer, et al., 1998; Gazzani, et al.,

1998). Abdul Hamid, et al. (2002), determined that various extracts from different parts

of pegaga exhibit antioxidant activity. Phenolic compounds were found out to be the

major contributor of antioxidant properties (Zainol, et al., 2003). Since quercetin and

kaempferol also appeared as part of major flavonoids components in pegaga (Radzali, et.

al., 2001; Koo and Suhaila, 2001), it is possible that these constituents may contributed

in the antioxidant capacity of pegaga drink. However, the specific phenolic components

that involves in antioxidant activity of pegaga are not clearly identified. In other study,

Shukla, et al. (1999a) investigated the role of asiaticoside as antioxidant property in

wound healing activity. Asiaticoside derived from pegaga has been attributed to

increase the antioxidant levels at an initial stage of healing. Beside, carotenoid and

ascorbate peroxidase are also present as antioxidative constituents in this herb (Yusuf, et

al., 2000). In fact, recent traditional applications indicated that a high intake of pegaga

is associated with the reduced risk of a number of chronic diseases (Brinkhaus, et al.,

2000).

Fruits and vegetable products are often subjected to heat treatments in order to

preserve their quality and prevent the microbial growth. The most important

commercial method of juice and drink preservation is pasteurization. This method is

5

based on time and temperature relationship (Moyer and Aitken, 1971). The standard

pasteurization process destroys harmful bacteria and deactivates detrimental enzymes

without adversely affecting the taste, quality and the nutritional value (Nagy and Shaw,

1970). Although, High Temperature Short Time (HTST) processing treatment or flash

pasteurization retained most quality and nutrient in processed foods, but the cost of the

equipments is high.

The traditional pasteurization processes or known as batch pasteurization often

heat the juice or drink for longer periods of time, at slower heat-up rates, using

considerably higher temperatures. Most of the vat or batch pasteurization of acidified

beverages applied at below 93 C in order to maintain the sensory quality and to reduce

the nutrient loss. For example, the mango puree heated under batch process in steam-

jacketed kettle until reaches 85 C (Luh, 1970).

The most important factor determining the minimum thermal process is the pH

of the product (Noraini, 1984). According to Pederson (1980), for highly acid drink and

juice (the pH is lower than pH 4.2) would normally be processed at 71.1 C to 100 C.

On the other hand, Chuah (1984) reported that the process of pasteurization usually

consists of a process whereby the food is heated to temperature 60-90 C either to

destroy the nonsporing pathogens or to prolong the shelf-life of the food, usually but not

conjunction with some added preservatives which prevent the spores of microorganisms

from germination. High temperature heat processes are unnecessary for acid juices

because the heated spores of spore-forming bacteria are unable to germinate at pH 4.2 or

lower (Pederson, 1980). The heat treatment of beverages held at 60 C for 10-20 minutes

is also recommended for the acidic products (Chuah, 1984). Scalzo (2004) studied the

effect of thermal treatments of blood orange juice at 80 C for 6 minutes on antioxidant

changes compared to non-thermally treated juice. After pasteurization at 80 C for 6

minutes, the inhibition DPPH (%) was reduced from 49.1% (unheated juice) to 43.2%.

The processing of pineapple and “asam jawa” drink at 85 to 90 C for 1 to 5 minutes still

6

maintained the sensorial quality of products (Che Rahani, 1998). The carrot juice heated

at 82 C for 5 minutes retained 57% of -carotene (Bao and Chang, 1994). The heating

temperature for canned fruit and vegetables beverage is depended on the microbial level

of the raw materials, the acidity of the products, the size of the can and the thermal

conductivity of the product. Canned mango puree was heated in open steam jacketed

kettle to 80 C for 10minutes. After hot-filling, the sealed cans were immersed in boiling

water for another 20 minutes (Godoy and Rodriguez-Amaya, 1987). In other processing

practice, the guava juice was heated to 87 C for 5 minutes, hot filled and sealed cans

pasteurized in boiling water for 30 minutes. (Padula and Rodriguez-Amaya, 1987). The

authors found that carotene content was maintained after heating at these processing

condition. In other report, Che Rahani (1998) recommended the heat processing of

guava drink at 82 C for 5 minutes, followed by canning and immersed in boiling water

(100 C) for another 10 minutes.

One of the issues in plant material processing is on the effect of processing

method on the phytochemical profile of the products. According to Nicoli, et al. (1999),

the health benefit of plant material is dependent on their processing methods. Food

processing procedures are generally believed to be responsible for the depletion of

natural antioxidant and at the same time it is expected to have a lower health protecting

capacity than fresh produce. Gazzani, et al., (1998) reported that processing steps

significantly influenced the antioxidant activity of plant materials. This is due to the loss

of antioxidant or the formation of compounds with pro oxidant action may lower their

antioxidant capacity. The naturally occurring antioxidant such as ascorbic acid and

phenolic compounds are generally degraded under thermal treatment (Mahanom, et al.,

1999; Makris and Rossiter, 2001; Fezah, et. al., 2000). Thermal treatment also

responsible for the reduction of antioxidant activity in processed products (Hunter and

Fletcher; 2002; Takeoka, et al., 2001). Pro oxidant compounds that formed in early

stage of Millard reactions significantly decreased the antioxidant activity (Nicoli, et al.,

1999).

7

Thermal treatments are also frequently used in the extraction of phytochemicals

substances from fruits and vegetables (Gazzani, et al., 1998). Some antioxidant

substances are well extracted during preparation of herbs extract at high temperatures.

For example, the maximum antioxidant capacity from in vitro studied is associated with

the drinking of green tea prepared at high temperatures (90 C) and with long infusion

time. However, Langley-Evans (2000) suggested that the black tea is ideally prepared

between 70-90 C with infusion times not exceeding 1-2 min for maximum antioxidant

recovery. According to Scalzo, et al., (2004), thermal treatment generally induced and

increased the extractability of the phenolic substances of orange juice, such as

anthocyanins and total cinnamates. The presence of intermediate oxidation state of

polyphenol is also reported to exert a higher antioxidant activity (Manzocco, et al.,

1998). On the other hand, alterations to the structure of existing antioxidants, as well as

the formation of novel antioxidant components may enhance the initial antioxidant status

(Gazzani, et al., 1998; Nicoli, et al., 1997b; Nicoli, et al., 1999). Heat treatment

accelerates the oxidation reactions responsible for the formation of compounds with pro

oxidant properties and compounds having antioxidant activity. Example of such

reaction is Maillard reaction products. The brown-coloured Maillard reaction products

formed in advanced stage of non-enzymatic browning reaction have clearly shown to

improve antioxidant activity in vitro. Complex relations between these variables are

generally obtained in multicomponent and in formulated foods (Manzocco, et al., 2000).

Thus, the heat processing treatment could caused negative effect as well as enhanced

their antioxidant activities on the herbalproducts.

The antioxidant potential of herbs dependent on many factors involved in it

preparations. The right choice of processing parameters of herbal products may help to

retain their phytochemicals content. In most cases, temperature control, minimizing

oxygen content and protection from light can help to ensure maximum retention of

antioxiants (Lindley, 1998). On the other hand, the eventual processing damage can be

minimized by the addition or enrichment of the product with natural antioxidants and/or

reconstituted with secondary antioxidants. According to Lindley (1998), the addition of

8

free radical chain breakers ( -tocopherol), reducing agents and oxygen scavengers

(ascorbic acid), chelating agents (citric acid) and ‘secondary’ antioxidant (carotenoids)

may be able to stabilize and prevented oxidation damage in fruits and vegetables.

Pokorny (2000) reported that modification of a recipe during preparation of food and

ready meals improved the stability against oxidation especially with the addition of

spices. Recent studies also indicated that the addition of sulphur dioxide (S02) or

sodium metabisulphite and vitamin C during processing of commercial food products

balanced the depletion of natural antioxidant (Tsai, et al., 2002; Majchrzak, et al., 2004).

The presence of metabisulphite has been demonstrated to control the spoilage and

promote the retention of the natural antioxidant. Sulphites were successfully used to

prevent the non-enzymatic browning in food and vegetables (Sapers, 1993), reduction in

decoloration of pigments, changes in texture and loss of nutritional quality (Lindley,

1998). Other food additives such as citric acid generally enhanced the antioxidant

activity via synergist effect with natural antioxidant like -tocopherol. Citric acid was

also used as metal chelators to inhibit oxidative reactions (Madhavi, et al., 1996). Citric

acid is widely used as acidulant and preservatives in food system. The high levels of

total soluble solid usually help to stabilize or reduce the deterioration rate of food

products. For example, high sugar concentrations are effectively to protect the

degradation of anthocyanin (Wrolstad, et al., 1990), the strong antioxidant compound in

Roselle (Tsai, et al., 2002) and berry fruits (Skrede, et al., 2000). The effect of sugar

concentration is most likely due to lower in water activity (Skede and Wrolstad, 2002).

The impact of food processing and handling on nutrients such as vitamins and

minerals are well established. However, the stability and the fate of phytochemicals in

processed food have not been investigated to similar extent. It is always believe that

phytochemical from pegaga are depleted by processing, particularly where thermal

treatments are employed. The level of antioxidant activity and the presence of

significant concentration of triterpene glycoside in pegaga are of interest to the herbal

industry. However, the effect of processing parameters on both antioxidant activity and

triterpene glycoside contents of products from pegaga is yet to be investigated

9

thoroughly. Since triterpene glycosides such as madecassoside, asiaticoside, madecassic

acid and asiatic acid have been reported to contribute to the pharmacological activities, it

is important to study the effect of processing treatment of pegaga on the fate of these

components.

1.1 Objective

The main objective of the study was to investigate the effect of heat processing

on the antioxidant activity and triterpene glycosides content of herbal pegaga drink

1.2 Scope

In order to achieve the objective, the scopes of the study are identified as

follows:

1. The herbal pegaga drink was prepared under three different heat

processing conditions; 65 C/15 minutes (A), 80 C/5minutes (B) and

canned process (heat at 80 C/5minutes followed by canning and boiling

at 100 C/10 minutes (C)). The unheated pegaga drink known as fresh

sample (F) and two commercial samples, CM1with no thermal treatment

and CM2, which heat processed at 90 C for 1 minutes were used as

comparison. All pegaga drink samples (F, A, B, C, CM1 and CM2) were

used for further assessment.

2. The physico-chemical characteristics of pegaga drink samples (F, A,B, C,

CM1 and CM2) including pH, total acidity, total soluble solid, colour,

proximate analysis, total polyphenol and ascorbic acid content was

10

studied. These assessments provide the basic data or information of

characteristics of sample studied.

3. The level of antioxidant activity in pegaga drinks prepared under

different heat processing conditions was assessed using two antioxidant

assays namely Ferric thiocyanate (FTC) method and Ferric reducing

ability of plasm (FRAP) methods.

4. The effect of addition of sodium metabisulphite and citric acid, and total

soluble solid of fresh herbal pegaga drink on antioxidant activities were

evaluated. The contribution of total polyphenol and ascorbic acid on

antioxidant activity was also evaluated.

5. The concentration of four components of triterpene glycosides in pegaga

drinks; including asiatic acid, madecassic asid, asiaticoside and

madecassoside were examined. The contribution of asiaticoside on

antioxidant activity of herbal pegaga drinks was also evaluated.

11

CHAPTER 2

LITERATURE REVIEW

2.1 Medicinal plants in Malaysia

Recently, there has been a worldwide interest towards the application of natural

products in the health care. There are 80% of world’s populations who are dependent on

the natural products for health care (Muhammad Idris, et.al, 1999).

In Peninsular Malaysia, there are about 1,230 plant species with medicinal value

have been recorded (Latif, 1983). In 2002 alone, Ministry of Health received about

22,493 applications for registration of herbal medicinal products. There are 10,758 of

traditional medicinal products that were registered until december 2002 and 146

premises were licensed (MOH, 2002).

Malaysian herbal remedies such as tongkat ali putih (Eurycoma longifolia),

pegaga (Centella asiatica), hempedu bumi (Andographis paniculata) and limau purut

(Citrus hystrix) have been recognised to provide health benefit. Their beneficial active

components have a potential to be developed into commercial products (Mohamad

Faisal, 2000).

12

2.2 Herbal Products in Food Industries

The global market for herbal products is estimated to be worth US$80 billion in

2000, and is expected to increase to US$200 billion in 2008 and US$5 trillion in 2050.

It is estimated that from about RM2 billion Malaysian herbal markets in 1999, only RM

100 million was locally produced while the reminder was imported (Business time,

2000). The herbal/natural product industry is considered to be one of the most dynamic

sectors with annual growth estimated at 20% a year (Mohamad Faisal, 2000).

Generally, Small and Medium Industries (SMI’s) contributed most of the

production of herbal food products in the market, however, they are low in technical

know how and managerial skills. Herbal food products attract a lot of interest from food

producer due to the changes in market trends. To date, no data has been reported on the

market value of herbal food products in Malaysia.

2.3 Plant Material (Centella asiatica)

2.3.1 Plant Description

Pegaga or Centella asiatica (L.) Urban is a genus of the plant family Apiaceae

(Umbelliferare). Medicinal herb, which has a mildly bitter taste also commonly known

as Hydrocotyle asiatica L., Indian Pennywort or Hydrocotyle asiatique in france (Ling,

et. al., 2000). Other names of pegaga include ‘Luci Gong Gen’ or ‘Tung Chain’ in

China, ‘Vallarai’ for tamil nadu in India and ‘Daun Kaki Kuda’ in Indonesia (Perry,

1980; Goh, et al., 1995).

13

Pegaga can be found easily in moist habitats or wet swampy area through out

India, Malaysia, Madagascar, China, Southern United State Amerika and Middle Africa

(Brinkhaus, et al., 1996; WHO, 1998; Perry, 1980).

Pegaga is a perennial creeping plant with cup-shaped of leaves, glabrous stems

and rooting at nodes. The leaves are thin, soft and green in colour. The whole plant

including leaves, stem and root are consumed as ‘ulam’ and therapeutic agents

(Brinkhaus, 2000; Indu Bala & Ng, 2000).

Plate 2.1: Pegaga (Centella asiatica)

2.3.2 Medicinal Applications

Pegaga is used for medicinal purposes since prehistoric time (Kartnig, 1988) and

it is used to treat a wide range of indications especially against gastrointestinal diseases,

gastric ulcer, indigestion, gastritis and inflammantory diseases of the liver (Brinkhaus,

2000; WHO, 1998).

14

Pegaga based products are available in the form of powder, infusions, soluble

and extract of fresh and dried plant, in both conventional and homeopathic preparation.

It is also prepared in the form of ointments and creams (Brinkhaus, 2000). Madecassol

(asiaticoside) in tablet, ointment and powdered form was used as anti-inflammatory

(Chen, et al., 1999) and autoimmune (Guseva, et al., 1998). In terms of cosmetic

application, it is used to promote skin regeneration and stimulate biosynthesis of

collagen through the formation of lipids and proteins. Pegaga extract is reported to be

effective on scar treatment (Faridah, 1998; Brinkhaus, et al., 2000).

2.3.3 Bioactive constituents in pegaga

The chemical constituents of pegaga are classified into main groups including

essential oil, flavone derivatives, triterpenic steroids, triterpenic acids and triterpenic

acid sugar ester or saponin (Brainkhaus, et al., 2000). Pegaga also contains various

important constituents for clinical and pharmaceutical uses (Bonte, et.al., 1994).

Chemicals that were previously investigated from pegaga are brahmic acid,

brahminoside, brahmoside, centellic acid, centelloside, hydrocotyline, 3-

glucosylkaempferol, 3-glucosyl-quercetin, indocentelloside, isobrahmic acid,

isothankunic acid, isothankuniside, madasiatic acid, madecassol, meso-inositol,

oxyasiaticoside, thankunic acid, vallerine; alkaloid, fatty acids, flavonols, polyphenols,

saponins, sterols, sugars, tannins, terpenoids, triterpenes (Goh, et al., 1995). Asiatic

acid, asiaticoside, madecossoside and madecassic acid are the biologically active

constituents in pegaga that have a potential to be promoted as commercial product (Indu

Bala and Ng, 2000).

15

2.4 Nutrient composition

Food is composed of several groups of constituents including carbohydrate,

protein, fat, inorganic mineral components and organic substances present in very small

amount. The organic components generally functions as flavour, pigments, enzymes,

emulsifier, acids, oxidant and antioxidants.

Epidemiological studies indicated that diets rich in fruit and vegetables are

associated with a low risk of several degenerative diseases. It has a potential to maintain

human health and prevent chronic diseases (Hunter & Fletcher, 2002). Nutritional

issues also highlight the relationship between diet and chronic diseases such as obesity,

heart disease, and cancer, especially with the high intake of fat (Zielinski, et al., 2001).

However, according to Nicoli, et al. (1999), the health-promoting capacity in fruits and

vegetables depends on its processing technology. Theoretically, processed fruits and

vegetables are expected to have a lower health benefit level then the fresh one.

The nutrients in foods required a balanced amount to promote and maintain

optimum health. They should consist of a broad group of carbohydrates, proteins, fats,

vitamins and minerals (Potter, 1986). Quantitatively, starch is the most important

carbohydrate in the human diet. It represents the primary energy source, contributing to

nearly 60–70% of the total energy consumed, of which nearly 75% of the starch is

derived from cereals, and pulses (Asp, 1995). Carbohydrate, protein and fat can be

oxidized to furnish energy. Dietary fibre display a wide range of physiological and

nutritional effects important to human nutrition and health. Dietary fibre improved the

diabetic problem (Monnier, et al., 1978), reduced risk of colorectal cancer (Klurfeld,

1992) and increased the digestion in gastrointestinal tract (Potter, 1999). There are also

increasing evidence that mental processes and behavioral attitudes are influenced by

nutritional status and specific nutrients. Beside the major components, some of organic

16

components present in small proportions functions as antioxidant, flavors, pigments,

acids, oxidants, emulsifiers and enzymes.

One of the major quality acceptances of foods is its content of vitamins and

minerals. The quantitative need for vitamins and minerals varies among the individuals.

The U.S Recommended Daily Allowance (RDA) of vitamin C, phosphorus, iron, zinc

and magnesium for adult is 60mg, 800-1200mg, 18mg, 15mg and 300mg, respectively.

Some of essential mineral may provide benefits for the body through their efficiency, as

miscellaneous antioxidant. Zinc and Selenium are function as an antioxidant. Zinc, one

of the essential nutrients, strongly inhibits lipid peroxidation, which is possibly due to

altering or preventing iron binding. Selenium generally used for the synthesis and

activity of glutathione peroxidase, a primary cellular antioxidant enzyme (Madhavi and

Salunkhe, 1996). It is also has a potential of protecting biomembrane, eradicating free

particles, enhancing immunity and inhibiting cancer (Zhiang Min, et al., 1983).

2.4.1 Nutrient composition of pegaga

Nutrient composition also plays an important role to promote health. Tee, et al.,

1988 presented a quantitative evaluation of proximate and nutrient composition of fresh

pegaga. Pegaga contained high potassium, calcium and phosphorus levels that

accounted for 391 mg, 171mg and 32 mg per 100g, respectively. Pegaga is not a good

source of protein, carbohydrate and fat. -carotene and ascorbic acid, known to have

antioxidative activities, are present at appreciable concentration (2649 g and 48.5mg,

respectively) in fresh pegaga. -carotene and carotenoids can act as antioxidant and are

effective quenchers in singlet oxygen. In terms of mechanism, they are preventing the

formation of hydroperoxides (Rajalakshmi and Narasimhan, 1996). Besides the more

popular phytochemical constituents in pegaga, these particular compounds also

17

contributed to the positive health. The nutrition composition in pegaga is shown in table

2.1.

Table 2.1: Nutritional composition of pegaga

%

E.P

Nutrient composition of edible portions (E.P),

per 100g sample

Proximate composition*

KclEnergy

gWater

gProtein

gFat

gCHO

gFiber

gAsh

Indian 37 87.7 2.0 0.2 6.7 1.6 1.8

Pennywort (pegaga);

44 Vitamin**

Hydrocotyleasiatica

gRetinol

gCarotene

gRE

mg B1

mg B2

mg Niacin

mg C

0 2649 442 0.09 0.19 0.1 48.5

Mineral**

mg Ca

mg P

mg Fe

mg Na

mg K - -

171 32 5.6 21 391

Sources: * Nutrient Composition of Malaysian Foods (Tee et.al., 1997)

** Nutrient Composition of Malaysian Foods (Tee et.al., 1988)

Note: RE (Total Vitamin A activity) is expressed as retinol equivalents and

calculated as g retinol + ( g carotene/6)

18

2 .5 Triterpene Glycosides

(Asiaticoside, Medacosside, Asiatic acid and Madecassic acid)

The bioactive constituent of therapeutic interest in pegaga is pentacyclic

triterpenoid group known as asiaticoside (De Lucia, et al., 1997) or saponin -containing

triterpene acids and their sugar esters, the most important being: asiatic acid, madecassic

acid and the three asiaticosides, asiaticoside, asiaticoside A and asiaticoside B (Sing and

Rastogi, 1969; Brinkhaus, et al., 2000). Pegaga contains not less than 2% triterpene

ester glycosides, asiaticoside and madecassoside (Kartnig, 1988). Asiaticoside and

asiatic acid were also reported to be found naturally in Schefflera octophylla (Sung,

et.al. 1992).

2.5.1 Chemical structure of triterpene glycosides

The chemical structure of triterpene glycoside is shown in figure 2.1. Glycosides

are compounds containing a carbohydrate and non-carbohydrate residue in the same

molecule. An acetal linkage at carbon atom 1 to a non-carbohydrate residue or aglycone

attaches the carbohydrate residue. In terms of chemical structure, the aglycone was

classified into several group including saponin, flavonol, phenol, tannins and lactone

group. Saponin glycosides are divided into 2 types according to chemical structure of

aglycone. The acid saponins possess triterpenoid structures as shown in figure 2.2.

Madecassic acid and asiatic acid are classified under miscellaneous triterpenoids,

whereas asiaticoside fall in a group of triglycoside (Jeffery, et al., 1999).

19

R4 R5

R3

HO

HO OR2

HOH2C R1

Saponins R1 R2 R3 R4 R5Asiatic acid -H -H -CH3 -CH3 H

Asiaticoside -H - -D-glc-(6-1)- -D-glc- (4-1)--L-rha

-CH3 -CH3 H

Madecassic acid -OH -H -CH3 -CH3 H

Madecassoside -OH - -D-glc-(6-1)- -D-glc- (4-1)--L-rha

-CH3 -CH3 H

Figure 2.1: Structure of triterpene glycoside: asiatic acid, asiaticoside, madecassic acid,

and madecassoside (Brinkhaus, et al., 2000)

Saponin

Glycone Aglycone

Sugar Sapogenin

Neutral saponins Acid saponins

Steroids Triterpenoids

Figure 2.2: The group of saponin glycosides (Duke, 1992)

20

2.5.2 Health-promoting effect of triterpene glycosides

From clinical point of view, there are numerous evidences on the effectiveness of

pegaga to alleviate diseases (Brinkhaus, et al., 2000). Asiaticoside is reported to have

positive effect to treat leprosy (Boiteau and Ratsimamanga, 1956). In fact, it is also used

as anti-inflammatory (Newall, et al., 1996), antimicrobial activity (WHO, 1998) and

antioxidant (Shukla, et al., 1999). The total triterpenoid fractions including asiaticoside,

asiatic acid, madecassoside and madecassic acid significantly influence the biosynthesis

collagen and improved the human skin problems (Indu Bala & Ng, 2000). Standardized

extracts of pegaga containing up to 100% total triterpenoids about 60mg once or twice a

day, are frequently used and suggested in modern herbal medicine (Murry, 1995; WHO,

1999). For example, in double-blind study, Pointel, et al. (1997) investigated the effect

of pegaga extract administrated at a dose of 60 mg/day and 120 mg/day to 94 patients

with chronic venous insufficiency. At both doses, significant improvements in affected

veins were observed.

2.5.3 Antioxidative activity of triterpene glycosides.

Among four triterpene glycosides derived from pegaga, only asiaticoside was

reported to have antioxidant activity. Asiaticoside is observed to improve healing of

surface wound. . Asiaticoside application (0.2%) twice daily for 7 days to wounds in rats

significantly increased the level of enzymatic and non-enzymatic antioxidants such as

superoxide dismutase, catalase, glutathione peroxidase, vitamin E and ascorbic acid

(Shukla, et al., 1999b). At lower concentrations (0.05% and 0.1%) asiaticoside were

found to have no significant effect on wound healing activity.

21

2.5.4 Methods for Assessing Triterpene Glycosides

To date, no studies have been done regarding the influence of food processing of

pegaga based products on it active ingredients especially their triterpene glycoside

content. Recently, the observation and determination of phytochemicals in pegaga only

focuses on pharmaceutical and cosmetic aspects (Shukla, et al., 1999b; Sairam, et al.,

2001; Sampson, et al., 2001; Morganti, et al., 1999).

The amount of triterpene acid and the glycoside of pegaga were previously

estimated by using titration method. Determination of asiaticoside and related triterpene

ester glycosides in pegaga and other plant extract were also done by thin-layer

chromatography (Meng and Zheng, 1988) and spectroscopic analysis (Castellani, et al.,

1981). TLC profile of triterpenoids distribution in pegaga was previously demonstrated

with the Rf values for madecassoside, asiaticoside, madecassic acid and asiatic acid was

28.7, 37.1, 91.6 and 93.7, respectively (Ling, et al., 2000). However, these methods are

non-selective, non-specific, lack of precision and accuracy (Inamdar, et al., 1996).

Thus, several methods have been developed to achieve the efficient result.

2.5.4.1 Extraction

Methanol and aqueous methanol effectively used for the extraction of triterpene

glycosides (Ling, et al, 2000; Inamdar, et. al., 1996). The extraction of asiaticoside is

efficient in methanol with the amount of 0.36% dry weight compared to chloroform

(0.30%), ethyl acetate (0.3%) and water (0.04%) (Verma, et. al., 1999).

22

2.5.4.2 HPLC Analysis

The determination of triterpene glycoside content in products containing pegaga

extract such as tablet and oinment (Inamdar, et al., 1996), and cosmetic product such as

anti-cellulite (Morganti, et al., 1999) has been studied. Due to the large difference in

polarity of the triterpene acids and their glycoside, a linear gradient is used to get the

separation under a single run. The efficiency of determination is up to 98.1% (Inamdar,

et al., 1996). Octadecylsilated silica column with wavelength of 220 nm is used to

detect the separation. Gunther and Wagner, 1996 also develop new HPLC method for

isolation and determination of triterpene. The detection is done using the reversed-phase

(RP) separation system with wavelength of 205 nm. A combination solvent of

acetonitrile and water is used on RP column. In other investigation, combination of

water (0.1%TFA), acetonitrile (0.1%TFA) and methyl tert-butyl ether (0.1%TFA) as

gradient mobile phase were applied using Phenomenex Aqua 5mu C18 (Schanebberg, et

al., 2003).

The quantitative determination of triterpene saponin and aglycone extract from

pegaga plant, which is used for treatment of cellulitis, is widely reported in many

studies. Phytochemical analysis is performed using reversed-phase high performance

liquid chromatography (HPLC) coupled with photodiode array detector at 200nm

(Morganti, et al., 1999; Burnouf-Radosevich and Delfel, 1996). The phosphoric acid

solution at 0.3% and acetonitrile has been used for efficient separation.

2.6 Ascorbic acid

Ascorbic acid is present in high amounts in fruit and vegetables, especially citrus

fruits. Ascorbic acid (figure 2.3) is well known as nutrient antioxidant and is important

for the maintenance of health and protection from coronary diseases and certain cancers

23

(Diplock, 1994). Ascorbic acid, in vitro, protects some flavonoids, such as antocyanins,

against oxidative degradation during processing and storage of juice (Kaack and Austed,

1998). The presence of ascorbic acid in processed food is considered as indicator for the

quality of product due to its relative instability to heat, oxygen and light (Birch, et al.,

1974). Ascorbic acid (Vitamin C) is usually added to fruit drinks, canned fruits and

vegetables with a headspace of air. It is increased the acidity of foods and prevent the

growth of aerobic bacteria. It is also widely fortified as an antioxidant or nutrient

supplement in many food products including processed fruits, vegetables, meat, fish,

dairy products, soft drink, and beverages. According to Food Act 1983 and Food

Regulation 1985, the maximum amount of L-ascorbic acid added as antioxidant in

canned food for infant and children are 0.05g per 100g. However, the amount of

2000mg/kg of ascorbic acid is permitted to be added in coconut cream and edible oil as

antioxidant.

HO OH

H

=O

H-C-OH

CH2OH

Figure 2.3: Structure of ascorbic acid (Madhavi, et al., 1996)

Ascorbic acid is a highly soluble compound that has both acidic and strong

reducing properties. At the same time, it is highly sensitive to various modes of

degradation including temperature, salt, sugar concentration, pH, oxygen, enzymes,

metal catalyst and initial concentration of ascorbic acid (Tannenbaum, et al., 1985).

24

Ascorbic acid also can be degraded by active oxygen and by reaction initiated by

transition metals. It removes oxygen in systems where oxygen is present in limited

amounts and gets oxidized to dehydroscobic acid (Jadhav, et al., 1996). Ascorbic acid is

easily destroyed through oxidation, especially at high temperature, and the amount

generally declined during food processing, storage and cooking. Sulfur dioxide

treatment can also affect the ascorbic acid losses during processing, as well as during

storage (Bolin and Stafford, 1974).

2.6.1 The contribution of ascorbic acid in antioxidant activity

Fruits like guava and apples, and vegetables such as kale, broccoli and asparagus

are valuable sources of ascorbic acid. According to Gardner, et al., (2000), ascorbic acid

was found as major contributor of antioxidant activity of fruits including orange (66%),

florida orange (100%) and grapefruit (89%). According to Majchrzak, et al. (2004) the

addition of lemon contains ascorbic acid on tea drink can positively influence the

antioxidant potential. The total antioxidant capacity in green tea extract increased

through the addition of ascorbic acid up to 30 mg/100ml of tea solution. Ascorbic acid

is also the major antioxidant in orange juice accounted about 87% of total antioxidant

activity (Miller, et al., 1997).

The addition of ascorbic acid to foods helps to maintain the antioxidant status

through their action as reducing agents and oxygen scavengers, which is to prevent

oxidation of oxygen-sensitive food constituents (Lindley, 1998). Ascorbic acid has also

the ability to regenerate phenolic or fat-soluble antioxidants, to act synergistically with

chelating agents, and or to reduce undesirable oxidation products such as enzymatic

browning (Madhavi, et al., 1996b). In fat and oils, ascorbic acid functions

synergistically with phenolic antioxidant such as BHA and propyl gallate (PG), and the

tocopherols in retarding oxidation. This nutrient antioxidant react directly with oxygen

25

to form dehydroascorbic acid and thus depletes the supply of oxygen available to effect

autoxidation (Jadhav, et al., 1996). Ascorbic acid can act as inhibitor of polyphenol

oxidase (PPO) activity due to a lowering rate of pH (Lindsay, 1985). In sliced fruits and

vegetables, the used of ascorbic acid is highly effective in preventing browning that

generally occurred due to the oxidation of phenolic compounds by PPO resulting in the

formation of orthoquinones.

2.7 Polyphenol

Polyphenols are group of chemical substances represented by more than one

phenolic groups. The structure of natural polyphenols varies from simple molecules,

such as phenolic acid, to highly-polymerized compounds, such as condensed tannins.

Phenolic compounds include the hydrocynnamic acid, which contains caffeic and ferulic

acid and the flavanoids and their glycosides (including flavones, isoflavones,

flavonones, anthocyanins, catechin, isocatechin). Flavonoids such as kaempherol,

quercetin, luteolin, and mycertin are low molecular weight polyphenolic compounds that

are widely distributed in vegetables and fruits.(Hertog, et al., 1992). In other

investigations, Bors and Saran (1987) reported that many flavonoids such as

kaempherol, quercetin, luteolin, mycertin, eridictyol, and catechin have been shown to

have antioxidant activities.

The biologically active phenolic compounds containing one or more aromatic

rings are found naturally in plant foods, where they provide much of flavour, colour and

texture. Phenolic compositions are also observed to be responsible in taste such as

bitterness and astringency (Lea and Arnold, 1978).

26

2.7.1 Phenolic compounds in pegaga

Total polyphenol was determined in all parts of pegaga including leaves, stem

and root and it shows the highest in the leaves for about 0.23 g/mg dried methanol

extract. The high concentration of polyphenol is thought to be responsible for the anti-

inflammatory activity in pegaga (Fezah, et. al., 2000). Zainol et al., (2003) studied the

amount of total polyphenol in four accessions of pegaga extract. The concentration of

total polyphenol varied from 3.23g to 11.7g per 100g of dry sample. They also

suggested that phenolic compounds are the major contributors to the antioxidative

activities of pegaga.

Flavonoid component including apigenin, kaempferol, quercetin and rutin have

been detected in different parts of pegaga by using Thin Layer Chromatography (TLC).

The yield of apigenin was found to be the highest followed by quercetin, kaempferol and

rutin (Radzali, et.al., 2001). However, only quercetin (423.5mg/kg dry weight) and

kaempferol (20.5mg/kg) was observed in pegaga using HPLC assay (Koo and Suhaila

Mohamed, 2001). The potential of dietary flavonoids has recently created an interest

among scientist for treating many diseases (Piskula and Terao, 1998). Flavanoid from

pegaga is used to assist strong, lustrous and healthy hair growth (Faridah, 1998).

2.7.2 The contribution of phenolic compounds in antioxidant activity

Flavonoids are widely occurring groups of secondary metabolites in plants. The

antioxidant activity of the flavonoids has been reported in a few experiments. Catechins

were found as major antioxidant in tea extract (Kikuzaki and Nakatani, 1993), phenolic

diterpenes (carnosic acid) in sage (Cuvelier, et al., 1994), proanthocyanins in grapes and

blackcurrants, and anthocyanins in Roselle extract (Tsai, et al., 2002). Flavonoids

function as primary antioxidants, chelators and superoxide anion scavengers

27

(Rajalakshmi and Narasimhan, 1996) and it has much stronger antioxidant activities

against peroxy radicals than vitamin E, vitamin C and glutathione (Cao, et al., 1996).

The quercetin was identified as the antioxidant property in Polygonum hydropiper, a

medicinal herb (Haraguchi, et al., 1992) and onion (Makris and Rossiter, 2001). This

compound has been effective in inhibiting copper-catalyzed oxidation.

The mechanism reaction of phenolic antioxidant is associated with their ability to

donate hydrogen atoms to free radicals. Epidemiological studies have shown that

consuming foods and beverages rich in phenolic content is related with reduced

incidences of heart disease (Muhammad Idris, et al., 1999). Phenolic antioxidant,

particularly flavanoids, exhibit a wide range of biological effects including anti-

inflammatory, anti bacterial, anti viral as well as anti allergic (Cook and Samman, 1996).

Phenolic compounds also have antioxidant activity in vivo. For example, the health

aspects of rooibos tea are mainly linked to its phenolic content and associated

antioxidant activity (Niwa and Miyachi, 1986). Donovan, et al., 1998 indicated that

polyphenol-containing fruits are potent inhibitors of the in vitro oxidation of low-density

lipoproteins (LDL). The protection of LDL by phenolic acids in a copper-induced

oxidation system could be due to both metal chelating and radical scavenging action. In

addition, the mechanisms of protecting effect on LDL by phenolic compounds are

through scavenging of various radical species in the aqueous phase, interaction with

peroxy radicals at the LDL surface and partitioning into the LDL particle and

terminating chain-reactions of lipid peroxidation by scavenging lipid radicals

(Laranjinha, et al., 1994).

Improvement of the antioxidant properties of naturally occurring antioxidants

seems to be related to the presence of polyphenols, the antioxidant properties of which

may change as a consequence of their oxidation state. Polyphenols with an intermediate

oxidation state can exhibit higher radical scavenging effect than the non-oxidized one

(Nicoli, et al., 1999). In terms of the mechanism, phenolic compounds is classed as

proper antioxidants or hydroperoxide stabilizer since it is able to inactivate the lipid free

28

radicals as well as prevent the decomposition of hydroperoxides into free radical

(Pokorny, 2001b). Several studies have also been made concerning relationship between

the phenolic structure and antioxidant activity (Kikuzaki and Nakatani, 1993).

2.8 Antioxidant Activity

The activity of antioxidant depends on their chemical reactivities towards peroxy

and other active species. The activity also changes according to many other factors such

as concentration of antioxidant, type of substrate, physical state of system, as well as the

number of microcomponents acting as pro-oxidants or synergists (Yanishlieva-

Maslarova, 2001).

Generally, antioxidant is defined as compounds that inhibit or delay the

oxidation of other molecules by inhibiting the initiation or propagation of oxidizing

chain reactions. Antioxidant is also called as oxidation inhibitor (Pokorny, et al., 2001b).

It is well established that lipid peroxidation is set in motion as a consequence of the

formation of free radicals in cells and tissue. Antioxidant plays an important role in its

ability against oxidation-reduction in lipids, natural pigments and other active chemicals

(Anese and Nicoli, 2001). In some processed foods, antioxidants are used to prolong the

shelf life as well as maintain the nutritional quality of lipid-containing foods.

The classifications of food antioxidants are shown in Table 2.2. Antioxidant can

also be divided into two categories namely the synthetic and natural antioxidant

(Hudson, 1990; Larson, 1988). Natural antioxidants are used because of their presumed

safety and potential nutritional and therapeutic effects (Heinonen, et al., 1998).

Recently, natural antioxidant extract from rosemary and sage is marketed in the form of

antioxidant additive or food supplement (Schuler, 1990). Synthetic antioxidants such as

butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) are widely used

29

as food preservative. BHT and BHA, in the group of primary antioxidants, terminate the

free-radical chain reaction by donating hydrogen or electrons to free radicals and

converting them to more stable products. However it is now been reported to be

dangerous for human health (Barlow, 1990; Ruberto, et al., 2000). Thus, the interest in

natural antioxidants has increased considerably (Lolinger, 1991).

Table 2.2: Classification of food antioxidant

Food Antioxidant Group / Mechanism of action Compounds

Phenols Gallates, Hydroquinone

Primary antioxidant ‘Hindered’ Phenols BHT, BHA, TBHQ

Miscellaneous Primary

Antioxidant

Trolox-C, Anoxomer,

Ethoxyquin

Oxygen Scavengers Sulfites, Ascorbic acid,

Ascorbyl palmitate

Secondary / Synergistic

Antioxidant

Chelating Agents EDTA, Polyphosphate,

Citric acid, Lecithin,

Tartaric acid

Secondary antioxidant Thiodipropionic acid,

Distearyl ester

Miscellaneous antioxidants Amino acid, Spices

extract, Flavonoids,

Vitamin A, -carotene,

Tea extract

Sources: Rajalakshmi and Narasimhan (1996).

30

2.8.1 Antioxidant activity in herbs

In traditional application, tea, herbs, fruit, vegetables and spices have been

widely used as major source of antioxidant (Cao, et.al.,1996; Rajalakshmi and

Narasimhan , 1996; Madsen & Bertelsen, 1995; Velioglu, 1998; Wang, et.al., 1996).

Most of tropical herbs are rich with antioxidant activities, for example Morinda

citrifolia, cucuma longa, zingiber officinale and lemon grass. There are wide range of

components identified as antioxidant compound in herbs. Several studies have been

made concerning relationships between the antioxidant activity and curcumin in C.

longa (Ruby, et al., 1995), carnosic acid in sage and rosemary (Cavelier, et al., 1994),

quercetin in Polygonum hydropiper (Haraguchi, et al., 1992), catechin in tea herb

(Wang, et al., 2000), vitamin E in green-leafy vegetables (Mallet, et al., 1994), total

polyphenol in Chrysanthemum morifolium and Hordeum vulgare (Duh and Yen 1997),

flavonoid (Makris dan Rossiter, 2001; Catarina, et al., 1999) and anthocyanin in roselle

(Tsai, et al., 2002). Phenolic components also appear to be major contributors to the

antioxidant potential of tea herbs and non-citrus juice (Wang, et al., 2000; Miller et al.,

1997).

2.8.2 Antioxidant activity of pegaga

Pegaga is well known to have a high antioxidant activity (Abdul Hamid, et al.,

2001). It has been established that the presence of polyphenol in pegaga extract is

contribute its antioxidative efficiency activity with the correlation of r2=0.9 (Zainol, et

al., 2003). The specific component of phenolic that contributed to antioxidant activity in

this herb is not reported clearly. Vimala, et al., (2003) reported that pegaga leaves were

found to have very high antioxidant activity in three different pathway including

superoxide free radical scavenging activity (86.4%), inhibition of linoleic acid

peroxidation (98.2%) and radical scavenging activity, DPPH (92.7%). The consumption

31

of pegaga was useful to protect the cells from oxidative damage, to destroy excess free

radicals and keep the oxidative stress state in balance. Shukla, et al., 1999 investigated

the role of asiaticoside as antioxidant properties in wound healing activity. Asiaticoside

derived from pegaga has been attributed to increase the antioxidant levels at an initial

stage of healing. Yusuf, et al., (2000) also observed the antioxidative axtivities of

carotenoid and ascorbate peroxidase in herb pegaga. The characteristics of antioxidant

activity in pegaga were previously studied. Pegaga exhibited optimum antioxidant

activity at neutral pH and the activity remained stable up to 50 C. The antioxidative

activities of pegaga extracts increased when concentration was increased from 1000 to

5000ppm(Abdul Hamid, et al., 2001).

2.8.3 The Role of Synergistic or Secondary Antioxidants

Synergistic antioxidant generally classified as chelators and oxygen scavenger.

Chelator such as citric acid is basically used as acidulant and stabilizer in some food

products. It is first suggested for the stabilization of edible oil where it acts as a

synergist of tocopherols. In food industries, it is commonly added in foods and

beverages (e.g. fruit and vegetable juice or drink) in order to produce sour taste as well

as lowering the pH in such products. In terms of antioxidant activity, citric acid

provides an acidic medium that improves the stability of primary antioxidants. It is also

act as metal-chelating agent in some food systems (Lindsay, 1985).

Heavy metals such as iron (Fe) and copper (Cu) are strong important promoter of

lipid oxidation as they catalyse the decomposition of lipid hydroperoxides into free

radicals. Chelating heavy metal, by chelating agents such as citric acid and EDTA, into

inactive complexes improved the stability of fats, oils and food lipids (Pokorny, 2001b).

32

2.8.3.1 Effect of citric acid

Chelators like citric acid and phosphate are not antioxidant, but highly effective

as synergists with both primary antioxidants and oxygen scavengers. For example, the

addition of citric acid generally enhances the activity of primary antioxidant such as

BHT and TBHQ, and the combination is used in vegetables oils, shortenings and animal

fats. The application of 0.02% citric acid with TBHQ is effective in the improvement of

oxidative stability of olive oil from 7 to 12 hours. In further investigation, addition of

citric acid was found to increase the stability to 58 hours (Sherwin, 1990). Mixtures of

citric acid and erythrobic acid are used to retard the browning of bananas. Santerre, et

al. (1988) reported that application of citric acid can prevent browning of sliced apple

and, thus, extend shelf life. Besides, the combination of citric acid with oxygen

scavenger such as ascorbic acid exhibited more beneficial effects (Pizzocaro, et al.,

1993). Citric acid prevents discoloration of some fruits and vegetables such as canned

pear, sliced beets, onions and potatoes. In meat products, the combination of citric acid

with BHA and phenolic antioxidants generally applied to increased stability, retarding

oxidative rancidity and preserved the flavour (Madhavi, et al., 1996b).

2.8.3.2 Effect of sulphites

Sulphites are weak antioxidant and are known as oxygen scavenger. Its also have

been used for food preservatives in commercial food production. Currently, the forms

employed include SO2 gas, and the sodium or potassium salt sulphite, bisulphite or

metabisuphite. It is most effective as an antimicrobial agent in acid media, which is

optimum from below pH 3.0. Generally, the production of brown pigments by enzyme

and catalyzed oxidation of phenolic compounds can lead to a various quality problem

during the handling of some fresh fruits and vegetables. However, the use of sulphite or

metabisulphite sprays or dips with or without added citric acid provides effective control

33

of enzymatic browning in prepeeled or presliced fruits and vegetables (Lindsay, 1985).

Sulphites are effective in preventing enzymatic browning and preserve freshness in raw

packaged and unpackaged fruits, vegetables, fruit juices and beer. According to Taylor,

et al. (1986), the mechanism protection of sulphites may involve several reactions

including directly inhibit the enzymes, interact with intermediates of reaction or act as

reducing agents promoting the formation of phenols from quinones. In the prevention of

non-enzymatic browning, sulphites react with carbonyl intermediates and preventing

their participation in reactions leading to the formation of brown pigments (Madhavi, et

al., 1996b). Sulphites functions as an antioxidant in variety of food products. For

example, the addition of sulphur dioxide (SO2) in dried apple cubes was contributed to

inhibit the development of enzymatic oxidation of phenols during the drying process.

SO2 has an ability to retain most of the original antioxidant activity (Manzocco, et al.,

1998). In other cases, the high antioxidant activity was also observed in the commercial

wine and juice sample, partially due to the presence of vitamin C or preservative such as

metabisulphite (Tsai, et al., 2002). The used of sulphites, however, associated with

asthma in some individuals. Since 1986, Food Drug Administration (FDA) banned the

use of synthetic preservative from sulphites in raw packaged or unpackaged fruits and

vegetables because of some adverse reactions reported in sulphites-sensitive individuals.

2.8.4 Effect of enzymatic oxidation on antioxidant activity

Chemical and enzymatic oxidations are the main caused of the reducing of

polyphenol antioxidant properties. Green tea was found to have higher phenol and

chain-breaking activity than those observed in black tea (Manzocco, et al., 1998; Yen

and Chen, 1995). The enzymatic oxidation of polyphenols during processing of black

tea was reduced the antioxidant properties. However, polyphenols with an intermediate

oxidation state have a higher radical scavenging efficiency than the non-oxidized

polyphenol. For example, antioxidant properties are increased and higher in semi

fermented tea as compared to fermented tea and non-fermented tea (Yen and Chen,

34

1995). Pokorny (1987) reported that oxidation of polyphenols leads to the formation of

stable intermediates or macromolecular compounds, which can still maintain strong

antioxidant activity. The chain-breaking efficiency during processing of beverages is

also attributed to the increased stability of partially oxidized polyphenols (Manzocco, et

al., 1998).

2.8.5 Effect of concentration and sugar content

Sugar is widely added in processed food partially to increase the product stability

via lowering the water activity (aw). Addition of sugar also increased the concentration

of products and generally measured by total soluble solid. It has long been recognized

that a relationships exists between water activity and concentration of food products.

Wrolstad, et al., 1990 reported that the concentration of sugar over 20% is preventing

the loss of anthocyanins. Jackman and Smith (1996) also found that the amount of

similar antioxidant compound is considered to be degrading at lower sugar level.

According to Takeoka, et al. (2001), the loss of antioxidant property in tomatoes such

lycopene content is increased at 25-30 Brix of total soluble solid. The longer processing

time required achieving the desired final solid levels also associated with increased

losses of lycopene. The enzymatic and/or chemical oxidation rate of phenolic

compounds are associated with some intrinsic food variables such as water activity (aw)

and it processing condition (Nicoli, et al., 1999). Wrolstad (2000) reported that the

stability of anthocyanin was increases with the decreased water content or with the

decreasing water activity.

35

2.8.6 Mechanism of antioxidant activity

Antioxidants help to prevent the occurrence of oxidative damage to biological

macromolecules caused by reactive oxygen species (Lindley, 1998). Reactive oxygen

species (ROS) are constantly generated in vivo, both by “accidents of chemistry” and for

specific purposes (Wang, et al., 1996). Active oxygen forms superoxide, hydrogen

peroxide (H2O2) and hydroxy radicals (OH) is a by- product of normal metabolism and

attacks biological molecules, leading to cell or tissue injury. Active oxygen and free

radicals are produced by certain chemical carcinogens and play a role in carcinogenic

process (Cerutti, 1985). The higher antioxidant properties of certain compounds are

related to their increased ability to donate a hydrogen atom to free radicals.

Antioxidants reduce the primary radicals to non-radical chemical species and are thus

converted to oxidize antioxidant (Gordon, 2001).

The mechanism protection by phenolic antioxidants as peroxy radical scavenger

is more effectives during the propagation stage of oxidation. It is prevent the formation

of hydroperoxides, so that it can stop the chain reactions and provide a longer shelf life

of the foods.

According to the chemical structure, antioxidant activities could be categorized

into four types including free radical chain breakers such as tocopherol, reducing agents

and oxygen scavengers such as ascorbic acid, chelating agents likes citric acid and other

secondary antioxidant such as carotenoids (Lindley, 1999).

Primary antioxidants, for example phenolic compounds react with peroxyl

radicals and unsaturated lipid molecules and converted them to more stable products.

Whereas, secondary antioxidant or preventive are compounds that retard the rate of

chain initiation by various mechanism. This antioxidant reduce the rate of autoxidation

of lipids by such processes as binding metal ions, scavenging oxygen and decomposing

36

hydroperoxides to non radical products (Gordon, 1990). Secondary or synergistic may

function as electron or hydrogen donors to primary antioxidant radicals, thereby

regenerating the primary antioxidant. Chelating agents remove prooxidant metals and

preventing metal catalyzed oxidations. The oxygen scavenger such as ascorbic acid is

able to scavenge oxygen and prevent oxidation of foods, regenerate phenolic or fat-

soluble antioxidant, maintain sulfhydryl groups in –SH form and act synergistically with

chelating agents (Madhavi, et al., 1996b).

The mechanism of Millard reaction products (MRPs) as antioxidant properties is

not clearly observed. In such cases, MRPs can act as metal ion chelators, which is bind

heavy metals into inactive compounds (Pokorny, et. al., 2001). Metal chelating is an

example of a secondary antioxidant mechanism by which many natural antioxidants can

influence the oxidation process. Metal chelators can stabilize the oxide forms of metals

that are reduced redox potential, thus preventing metals from promoting oxidation (Hall,

2001).

2.8.7 Assessment of antioxidant activity

Herbs and other natural products contain many hundreds compound of natural

antioxidant. Therefore, several methods have been developed to quantify these

compounds individually. The techniques are different in term of mechanism of reaction,

effectiveness and sensitivity (Khal dan Hildrbrant, 1986; Frankel, 1993; Koleva, et al,

2002). Methods that are widely used to measure the antioxidant activity level in herbal

sample, fruits and vegetables, and their products are thiobarbituric acid reactive species

(TBARS) (Roberto, et al, 2000), oxygen radical absorbance capacity (ORAC) (Tsai, et

al. 2002; Wang, et al, 1996; Zheng and Wang, 2001), -carotene bleaching test (BCBT)

(Markin dan Rossiter, 2001; Gazzani, et al., 1998), ABTS radical-cation (Arena, et al,

37

2000; Miller, et al, 1995), DPPH titration (Imark, et al, 2000), Folin-Ciocalteu

(Donovan, et al, 1998) as well as FTC and FRAP.

2.8.7.1 Ferric Reducing Ability of Plasma (FRAP)

Ferric Reducing Ability of Plasma (FRAP) is a novel method for assessing

antioxidant power through their reduction of ferric to ferrous ion at low pH. The

combination or complex of ferrous-tripyridyltriazine is caused to formation of blue

colour and it is detected at wavelength of 593 nm (Benzie and Strain, 1996).

The antioxidant activity has been detected on fresh plasma. Besides, this method

also applied on beverages such as roselle (Tsai, et al., 2002) and vegetable sample

(Hunter and Fletcher, 2002). This assay offers a putative index of antioxidant defense of

potential used to. It is simple assay and gives a highly reproducible result over a wide

range of studies. The FRAP assay is inexpensive, reagents are simple to prepare, and

the procedure is straightforward and speedy (Benzie and Strain, 1996). Furthermore,

this method also gives a linear response over a large concentration range and can be

made applicable to both water- and lipid-soluble components (Hunter and Flatcher,

2002).

2.8.7.2 Ferric thiocynate (FTC)

Ferric thiocynate (FTC) method has widely been used to determine the

antioxidant activity on essential oil and oleoresin (Kikuzaki and Nakatani, 1993 ; Yumi

Yuhanis, 2002), and plants extract (Yen and Chen,1995; Mohd Zin, et al., 2001). The

FTC method is used to measure the amount of peroxide in initial stages of lipid

38

oxidation. During the oxidation process peroxide is gradually decomposed to lower

molecular compounds. Linoleic acid acts as the substrate in ethanol-phosphate buffer

solution while the present of antioxidant compounds in sample are delayed oxidation of

linoleic acid and exhibited the antioxidative activity (Kikuzaki and Nakatani, 1993).

Briefly, the assay evaluates the inhibitory activity of the sample against lipid

peroxidation (oxidation of fatty acids) caused by hydrogen peroxides. The absorbance

of the red colour developed is measured at 500nm.

Zainol, et al., (2003) studied the correlation between two different methods

namely FTC and TBA. Results from both methods showed different pattern that

probably due to several factors including the different mechanisms involved and

structures of the different phenolic compounds.

2.9 Heat Processing of Food and Beverages

The main concern of the food industry in thermal processing is to prevent the

growth of bacterial pathogens. The quality and the uniformity of beverages will largely

depend on the degree of control during the heat process, because over-processing may

lead to undesirable changes in flavour, texture and nutritive value. Conversely, under-

processing, which may not destroy all the organisms, leads to spoilage and is a potential

health-hazard. It is therefore important that suitable heat processing schedules be

obtained, taking into consideration the effect of the water activity, pH and thermal

conductivity of the product (Desrosier and Desrosier, 1977). Blanching, dehydration,

sterilization and pasteurization are an example of thermal treatment that commonly

practice in food industry (Pokorny, 2001).

39

The pasteurization process is firstly established for the preservation of milk.

They are two types of pasteurization procedures namely batch or vat pasteurization and

flash pasteurization. Flash pasteurization is used to designate High Temperature Short

Time treatment or HTST (Potter, 1986). Recently, the flash pasteurization at 88 C for 1

minutes, 100 C for 12 seconds and 121 C for 2 seconds are common practice in fruit

juice industry, where the bacterial destruction effect is very nearly equivalents

(Veldhuis, 1971). HTST practice is effectively retained the flavour and nutritional value

of juices. However, the short holding time required special equipment, which is more

expensive than the batch process. The flash pasteurization of milk at 71.1 C for 15 sec

is equivalent in bacterial destruction to batch method at 62.8 C for 30 minutes (Potter,

1986).

The time and temperature relationship of pasteurization process required the

knowledge D and Z value for the destruction of the target organisms. The D value is a

measure of heat resistance of a microorganism. It is the time in minutes at a given

temperature required to destroy 1 log cycle (90%) of the target microorganism. The Z

value reflects the temperature dependence of the reaction. It is defined as the

temperature change required to change the D value by a factor of 10 (Potter, 1986;

Desrosier and Desrosier, 1977). The times and temperatures however vary according to

the heat sensitivities of the foods and the effects of the different foods on microorganism

survival (Noraini, 1984; Potter, 1986). For example, a study done by Mazzota (2001)

has resulted in a recommended general thermal process of 3 seconds at 71.1 C for

achieving a 5-log reduction for E. coli, Salmonella and Listeria monocytogenes in apple

juices with the pH adjusted to pH of 3.9. However, a study done by Mak, et al. (2001)

has shown that treatments of 68.1 C for 14 seconds and 71.1 C for 6 seconds are

capable of achieving a 5-log reduction of acid adapted E. coli in apple cider (pH 3.3).

The heat resistance of the microorganisms and their spores is affected by a

number of factors which include; 1) Age and previous history of the organisms or

spores, 2) composition of the medium in which the organisms or spores are grown,

40

heated and recovered, 3) pH and water activity of heating medium 4) heating

temperatures and 5) initial concentration of organisms and spores (Chuah, 1984). As

shown in Figure 2.2, Desrosier and Desrosier (1977) were reported the effect of pH on

heating temperature and the time required to kill the heat resistance of spores. Thermal

processes for low acid foods are designed to in activate the spore of Clostridium

botulinum. Low acid foods usually processed in steam under pressure at temperatures of

116 C or 121 C and sometime in steam at temperatures of about 140 C (Noraini, 1984).

Several low acid beverages are acidified with organic acid, such as citric acid and malic

acid, to reduce their pH to less than 4.6 so that pasteurization processes may be used.

However the addition of acid promotes the formation of coagulation of suspended solids.

Thus, thickening agents such as methyl cellulose have been used to increase stability via

holding the solids in suspension (Pederson, 1986).

In pasteurization certain acid juices, the industry formerly used treatments at

63 C for 30 minutes (Potter, 1986). The high acid (below pH 4.2) beverages could also

be process at the temperature as low as 60 C for about 10-20 minutes (Chuah, 1984).

According to Pederson (1980), the heating treatment at the temperature as low as 71.1 C

is high enough to kill the vegetative bacteria, since the spore-forming bacteria are unable

to germinate at pH 4.2 or lower. In this type of beverages, the preservatives such as

benzoic acid, sodium metabisulphite and sobic acid, sometime is added (Moyer and

Aitken, 1971).

41

100

KILLINGTIME pH 5 to pH 7 (min)

10

pH 4.5 1.0 pH 3.5

0.1 99 110 121

TEMPERATURE ( C)

Figure 2.4: Influence of pH of heating medium on heat resistance of spores

(Desrosier and Desrosier, 1977).

Depending on the types of beverages, the pasteurization process is applied at

different combination of time and temperatures. For example, the pineapple and “asam

jawa” drink was prepared at 85 to 90 C for 1 to 5 minutes (Che Rahani, 1998), but the

orange juice was thermally treated at slightly low temperature, 80 C for 6 minutes

(Scalzo, 2004). For the heat processing of guava drink and carrot juice, the

pasteurization at 82 C for 5 minutes was recommended (Bao and Chang, 1994). In

other study, the mango juice was processed by 4 different methods by Muhammed, et al.

(1965). Of their processing methods, the one employing the least heat (pasteurize at

87.8 C for 1 minutes) gave the best quality.

The pasteurized of acid juices and drinks may be filled into plastic bottles, glass

bottles or into cans. Previously pasteurized or sterilized beverages are hot filled between

78-93 C and held in this temperature for 1-3 minutes in containers before cooling

(Noraini, 1984). In other practices, the unheated juice was put in glass bottles, which

were then crowned and pasteurized at 77-82.2 C for 20 to 30 minutes (Pederson, et al.,

1980). According to Mehrlich and Felton (1971), the pasteurization of canned pineapple

42

juice may be handled according to either of two alternatives. The first alternative

procedure for handling the juice is pasteurized the product to approximately 90 C. The

cans are filled with the juice at this temperature and held for 1-3 minutes. In the second

alternative, the juice was first pasteurized at 60 C, filled into cans and the can sealed

was then boiled for certain time according to the size of the can. In canning process of

some juices and drinks, the products are commonly heat-treated at the temperature of 80

to 87 C for 1-10 minutes, filled into cans, sealed and immersed in boiling water in the

range of 10 to 30 minutes (Godoy and Rodriguez-Amaya, 1987; Padula and Rodriguez-

Amaya, 1987; Che Rahani, 1998). According to Luh (1980), the mango juice should be

heat processed at 87.8 C, followed by filling, sealing in processed in water bath.

Processing in the boiling water sterilizes the inner surfaces of the can and lids and

prevents contamination of the product from those surfaces. Although canning processes

result in the losses of sensorial and nutritional quality attributes, the processes are still

widely used, and could be optimized to improve quality retention regarding the specific

of any particular commodity.

2.9.1 The retention of nutrient and phytochemical during processing of foods

Exposure of food components to temperature above ambient condition during

heat processing) is a major cause of detectable changes, no only on nutritional quality,

but also of phytochemical contents (Pokorny, 2001b). Phytochemical is a food

components that are derived from natural occurring ingredients and are actively being

investigated for their health-promoting potential (Bloch and Thomson, 1995). The

phytochemicals and/or health preserving elements are present in number of frequently

consumed foods, especially fruits, vegetables, legumes and seeds, and in less frequenly

consumed foods such as green tea and herbs. It is usually identified as antioxidant

properties, which are responsible to prevent various diseases (Hunter and Fletcher, 2002;

Velioglu, et al., 1998; Zheng and Wang; 2001; Mahanom et al., 1999). Phytochemicals

are divided into different classes including polyphenols, terpenoids, glucosinolates,

43

organic acids, fibres and minerals (Dillard and German, 2000). The subject of

phytochemistry deals with the chemical structures of the substances, their biosynthesis,

turnover and metabolism, their natural distribution and their biological function

(Harborne, 1998).

It is very important to preserve the phytochemical during processing of foods

because of their in vitro and in vivo functions such as towards the antioxidant activity,

anticancer activity, antimicrobial activity etc. Although most of the phytochemical

amounts are generally reduced after heat treatment, there is no evidence that processing

had any detrimental effect on the nutritional status of the population (Bender, 1987).

Surprisingly, the bioavailability and the human uptake are found to be higher in

thermally processed food. For example, lycopene serum concentration is higher by

consuming heat-processed tomato-based food rather than after the consumption of

unprocessed fresh tomatoes (Gartner, et al., 1997). Hussein and El-Tohamy (1990)

suggested that cooking practices is able to increase bioavailability by physically

disrupting or softening plant cell walls.

Deterioration of foods, subjected to chemical, physical and biological changes, is

always influenced their organoleptic properties, nutritional value, safety and health

benefits. The loss of phytochemicals depends upon many parameters during food

processing and storage. Heat, cold, light and other radiation, oxygen, moisture, dryness,

natural food enzymes and microrganism, all can adversely affects the quality and

wholesomeness of foods (Potter, 1986). Some foods are heated to drive off moisture,

develop flavors as during the roasting of coffee, and to inactive natural toxic substances.

These processing techniques often result in loss of nutritional quality, and in some cases,

in losses of their resistance against lipid oxidation. The phytochemical retention of food

products during processing procedures and storage are really depends on the nature of

raw materials such as their content and oxidation state (Anese and Nicoli, 2001).

According to Tannenbaum, et al. (1985), the optimization of nutrient retention in foods

can be achieved through (1) High Temperature Short Time (HTST) processing

44

combined with aseptic canning and (2) prediction of vitamin losses in storage, which is

required information of the nutrient content of a processed food at various time during

distribution. Low storage temperatures, low oxygen contents and protect the product

from light in storage are also suggested to increase the retention of these compounds

(Shi, et al., 2002).

2.9.2 Effect of food processing on nutrient composition

Carbohydrate can be hydrolyzed under different conditions such as pH,

temperature and anomeric configuration or structure of the material. For example, -D-

glycosides hydrolyzed less rapidly than -D-glycosides. During processing, the starch

molecule undergoes several physical modifications depending on the type of contained

starch and severity of the conditions employed (Goni, 1996) leading to the formation of

resistant starch (RS) that escapes digestion and absorption in the intestine (Annison and

Topping, 1994). Oxidation or degradation of lipid and protein leads to the development

of off-flavour, rancidity, softening, loss of solubility and loss of nutritive value (Cheftel,

et al., 1985).

The effect of storage and food processing on nutrients particularly in vitamins

and minerals are well known. The Arrhenius activation energy (Ea) for ascorbic acid

degradation in canned peas at 110-132 C was calculated to be 41 kcal/mol (Lathrop and

Leung, 1980). The amount of potassium, sodium and phosphorus in spinach were

reduced by 56%, 43% and 36%, respectively after blanching treatment (Bengtsson,

1969). Similarly, Meiners, et al. (1976) reported that cooking process of navy bean

caused to the decrease the amount of iron, zinc, magnesium and phosphorus in the range

of 50-65%. Extrusion of cereal at high temperatures caused a significant decreased the

amount of biologically active compounds including tocopherols, reduced glutathione,

45

melatonin, as well as trace elements such as Cu, Zn, Mn and Se (Zielinski, et al., 2001).

According to Min. et al., (2004), the loss of total selenium content caused by blanching

treatment is greater than the effect of sterilization. The application of moderate

temperatures, up to 100 C, reduces the negative changes of nutritional quality (Pokorny,

2001).

2.9.3 Effect of heat processing on natural antioxidant

There are many evidences found that industrially processed food and home

prepared significantly change the natural antioxidant. This is based on fact that most of

chemical constituents in food are unstable (Erdman Jr, 1979; Hurt, 1979). Few studies

on the phytochemicals retention including natural antioxidant of processed foods have

been published. The stability of ascorbic acid and some phenolic compound during

processing of foods and beverages are discussed as follows.

Ascorbic acid level in foodstuffs depends not only on the raw material

composition but also on the processing method employed (Marin, et al., 2002). There

are many studies for determining the ascorbic contents under different processing

parameters and storage conditions (Kabasakalis, et al., 2000; Hunter and Fletcher, 2000;

Franworth, et al., 2001; Wong, et al., 2000). The amount of this particular

phytochemical is significantly destroyed in canned peas, pasteurized pineapple and

orange juice as well as processed roselle juice (Lathrop and Leung, 1980; Akinyele,

et.al., 1990; Wong, et al., 2001). Lea (1992) reported that, fresh apple contain up to 100

ppm of vitamin C, but during processing into juice it is rapidly lost. The loss of ascorbic

acid was also found to be highest in medicinal plants dried at 50 C for 9 hour (75.60%)

compared to freeze drying (21.13%) (Mahanom, et al.,1999). Mild (75 C for 30 sec)

and standard pasteurization (95 C for 30 sec) slightly increased the total vitamin C of

orange juice from 143.5 mg to 160.5 and 131.2 to 155.7 mg, respectively, probably due

46

to the contribution from the solid parts (pulp) as a consequence of heat treatment (Gil-

Izquierdo, et al., 2002).

Several studies on the effect of home and industrial processing on polyphenol

stability have been carried out (Gil-Izquierdo, et al., 2001; Mannzocco, et al., 1998;

Takeoka, et al., 2000; Wang, et al., 2000). Their influence on total polyphenol is

dependent on the types of processing employed and the stability of individual phenolic

compounds. In fact, the concentration of phenolic compounds is not necessarily reduced

as a consequence of heat processing. For example, Spanos, et al. (1990) reported that a

high temperature during initial processing of apple juice produced up to a 5-fold

increased in amount of phloretin glucosides as compared to that obtained in pressed

juice without temperature elevation. The concentration of anthocyanins of pasteurized

(80 C/1minutes) blood orange juice was higher than non-thermally treated juice (Scalzo,

et al., 2004). They also reported that a higher antioxidant capacity of thermally treated

juice can be ascribed to the extraction, during processing, of antioxidant compounds,

such as free and bound hydrocinnamic acids and anthocyanins. After drying at 75 C and

storage for 15 weeks at 40 C, 85% of the total phenolic in Roselle extract remained

(Tsai, et al., 2002).

Many studies, however, indicated that most food processing procedures

significantly reduced the concentration of phenolic compounds. Boilling for 60 min

caused overall flavonol losses of 20.6% and 43.9% in onions and asparagus, respectively

(Makris and Rossiter, 2001). The level of polyphenol content in air-dried sample of

pegaga is also lower than the fresh sample. The difference is expected due to oxido-

reduction of polyphenol compound during processing and storage (Fezah, et. al., 2000).

During extraction process, phenolic compounds are usually sensitive to acidic solution

and high temperature. According to Julkunen-Garcia (1997), drying at below 50 C

yields the highest amount of total phenolics in the sample. Increasing the temperature

above 60 C, however, lowered the phenolic compounds considerably. The negative

effect of thermal treatments on some phenolic antioxidant is also widely reported in a

47

few experiments. The greater loss of total lycopene (35%), major carotenoid pigment

and antioxidant in tomato, was reported when the temperature was increased from 90 to

150 C. The duration of heating below 100 C, however, had little or no effect on the

degradation of lycopene (Shi and Le Maguer, 1999). Thermal processing of tomatoes

into paste partly decreased the concentration of lycopene of 9-28% and it is believed to

be due to longer processing time required to achieve the desired final solid levels

(Takeoka, et al., 2001). Heat is also observed as one of the most destructive factors of

anthocyanins in berry fruit juices (Jackman, et al., 1987). The degradation of

anthocyanin is increased from 30% to 60% after 60 days storage when storage

temperatures were increased from 10 C to 23 C (Cabrita, et al., 2000). Wang, et al.

(2000) also reported that after heat processing and 12 days of storage about 86% of

epigallocatechin gallate, 79% of epigallocatechin and 57% of epicatechin in green tea

extract were lost. Again, carotenoid content in 8 medicinal plants is loss by 27% and

20% after oven drying at 50 C for 9 hours and 70 C for 5 hours (Mahanom, et al.,

1999). The fate of most phytochemicals in processed food products are also notably

influenced by storage conditions. Storage of concentrates of apple juice for 9 months

resulted in 50-60% loss of quercetin and phloretin derivatives (Spanos, et. al., 1990).

In other investigation, blanching and boiling treatment significantly affected the

amount of quercetin and kaempferol in onion. The amount of quercetin in onion was

reduced from 41mg/100g in fresh to 25mg/100g after steam blanching and 22mg/100g

after boiling for 3 minutes. Since quercetin previously has been reported to be heat

stable, the great loss of these compounds may occur during pre-processing like peeling,

chopping and trimming (Ewald, et al., 1999). Similarly, chopping significantly reduced

the amount of rutin in asparagus up to 18.5% (Makris and Rossiter, 2001).

48

2.9.4 Effect of heat processing on antioxidant activity

The change in antioxidant activity, particularly during thermal processing, is

mainly due to the loss of naturally occurring antioxidant properties, the presence of very

heat stable natural antioxidant, the presence of polyphenols and formation of novel

compounds having pro-oxidant and antioxidant activity (Nicoli, et al., 1999). Thus, the

antioxidative activities processed foods can be loss, remain stable or unchanged and

even enhanced.

During processing of fruits and vegetables, several oxidation-reduction may

occurred in which electron is removed from atom or molecule and presence as oxidized

form. It is known that, this particular reaction is susceptible to colour such as browning,

flavour, odor, texture and nutritional change if processed and stored in high temperature.

For example, during pasteurization, the colour deterioration in fruit juice is mainly due

to enzymatic browning of polyphenolic, catalysed by polyphenoloxidases in the

presence of dissolved oxygen (Pokorny, 2001b). Generally, the physico-chemical

change was indicated through the degradation of vitamins and essential fatty acid

(Dziezak, 1986). Antioxidant activity depends on many factors such as lipid

composition, antioxidant concentration, temperature, oxygen pressure and presence of

other antioxidant and many common food components, for example, protein and water

(Pokorny, et al., 2001b). Oxidation reaction has a deleterious effect on antioxidant

activity. The oxidation level is influenced by temperature, light, air and physico-

chemical as well as the presence of catalyst (Frankel and Meyer, 2000). Heating and a

high oxygen pressure cause an acceleration of the chain initiation and propagation of the

oxidation process, and hence a decrease in the oxidation stability, or in the activity of the

present antioxidant (Yanishlieva-Maslarove, 2001).

Hunter and Flatcher (2002) investigated the antioxidant activity, total polyphenol

and ascorbic acid content in peas and spinach during microwave heating, boiling

treatment for 3 minutes and boiling treatment for 8 minutes (overcooked). The ABTS

49

and FRAP method is used in their assessment. The also studied the antioxidant activity

of peas and spinach at frozen storage and after blanching treatment (97 C for 85 seconds

and 97 C for 90 seconds, respectively). Blanching treatment is found to be useful to

prevent the enzymatic oxidation that usually responsible to the loss of natural

components in raw material or plants (Nicoli, et.al., 1999). However, after blanching of

peas and spinach the level of their antioxidant activity is reduced for about 50% and

20%, respectively, subjected to ABTS assay. The antioxidant activity remained constant

and stable at frozen storage. Boiling peas (100 C for 8 minutes) caused losses in water-

soluble antioxidant activity and ascorbate content of 34% and 61% respectively (Hunter

and Platcher, 2000). The reduction of antioxidant activities in pegaga extract at 70-

90 C is also may associated with the loss of naturally occurring antioxidant (Abdul

Hamid, et al., 2002). Gil-Izquierdo, et al. (2002) studied the effect of pasteurization at

75 C and 95 C on antioxidant activity towards the DPPH method. The antioxidant

activity equivalent to mg L-Ascorbic acid of orange juice increased from 126.8 mg

(before pasteurization) to 135.3 mg after pasteurization at 75 C. However, the activity

was decreased from 150.1 mg to 143.7 mg after standard pasteurization at 95 C for 30

sec.

Processing treatment sometimes did not affect or caused insignificant change to

the content and activity of naturally occurring antioxidant. As previously observed,

carotenoids content such as lycopene and –carotene, are very heat stable even after

prolonged heat treatments (Elkin, 1979; Miki & Akatsu, 1971).

2.9.4.1 Development of pro-oxidant during heat processing

The loss of antioxidant activity in food products not only associated with the

degradation of natural antioxidant but also due to the formation of compounds with pro

oxidant properties. Pro-oxidant generally appeared in early stages of non-enzymatic

50

browning (Nicoli, et al., 1999). Gazzani, et al. (1998) investigated the effect of thermal

treatment at 2, 25 and 102 C for 10, 20 and 30 minutes on antioxidant activity of

vegetable juice based on -carotene bleaching test. When prepared at 2 C for 10

minutes, most vegetables juice showed initial pro-oxidant activity. The pro-oxidant

activity was very high in eggplant (-307%), tomato (-621%) and yellow bell pepper (-

432%).

2.9.4.2 Development of heat-induced antioxidant

Food processing may also result in the formation of antioxidant compounds such

as Millard reaction products (MRPs) (Madhavi, et al., 1996b). These particular

compounds having antioxidant activity that influenced the antioxidant properties of

food. Formation of advance MRPs during prolonged heating time and storage generally

exhibited strong antioxidant properties (Eichner, 1981; Nicoli, et al., 1999). Millard

reaction products were identified to be active as oxidation inhibitors in tomato puree

(Nicoli, et al., 1997b). The development of non-enzymatic browning reactions, as

occurs in the production of Marsala-type wine, resulted in a great increase in its chain-

breaking activity (Monzocco, et al., 1999b).

The development of non-enzymatic browning (NEB) in foodstuffs is caused

positive and negative impact to the food industry and consumers. For example, it is

important for some types of food processing (baking, cocoa and coffee roasting) but

often has negative impact due to changes in sensorial aspects (colour and aroma) in

other food products such as fruit juice (Carabasa-Giribet and Ibarz-Ribas, 2000).

Browning due to thermal treatments are the results of several reactions. Non-enzymatic

browning reactions between amino acids and reducing sugars are the basis of the Millard

reaction, which usually appears during thermal process (Whistler and Daniel, 1985).

Beside, the reactions are included caramellisation, ascorbic acid browning process

51

(Cornwell and Wostad, 1981) and pigment destruction (Beveridge, et al., 1986). The

rate of NEB is depends on water activiy, pH, temperature and chemical composition of

the food system (Whistler and Daniel, 1985; Potter, 1986). The brown colour is

developed in c. asiatica drink during heat processing but it is not clear which reactions

are involved to enhance NEB. The influenced of NEB to antioxidant capacity is already

discussed in a few papers (Manzocco, et al., 2000; Nicoli, et al., 1999; Morales and

Jimenez-Perez, 2001; Manzocco, et al., 1999). Although the concentration of natural

antioxidant is significantly reduces as a result of thermal treatments, the overall

antioxidant properties of process products are maintained by the development of NEB

such Millard reactions (Nicoli, et al., 1997b). They also described the correlation

between the developments of Millard reaction products with relative antioxidant activity.

The correlation of relative antioxidant activity with heating time and heating temperature

is shown as figure 2.5.

Gazzani, et al. (1998) reported that heat treatment of carrot juice, cauliflower

juice and zucchini juice at 102 C for 10 minutes exhibited higher antioxidant activity.

The antioxidant activity (based on -carotene bleaching test) also increased with the

increasing of heating time and heating temperature. For example, the antioxidant

activity of carrot juice at 25 C for 10 minutes was 24% and it was increased to 75% after

30 minutes of heating. They suggested that pro-oxidant activity, which is due to

peroxidases, are inactivated at high temperature. Wang, et al. (1996), have observed

that commercial tomato and grape juice had much higher antioxidant activity than fresh

materials but the reason for the increase in antioxidant as a consequence of food

processing was not evaluated.

52

Heating time

Rel

ativ

e an

tioxi

dant

act

ivity

T1

T2

T3

3

4

5

6

7

8

9

10

11

12

Figure 2.5: Changes in overall antioxidant activity due to development of different

stages Millard reaction at different temperatures: T3>T2>T1 (Nicoli, et al., 1999)

The antioxidant activities of polyphenol-containing food can be improved

depends on it processing condition such as aw, pH, time and temperature, and oxygen

availability (Nicoli, et al., 1999; Kikugava, et al., 1990). In some cases, food processing

is resulted to increase resistance against oxidation through the transformation of

glycosides to active compound such as aglycones, inhibition of oxygen access and

formation of novel compounds (Pokorny, 2001b).

2.10 Effect of heat processing on triterpene glycosides

Although the study of the effect of food processing on phytochemical content has

been employed by a number of investigators, no data has been documented on the fate of

triterpene glycosides. However, the stock solution of asiaticoside was found to be stable

under refrigeration with the percentage was remained at 99.2% after 90 days of storage

53

(Qi, et al., 2000). The effect of heat was previously observed in other saponin

components. According to Lau, et al., (2003), the notoginsenoside R1, ginsenoside Rg1,

Re, Rb1, Rc and Rd, saponins components in Panax notoginseng was degraded after

exposure at high temprature during steaming process. The amount was significantly

declined upon prolong steaming duration.

54

CHAPTER 3

MATERIAL AND METHODS

This chapter presents the material and methods used for the overall experiments.

This work was aimed at investigating the antioxidant activity and the fate of triterpene

glycosides content of herbal pegaga drink as affected by heat treatment. The physico-

chemical characteristics of pegaga drinks were also observed. The information obtained

from the study could be used as a guideline for designing thermal processes to reduce

the phytochemical degradation of the products. Besides, the factors that may contribute

to the antioxidant activity in unheated pegaga drink were also studied.

3.1 Introduction

Thermal treatment is generally applied to extend shelf life of fruit and vegetable

products. However, heating processes can affect the nutrient and phytochemical loss,

which leads to consumer dissatisfaction. In this study, the three different heat

processing treatment applied on pegaga drink were 65 C/15 minutes, 80 C/5 minutes

and in canning process (heat at 80 C/5minutes, canned and followed by boiling at

100 C/10 minutes before cooling process). The heat processing parameters were based

on pasteurization methods of acidified foods (Chuah, 1984; Scalzo, et al., 2004; Che

Rahani, 1998). The canning process of herbal pegaga drink was followed the procedures

55

of high acid canned beverages as previously done on fruit and vegetable juice. (Godoy

and Rodriguez-Amaya, 1987; Luh, 1980; Che Rahani, 1998).

Traditionally, pegaga juice was prepared by blending the whole parts of pegaga

with certain amount of water, before it is consumed fresh as cooling drink. In this study,

the untreated drink, known as fresh sample, was used in order to compare the status of

antioxidant activity and triterpene glycosides content before heating and without

addition of any food additives and food ingredients.

Recently the demand of herbal pegaga drink by the consumers is on the increase

mostly due to the health benefit and the phytochemical presence in the drinks.

Therefore, the current status of nutrient content, antioxidant activity and active

constituents in commercial pegaga drink available in the market are important to be

studied. The results obtained are useful as a reference for consumers and researchers.

The factors influence to the antioxidant activity was also investigated. The used

of citric acid (Dziezak, 1986; Sherwin, 1990) and sodium metabisulphite (Tsai, et al.,

2000) is reported to increase the antioxidant activity in several food products. However,

the effect of these food additives and total soluble solid via sugar addition on antioxidant

activity of pegaga drink is still unclear. The range of citric and total soluble solid used

in this study was based on consumer acceptances as previously reported in many

research works (Pederson, 1980; Lea, 1991; Henrix, 1995), while the range of sodium

metabisulphite was followed the level permitted in Malaysian Food Act 1983 and Food

Regulation1985. The study on effect of addition of citric acid (0-0.3%w/v), sodium

metabisulphite (0-350ppm) and sugar (in the range of 1 to 15 Brix) on antioxidant

activity of fresh pegaga drink was carried out. Citric acid addition varied in accordance

with acidities of raw materials and consumer acceptance. Citric acid in the range of

0.1%-0.3%w/v are usually added into fruit and vegetable juices to increase the acidity

for the flavour and preservative purposes (Pederson, 1980). Vegetable juice acidified

56

with 0.4%w/v citric acid was too sour. According to Malaysian Food Act 1983 and

Food Regulation1985, the maximum level of sodium metabisulphite permitted in fruit

and vegetable drinks is about 350 part per million (ppm). Therefore, the effect of

sodium metabisulphite at concentration of 0-350ppm on antioxidant activity was used in

this study. The amount of sugar added to fruit and vegetable drink mainly based on

sensory test or consumer acceptance. However, the total soluble solid in ready-to-drink

of fruit beverages is widely varied from 5-15 Brix (Lea, 1991; Henrix, 1995)

3.2 Material and sample preparation

3.2.1 Juice extraction

C. asiatica from species ‘pegaga ubi’ or also known as ‘pegaga biasa’ that was

recommended for commercial production (Indu Bala & Ng, 2000) was used in

preparation of pegaga drink. Local supplier from Johor Bahru supplied the plant material

for this study. 400 g of pegaga including leaves, stolon and root was cleaned under

running tap water. The clean sample was blend with 2 litre deionised water by using

food processor. The juice extract then was filtered using muslin-cloth.

3.2.2 Preparation of pegaga drink

Pegaga drink was prepared based on formulation that was developed by

Malaysian Agricultural Research & Development Institute (MARDI). The juice extract

was mixed with 11% (w/v) sugar, 0.12% (w/v) citric acid, 0.11% (w/v) carboxyl methyl-

cellulose (CMC) and made up to a volume of 2.3 L with deionised water. The flow chart

57

for the preparation of pegaga drink is shown in Figure 3.1. The product was pasteurized

at three different heat-processing temperatures; 65 C/15 minutes, 80 C/5 minutes and in

canning process (heat at 80 C/5minutes, canned and followed by boiling at 100 C/10

minutes before cooling process). Fresh sample (F) or non-thermally treated drink

without added sugar and food additives was also prepared. Each product was then kept

at 4 C.

Pegaga sample (Centella asiatica)

Cleaned and washed under running tap water

Homogenized in deionized water

Filtered through muslin-cloth

Pegaga juice extract Commercial sample

Add water Add sugar, CMC, citric acid CM1 CM2

and water (unheated) (90 C/1min)

Fresh sample (F) Heat treatment

65 C/15 min (A) 80 C/5 min (B) Canning process (C)

(heat at 80 C/5 min, canned,

boiled at 100 C/10 min)

Packed in LDPE bottle

Analysis

Figure 3.1: Flowchart of the preparation of pegaga drink

Sam

ples

Ass

essm

ents

Trip

er

Out

put

Figu

re 3

.2: E

xper

imen

tal l

ayou

t

pH, T

otal

aci

dity

, Col

our i

ndex

L*,

a*

and

b* v

alue

s, To

tal s

olub

le so

lid,

Prox

imat

e co

mpo

sitio

n, M

icro

elem

ent,

Tota

l pol

yphe

nol a

nd A

scor

bic

acid

co

nten

t

Ferr

ic th

iocy

anat

e as

say

(FTC

) and

Fer

ric R

educ

ing

Abi

lity

of P

lasm

a (F

RA

P)

assa

y

Asi

atic

osid

e co

nten

t, M

adec

asso

side

con

tent

, Asi

atic

ac

id c

onte

nt a

nd M

adec

asso

side

co

nten

t

Phys

ico-

chem

ical

char

acte

ristic

s A

ntio

xida

nt a

ctiv

ity

Trit

erpe

ne g

lyco

side

s

con

tent

Con

tribu

tion

of to

tal p

olyp

heno

l an

d as

corb

ic a

cid

on a

ntio

xida

nt

activ

ity u

sing

cor

rela

tion

coef

ficie

nt,r

Cor

rela

tion

of F

RA

P an

d FT

C

mea

sure

men

t of a

ntio

xida

nt a

ctiv

ity

(cor

rela

tion

coef

ficie

nt, r

)

Con

tribu

tion

of a

siat

icos

ide

on

antio

xida

nt a

ctiv

ity

Fres

h sa

mpl

e (S

ampl

e F)

H

eat-t

reat

ed sa

mpl

es

(Sam

ple

A, B

and

C)

Com

mer

cial

sam

ple

(Sam

ple

CM

1 an

d C

M2)

59

3.2.3 Commercial pegaga drink samples

The two commercial samples were obtained from Loo Pegaga Enterprises,

Taman Anggerik Johor Baharu (CM1) and HPA Sdn Bhd, Kuala Perlis, Perlis (CM2). .

The pegaga drink of CM1 was prepared without any thermal treatment. The second

commercial sample (CM2) was prepared in squash form and pasteurized at

90 C/1minutes. This sample was first diluted into drink prior to analysis. The squash

sample was diluted three times according to direction on the label. All samples were

kept at 4 C.

3.3 Experiments and Analytical Methods

The heat-treated sample (A, B and C), fresh sample or non-thermally treated (F)

sample and two commercial samples (CM1 and CM2) were subjected to analysis of

physico-chemical characteristic, antioxidant activity and triterpene glycosides content.

Three replicates sample of pegaga drink for each treatment were used for each analysis.

The data was presented as means and were analyzed by ANOVA. Figure 3.2 presented

the layout of experiments.

3.3.1 Physico-chemical characteristics

3.3.1.1 Colour Index

Colour analyses were carried out pegaga drink samples using Minolta

Chromameter CR-300 (Minolta Camera Co. Ltd., Osaka, Japan). The instrument was

60

standardized against a white tile before each measurement. Colour was expressed in L*,

a* and b* Hunter scale parameters (Nicoli, et al., 1996). Hunter L* denotes lightness

with 0 being black and 100 being white, while a* denotes a red hue when positive or a

green hue when negative, and b* denotes a yellow hue when positive and blue hue when

negative.

3.3.1.2 Total Soluble Solid (TSS) and pH

Total Soluble Solid (TSS) and pH was measured with a hand held refractometer

(Atago) and pH-meter (EcoMet), respectively.

3.3.1.3 Total Acidity (TA)

Total acidity, expressed as citric acid monohydrate was calculated in percent

after titration of 10 mL sample against 0.1N NaOH to end point, pH 8.2 (AOAC, 1980).

% TA = Titrate (ml) x 0.007009 g/ml x 100 (3 .1)

Sample (ml)

3.3.2 Proximate Composition and Microelements Analysis

3.3.2.1 Moisture

10-15 g of homogenized sample was placed into glass dish before dried in a

105 C oven for five hours. The dish was then removed from oven (Memmert,

61

Germany), cooled in dessicator and weighed soon after attaining room temperature. The

steps were repeated until constant weight was obtained (AOAC, 1984).

% Moisture by weight = loss of weight in gramme of the sample x 100 (3.2)

Weight in gramme of sample

3.3.2.2 Ash

2.5-3 g sample was weighed into crucible. The sample was charred on heating

mantle until no smoke evolves. Ashing was carried out in muffle furnace (Memmert,

Germany) at 550 C for about 8 hours or until grey ash was obtained. Sample was then

cooled in dessicator. The ash was calculated after constant weight was obtained

(AOAC, 1984).

% of Ash = Weight of ash / Weight of sample x 100 (3.3)

3.3.2.3 Protein

The Kjeldahl method for determining total nitrogen was based on Tecator Kjeltec

System 1026 and David Pearson (1976) was used.

Reagent: Concentrated sulphuric acid (A.R Grade), Sodium hydroxide (A.R Grade

40%), 0.05M Hydrochloric acid, 4% Boric acid with bromocresol green indicator and

catalyst, Kjeltabs (1.5 g K2S04 and 0.0075 g Se) were used.

Assay: 0.2-1 g sample was weighed and mixed with 2 pieces of Kjeltabs and 10 ml of

sulphuric acid in digestion tube. The mixture was digested for 1 hour or until a clear

solution was obtained at 420 C. The sample was cooled and distilled using Kjeltec 1026

Distilling Unit with 25 ml of 4% boric acid solution. Bromocresol indicator was placed

62

on receiver flask. The sample was then titrated with 0.05M Hydrochloric acid (HCL) to

neutral grey.

Calculation:

% N = 14.01 x (ml of titrant of sample – ml of titrant of blank) x conc. of standard acid

g of sample x 10

% Protein = % N x factor specific for different product (6.25) (3.4)

3.3.2.4 Fat

Reagent: Petroleum ether BP 40 – 60 C

Assay: Sample (3-4g) was placed into an extraction thimble. Thimble was then placed

in a beaker and dried in an electric oven for 5 hours at 70-80 C. Dried sample was

extracted with petroleum ether using Soxhlet extraction apparatus for 6-8 hours. The

solvent was evaporated and the residue was dried in an electric oven for 30 minutes at

105 C. The sample weight was then measured (AOAC, 1980).

% Fat = (W2-W1) x 100 (3.5)

Sample weight in g

W1 = weight of evaporating flask

W2 = weight of evaporating flask + content after drying

3.3.2.5 Fibre

Reagent: 0.255N Sulphuric acid (A.R Grade), 0.313N Sodium hydrochloride (A.R

Grade), Hydrochloric acid (1% in water v/v) were used.

63

Assay: Defatted sample (1-3g) was weighed (W0) and placed in beaker. 200ml of

sulphuric acid was added and boiled for 30 minutes. The sample was filtered with

Whatman paper no. 1 and the residue was washed with hot water until free from acid.

The residue was then washed with 200 ml of warmed sodium hydroxide (0.313N),

boiled for 30 minutes and filtered through crucible. The residue was washed with hot

water, 1% HCL and hot water again until neutral, then followed by ethanol. The sample

was dried in oven at 105 C for 1 hour. The crucible with residue was weighed (W1) and

ignited in muffle furnace at 450 C for 4 hours. The cooled crucible was weighed again

(W2) (AOAC, 1984) .

% Crude fiber = W1 - W2 / W0 x 100 (3.6)

3.3.2.6 Carbohydrate and Energy

Total carbohydrate was estimated according to Nergiz and Otles (1993). Energy was

calculated using the factors 4.0, 4.0 and 9.0 kcal/g for protein, carbohydrate and fat,

respectively (Abdurahman, et al., 1998).

Calculation:

Total carbohydrate (%) = 100% - (moisture content (%) + ash (%) + fat(%) + protein(%)

+ crude fiber(%) ).

Energy (Kcal)= (4 kcal/g x amount of protein, g) + (4 kcal/g x amount of carbohydrate,

g) + (9 kcal/g x amount of fat, g)

3.3.2.7 Microelement

Instrument: The analysis of micronutrient element (selenium, aluminium, plumbum

and magnesium) was conducted using Elan 6100 Inductively Coupled Plasma Mass

64

Spectrometer Method (Perkin Elmer, Canada) and multi-element calibration standard

diluted into 10ppm.

Assay: 1 ml of sample was digest with 5 ml of Aqua Regia solution for 30 minutes at

70-80 C. Aqua Regia solution was prepared from mixing of 1N Nitric acid and 1N

Hydrochloic acid (3:1). The sample was then filtered using Wathman no. 540 Hardened

Ashless. The filtrate was added with deionized water and make up to 100ml. The

sample was again filtered through a nylon filter Wathman 0.2 m before injected on

Mass Spectrometer. The calculation of microelement was based on multi-element

calibration standard.

3.3.3 Ascorbic acid assay

Ascorbic acid content (mg per g sample) was determined using direct titration

method according to Suntornsuk, et al., 2002. Each sample of pegaga drink was filtered

through a Whatman paper number 4 filter paper. The filtrate was used for analysis.

65

Preparation and standardization of 0.1N iodine: Iodine (14g) was dissolved in

potassium iodide solution (100ml). The solution was acidified with hydrochloric acid

(1N). The acidified solution was diluted with water to 1000ml and standardized with

primary standard arsenic trioxide prior use. Standard arsenic trioxide (150mg) was

dissolved in 1N sodium hydroxide (20ml) and diluted with water to 40ml. The solution

was acidified with dilute hydrochloric acid using methyl red as an indicator. Sodium

bicarbonate (2g), water (50ml) and starch soluble (3ml) was added into the acidified

solution prior to titration with iodine solution. Each ml of 0.1N iodine was equivalent

to 4.946 mg arsenic trioxide

Assay: Each 25 ml of the sample was transferred into a 250 ml Erlenmeyer flask. 25 ml

of 2N sulphuric acid was added. It was further diluted with 50 ml of water and finally 3

ml of starch soluble was added as an indicator. The solution was directly titrated with

0.1N iodine. A blank titration was performed prior to titration of each sample. Each ml

of 0.1N iodine is equivalent to 8.806 mg ascorbic acid.

3.3.4 Total polyphenol assay

The content of total phenolics was determined according to the Folin-Ciocalteu

assay (Ragazzi and Veronese, 1973). The sample was centrifuged at 4000 rpm/min for

about 15 min. 1 ml of sample was added to 10 ml of deionized water and 2 ml of Folin-

Ciocalteu phenol reagent (Merck-Schuchardt, Hohnenburn, Germany). The mixture was

than allowed to stand for 5 min and 2 ml of sodium carbonate were added to the mixture.

The absorbance was measured using UV-Vis spectrophotometer (Interscience). The

absorbance of blue complex was analysed at 750 nm in a cuvette of 1 cm. The total

phenolic content of pegaga drink was calculated from the calibration curve prepared

from the absorbance of gallic acid standard (Fluka, Chemicals) solutions and ferullic

acid standard (Fluka, Chemicals) solutions. Results are expressed as equivalents mg of

gallic acid (GAE) per 100ml of sample.

66

3.3.5 Antioxidant Assay

Preparation of sample: Each sample of pegaga drink was filtered using Bunchner

funnel with Whatman no.4 filter paper. The extract was kept at 4 C before assay.

3.3.5.1 Ferric reducing ability of plasm (FRAP) Assay

Reagent: 300 mmol/litre buffer acetate, p.H 3.6 ; 10 mmol/litre TPTZ (2,4,6-

trypyridyl-s-triazine, Fluka Chemicals) in 40 mmol/litre HCL (BDH); 20 mmol/litre

Fe3.6H2O (BDH). FRAP reagent was prepared by mixing 25 ml buffer acetate, 2.5 ml

TPTZ solution and 2.5 ml Fe3.6H2O solution (Fluka, Chemicals).

Assay: Antioxidant activity was analyzed according to procedure of Benzie and Strain

(1996) with slight modification as described by Gardner, et al. (2000). Freshly prepared

FRAP reagent was warmed to 37 C. The Reagent blank reading was taken at 593 nm. 1

ml of diluted 10-fold sample was added into 3 ml of FRAP reagent. Absorbance reading

was taken after 4 minutes. Results were calculated from calibration curve prepared from

Fe2SO4.7H2O (Fluka, Chemicals) solution in the range of 0.1mM to 10mM.

3.2.5.2 Ferric thiocyanate method (FTC)

The FTC model method will be used according to modified method of Kikuzaki dan

Nakatani (1993).

67

Reagent: 2.51% linoleic acid in 99.8% ethanol. 0.05M phosphate buffer (pH 7). 30%

ammonium thiocynate. 0.02 M ferrous chloride in 3.5% HCl.

Assay: 1 ml sample (0.02% in 99.5% ethanol) was mixed with 2 ml of linoleic acid, 4.0

ml phosphate buffer (pH 7.0) and distilled water (3.0 ml). The sample was kept in cap

screwed container in dark condition at temperature of 40 C.

0.1 ml of sample was added with 75% ethanol (9.7ml) and 0.1 ml of 30% ammonium

thiocyanate. 3 minutes after addition of 0.1 ml of ferrous chloride to the reaction

mixture, the absorbance of red colour was measured at 500 nm until absorbance of

control (blank reagent) reach maximum. -tocopherol and ascorbic acid were used as

standard sample.

% Inhibition = 100 – Absorbance increase of sample (max) x 100 (3.7)

Absorbance increase of control (max)

3.3.6 Study on factors influence to the antioxidant activity of pegaga drink

This experiments consisted of the effect of addition of citric acid, effect of

sodium metabisulfite, effect of total soluble solid on antioxidant activity of pegaga drink

using FTC assay and FRAP assay. Fresh pegaga drink was added with following

ingredients;

1. Citric acid at concentrations of 0% (control), 0.1%w/v, 0.2%w/v, 0.3%w/v)

2. Sodium metabisulphite at concentrations of 0 ppm (control), 200ppm,

250ppm, 300ppm and 350ppm

3. Sucrose was also added onto fresh pegaga drink up to total soluble solid

5 Brix, 10 Brix and 15 Brix. Fresh drink (1 Brix) without sucrose added

was used as control.

68

3.3.7 Determination of Triterpene glycoside

Determination of bioactive constituents such as triterpene acid (asiatic acid,

madecassic acid) and its glycosides (madecassoside and asiaticoside) were according to

modified method as reported by Inamdar, et.al. (1996).

HPLC Conditions: Isocratic HPLC system by Waters 2487 Dual Absorbance

Detection was used. Chromatographic separation was performed with a Genesis, C18, 4

cm 120 with a methanol-water mobile phase (90:10) for triterpene acid and (80:20) for

its glycosides, UV detection at 220nm, flow rate at 0.4ml/min. A 10 l volume of

sample was injected onto the column.

Preparation of Standard Triterpene Glycosides: Standard triterpene acids;

madecassic acid (18449-41-7) and asiatic acid (464-92-6) (Estersynthase, France) and its

glycosides; asiaticoside (16830-15-2) and madecassoside (34540-22-2) (Indofine

Chemical Co., France) were used in this experiment. Stock solution was prepared at

concentration of 0.4 mg/ml each in methanol:water (90:10). Methanol with HPLC grade

(99.99%) (Merck, Germany) was used. The standard solution was then diluted into

0.05-0.4 mg/ml to give a linear range for the preparation of standard curve. The

solutions were filtered through 0.45- m membrane filter and 10 l of each standard was

injected into the HPLC.

Sampel preparation:

Water extract: Pegaga drink (20ml) was centrifuged at 4000 rpm for 15 min. The

supernatant was filtered through a Milipore filter (0.45-µm) before injection into the

High Performance Liquid Chromatography (HPLC) (Shui & Leong, 2002).

Methanol extract: Pegaga drink (20ml) was centrifuged at 4000 rpm for 15 min. The

sample was then concentrated using vacuum evaporator and dissolved in 20 ml of

69

methanol-water (90:10) (Inamdar, et al., 1996). The sample was vortexed for 5

minutes, centrifuged and filtered using a Milipore filter (0.45-µm) and a known amount

of extract was subjected to HPLC under the above conditions. The contents of triterpene

glycosides were calculated based on water extract and methanol extract with the aid of

calibration graph obtained using a stock solution of each component.

3.4 Statistical analysis

Experimental data was analyzed by analysis of Variance (ANOVA) and the

significant differences among means was determined by Duncan’s multiple range test

(DMRT) using the Statistical Analysis System (SAS V.8) computing program (SAS,

Cary,NC)

70

CHAPTER 4

RESULTS AND DISCUSSION

4.1 Introduction

This chapter presents the results on the effect of thermal processing on the

physico-chemical characteristics of pelage drink. The thermal processing parameters

considered in this study were the preservation temperature and time of the treatment and

canning process. The physical and chemical analysis was carried out to determine the

product characteristics including acidity, soluble solid content and the color index in

herbal pelage drinks and, hence, compare them with some other commercial samples,

which is highly consumed locally. The level of nutrient compositions and trace

elements were also examined. The antioxidant activity was assessed using two different

methods and their correlation was discussed. The factors influenced to the antioxidant

activity in pelage drink were also investigated. In addition, the concentration of total

polyphenol and ascorbic acid was demonstrated and their contribution to antioxidant

activity was predicted through the coefficient of correlation (r). Herbal pegaga drink

prepared by different heat treatments was analyzed for their triterpene glycosides content

using High Performance Liquid Chromatography (HPLC).

71

4.2 Physico-chemical characteristics of pegaga drink

The results for pH, total soluble solids (TSS), % of total acidity (TA) expressed

as citric acid, L*, a* and b* values of different samples are shown in Table 4.1. The low

pH (3.72-3.79) of the heat-treated drink (sample A, B and C) was accompanied by a

high acidity (14.37-14.72%) calculated as citric acid. The addition of citric acid (0.12%)

in heat-treated pegaga drink is responsible for the low of pH and by a high acidity as

compared to untreated sample (F). The pH and total acidity (TA) of fresh or untreated

sample are 5.93 and 3.85%, respectively. The pH was higher than those obtained in two

commercial samples (CM1 and CM2). The organic acid may added to both commercial

samples as preservative to extend the shelf life of products. For comparison, the titrable

acidity of apple juice is 0.2-0.7% (Lea, 1991). The herbal pegaga drink had a rather low

pH. However, it is higher than apple juice (3.5-3.8) (Lea, 1991), orange juice (3.3-3.8)

and grape juice (2.8-3.0) (Henrix, 1995). The content of soluble solids in pegaga drink

was between 1.0 Brix (fresh) and 11.2-11.8 Brix (heat-treated drink), which is almost

similar to orange juice (9-15 Brix) and apple juice (11-14 Brix). The total soluble solid

in CM2 was significantly lower (7.6 Brix) than CM1 (12.6 Brix) and heat-treated drink.

For heat-treated samples (65 C/15 minutes, 80 C/5 minutes and canned, the parameters

of pH, TSS and %TA shows small changes. According to Kaanane, et al. (1988), the

minimal change in pH can be explained by relationship existing between pH and free

acid content.

To elucidate the formation of browning, absorbance (A) at 280 and/or 420 nm on

a UV-Vis spectrophotometer has been extensively used by the other researchers for

measurement of brown colour in fruit juices (Buedo, et al., 2000). The formation of

advanced Millard products was also monitored by CIE values such as L* (ligntness), a*

(redness) and b* (yellowness) values of the sample (Carabasa-Giribet and Ibraz-Ribas,

1999). Since original colour of pegaga was dark green, the absorbance at 280 and/or

420 nm, as well as the L* value were not suitable in indicating the colour changes. In

this cases, the changes in green colour of pegaga drink during processing was expressed

72

as b* values. The increase in b* value was used to indicate the development of a brown

colour. The data on L*, a* and b* values are shown in table 4.1. Results shows that all

heat-treated samples (65 C/15 minutes, 80 C/5 minutes and canned) gradually turned

brownish during processing and their b* values steadily increased from 4.88 + 0.06

before heating (F) to in the range of 6.03 + 0.18 - 6.88 + 0.18 after heat processing.

Heating at 65 C/15 minutes shows higher development of browning followed by canned

and pasteurization at 80 C/5 minutes. The results showed that heat treatments

significantly increased (P < 0.05) the brown colour development. CM1 shows greenish

in colour as good as fresh sample (F). Both sample were not involved heating process.

The whiteness value (L*) of the products was significantly different between F and

sample A, B and C. This shows that heat treatment affects the colour of the products.

Table 4.1: Physico-chemical characteristic of pegaga drink

Samples pH TSS (Brix)

TA(%)

L*+sd

a*+sd

b*+sd

F (Fresh) 5.93 1.0 3.85 24.43+0.21c 2.83+0.04a 4.88+0.06bAA (65 C/15min) 3.72 11.2 14.37 27.84+0.31ab 2.17+0.08b 6.88+0.18a

B (80 C/5min) 3.79 11.8 14.72 28.86+0.48a 2.09+0.08bc 6.03+0.18abC (Canned) 3.72 11.2 14.38 27.04+2.57ab 2.67+0.24a 6.56+2.27aCM1 3.89 12.6 7.71 26.90+0.77ab 2.22+0.16b 4.80+0.21bCM2 4.86 7.6 4.21 26.35+0.21bc 1.96+0.03c 5.40+0.03ab

Mean values in each column with the same letter (a, b, c) are not significantly different

(p>0.05) according to LSD test; sd = standard deviation

According to Labuza and Baisier (1992), the rate of formation of brown pigment

is increased with the increase of the heating temperatures. The longer heating time and

other complex reaction between components during initial stages of browning, may be

associated with the increase of colour.

73

Decolouration and browning due to thermal treatments involved several

reactions. These include Millard condensation between reducing sugars and amino

asids, ascorbic acid browning process (Cornwell and Woodstad, 1981), and pigment

destruction (Beveridge,et al., 1986). Millard browning is observed due to the presence

of carbohydrate; particularly reducing sugar, water with the increasing of heating

temperatures and pH (Cheftel, et al., 1985). Several investigations indicated that

browning formation is attributed to L-ascorbic acid loss (Clegg, 1966; Roig, et al.,

1999). Chlorophyll is unstable at elevated temperature and change colour to olive green

or brown. This colour change is believed to be due to conversion of chlorohyll to

pheophytin and it is favoured by high acid (Potter, 1986; Francis, 1985).

From quality point of view, the development of browning in pegaga drink is

undesirable due to less desirable sensorial characteristics including appearance and

aroma. However, some non-enzymatic browning (NEB) reactions, such as Millard

reaction, are reported to have positive correlation to the formation of compounds with

antioxidant capacity (Manzocco, et al., 2000). Non-enzymatic browning and its relation

to free radical scavenging capacity has been the subject of numerous studies and review

articles (Morales and Jimenez-Perez, 2001; Manzocco, et al., 1999; Nicoli, et al.,

1997b). Morales and Jimenez-Perez (2001) also indicated that browning was not

directly related to the free radical scavenging properties of MRPs formed at prolonged

heating condition. The mechanism of browning is complex and not yet fully understood

but in some food processing, Millard reactions produce chelating macromolecules,

which were attributed to the high antioxidant activities in aqueous solutions and

emulsions (Pokorny, 2001b). There are evidences showed that MRPs were found to act

as oxygen scavenger (Hayase et al., 1989; Lingnert and Waller, 1983). Besides, these

particular components are effective as metal chelating agents and have an ability to

reduce hydrogen peroxide to non-radical products (Eichner, 1981).

74

4.3 Nutrient composition

Development and production of value-added pegaga drink were conducted

mainly to increase the usage of local herbal as well as for their health benefit. However,

methods of preparation, product formulation, the nature of raw materials as well as the

temperature applied may cause different effect. Some processing treatment is causing

rapid degradation of chemical composition. A balance human diet is required to

maintain optimum health (Potter, 1996) and to protect from chronic diseases (Hunter and

Flatcher, 2002). Thus, the changes in nutritional quality also associated with greater

changes in consumer acceptance. The effects of thermal treatment during preparation of

herbal pegaga drink on the proximate composition and trace elements content were

investigated.

Table 4.2 shows the proximate values of pegaga drink in all sample tested.

Generally, proximate values and elements of heat-treated samples were almost higher

than those obtained in fresh drink, except for moisture. Fresh drink contained 99.62% of

moisture that is significantly higher than heat-treated samples (approximately 88%).

This indicate evaporations of water occurred during the heating process as well as sugar

addition. A higher amount of carbohydrate was detected in all heat-treated samples (in

the range of 10.99% to 11.40%), which mostly due to addition of sugar. The fresh drink

provide only about 0.22% of carbohydrate. Most of metabolizable carbohydrate used by

humans comes from sucrose or starch. However, sucrose is present in relatively minor

quantities in most plant foods and sucrose isolated from sugarcane generally added to

commercial foods (Whistler and Daniel, 1985). Similar results were found in crude fiber

content that only 0.01% detected in fresh and approximately 0.015% in heat-treated

samples, respectively. The nondigestible polysaccharides (fiber) are beneficial for a

healthy intestinal activity. There were no significant effect of ash and protein content

after heating processed that the amount in heat-treated was approximately 0.07% and

0.1%, respectively. As can be observed, the amount of nutrient components in pegaga

drink was very low and/or below human requirements. For example, staple foods with

75

protein content below 3% do not meet the protein requirements in human, but a diet of

cereals with an 8-10% protein content, provided enough to supply caloric requirements

of adults (Cheftel, et al., 1985). Fats serve as concentrated source of energy compared

to protein and carbohydrate. Unfortunately, no fats were detected both in fresh and heat-

treated samples. Similarly, Prasad, et al., (2000) reported that the fruit based products

such as pineapple beverage powder contained negligible amounts of both protein and fat.

Fresh pegaga drink contained only 0.06% amount of total ash, which was 0.01% less

than other samples.

As shown in the data, herbal pegaga drink provides a good source of mineral and

trace elements. Potassium was found as major components (347.99-469.91mg) in herbal

pegaga drink, followed by sodium (12.06-82.01 mg) and phosphorus (28.91-40.70mg).

Generally, the amount of minerals and trace elements in fresh and heat-treated samples

were greater than commercial samples. According to food U.S RDA (1980), the

optimum daily dietary intakes of adults for phosphorus, magnesium, iron, zinc, sodium

and potassium are about 800mg, 300-350mg, 10-18mg, 15mg, 1100-3300mg and 1875-

5625 mg, respectively. Consumption of one liter of herbal pegaga drink daily could

contribute appreciable amounts of minerals to the body. The calculation indicates about

9.3%-12.5% of RDA for potassium being contributed from 500 ml of sample, followed

by phosphorus (1.8%-2.5%) and sodium (0.5%-3.7%). Potassium (intracellular cation)

and sodium (extracellular ion) are regulated osmotic equilibrium and pressure, and also

maintained body-fluid volume. Phosphorus is involved in the enzymes-controlled

energy-yielding reactions of metabolism and helps control the acid-alkaline reaction of

the blood (Potter, 1986). 500 ml of pegaga drink also provided about 6.7%-11.2% of

iron for daily requirement.

Table 4.2 also demonstrated that the amount of zinc traced in pegaga drink was

in a range of 1.08-1.83 mg. The amount of zinc in pegaga drink was accounted about

7.2-12.2% of Recommended Daily Allowance (RDA).

76

Table 4.2: The nutritional value and trace element of pegaga drink

Sample of pegaga drink

Proximate value Fresh A B C CM1 CM2 Calorie (Kcal) 1.20 45.50 46.00 44.30 1.21 49.56 Moisture (%) 99.62 88.53 88.41 88.83 99.64 87.50 Ash (%) 0.063 0.070 0.073 0.070 0.055 0.089 Protein (%) 0.093 0.100 0.101 0.091 0.083 0.090 Crude fiber (%) 0.009 0.015 0.014 0.015 0.008 0.020 Fat (%) ND ND ND ND ND ND Carbohydrate (%) 0.215 11.285 11.402 10.994 0.220 12.301

Minerals (mg/L) Zinc 1.09 1.83 1.16 1.41 1.28 1.63 Phosphorus 33.74 30.71 28.91 40.70 18.53 16.60 Iron 4.04 3.17 2.55 2.41 2.81 2.58 Sodium 12.06 82.01 71.47 68.24 8.43 52.75 Potassium 469.91 446.10 372.42 347.99 273.28 131.94

Element (mg/L) Selenium ND 0.01 ND 0.01 ND ND Aluminium 149.38 3.45 3.24 1.13 0.97 6.55 Plumbum 0.44 2.18 0.87 0.56 0.45 0.33 Magnesium 10.27 10.19 9.38 8.28 6.86 4.69

* ND – Not detected

No selenium was detected in most samples except for sample A and C. The

concentration of selenium in sample A and C was only 0.01mg each. At low levels of

occurrence, zinc, selenium and manganese are essential to life, which usually function as

miscellaneous antioxidant. Zinc, one of the essential nutrients, strongly inhibits lipid

peroxidation, which is possibly to be due to altering or preventing iron binding. On the

other hand, selenium plays a major role in the synthesis and activity of glutathione

peroxidase, a primary cellular antioxidant enzyme (Madhavi and Salunkhe, 1996).

Since the intakes of trace elements may caused toxicity, the maximum levels of

selenium for adults should not exceeded 0.05-0.2 mg (Potter, 1986). Potentially harmful

77

metals such as lead, mercury, cadnium, zinc and selenium naturally present in soil, water

and plant foods. However, according to Potter (1986), some undesirable minerals and

certain natural toxicants are largely removed or inactivated when foods are processed.

4.4 Total Polyphenol

Naturally occurring phenolic compounds in fruit and vegetables mostly exhibit

antioxidative activity. Thus, the protecting effects of diet from fruit and vegetables

products have also been attributed to the presence of these compounds. Commercially

produced herbal pegaga drink can be rich in total polyphenol, however it is mainly

depend on the quality of raw materials and the processing conditions. The changes in

total polyphenol concentration as a consequence of heating treatment of herbal pegaga

drink were observed.

The data on total polyphenol, determined according to method by Ragazzi &

Veronese (1973), expressed as gallic acid equivalent (GAE) and ferulic acid equivalent

are plotted in figure 4.1. In general, the phenolic compounds of pegaga drinks declined

with the increasing of heat processing temperature. Fresh drink showed the highest

quantity of total polyphenol (1470.14mg/100ml of GAE equivalent) compared with

other samples tested. After heat treatment the levels of total polyphenol declined to

lower than found in unheated or fresh drink. In this study, the concentration of total

phenolic compounds of heat-treated sample (A, B and C) gradually decreased by 903.23,

805.54 and 730.27 mg/100ml, respectively. Similar trend was observed in total phenolic

compounds of all sample tested expressed as ferullic acid equivalents. The total

polyphenols are in the range of 147.92-1413.49mg/100ml. The amount of total

polyphenol in commercial sample (CM1) was higher (1140.24 mg/100ml), than sample

A, B, and C but the concentration in CM2 (797.53 mg/100ml) was insignificant with

sample B. Generally, it can be observed that all pegaga drink sample contained high

78

amount of phenolic compounds as compared to orange juice (75.5 mg/100ml), pineapple

juice (35.8 mg/100ml) and vegetable juice (29.3 mg/100ml) (Gardner, et al., 2000).

However, based on other finding reported by Manzocco, et al. (1998), the amount of

phenolic compounds in heat-treated drink was still lower than observed in green tea and

black tea beverages (953.84 mg/L and 801.16 mg/L GAE equivalents, respectively).

Heat treatment applied during preparation of herbal pegaga drink still retains

appreciable amount of total polyphenol. After canning processed with the temperature

up to 100 C, about 50% of total polyphenol in pegaga drink remain. The loss of total

polyphenol content under heat processing treatment at 65 C for 15 minutes and 80 C for

5 minutes was 45% and 49%, respectively. In agreement with previous investigation,

phenolic compounds contained in food were significantly loss during heat processing.

This finding supported by Fezah, et al. (2000), who noted that the air-dried treatment at

room temperature of pegaga leaf contained about 0.111 mg pyrogallol per mg dried

MeOH extract, which is significantly lower than fresh sample. The total polyphenol in

leaf and underground part of pegaga was reduced by 52% and 50%, respectively. Gil-

Izquierdo, et al. (2002) reported that pasteurization led to degradation of several

phenolics such as caffeic acid, vicenin 2 and narirutin in orange pulp. Boiling of onion

bulbs considerably affected the content of quercetin, yielding losses of 43.2%. The 60-

min boiling had more severe effects in terms of flovonol loss in onion and asparagus.

This treatment resulted in 20.5% and 43.9% decrease in total flavonol content,

respectively (Markis & Rossiter, 2001). Similarly, Crozier, et al. (1997) reported that

cooking lowered the quercetin content of both tomatoes and onion. In contrast, 80% of

total phenolics in Roselle remained, even after drying at 75ºC and storage for 15 weeks

at 40ºC (Tsai, et al., 2002). Since the amount of total polyphenol was significantly

reduced after thermal treatment, the unstable phenolic compound may present in pegaga

drink as major component.

For comparison, Zainol, et al. (2003) reported that 100g of pegaga leaf extract

contained 8130-11700mg of total polyphenol in all accession tested. Fezah, et al. (2000)

79

observed slightly high total polyphenol content (23000mg per 100 g) in similar herb. It

can also be monitored that the phenolic content of pegaga drink was significantly lower

than raw material. This is mainly due to dilution process and the reduction of naturally

occuring phenolic compounds during preparation of raw material into drink.

Nevertheless, polyphenols composition in foods and processed products, is influenced

by the source of raw materials, variety and procedure used of sample preparation as well

as by the analytical methods employed to quantify polyphenols (Peleg, et al., 1991). As

previously observed, processing treatment of pegaga drinks at the high temperatures,

potentially causing thermal decomposition of some phenolic antioxidant. On the other

hand, processing steps such as cutting, blending and storage are expected to contribute

the degradation and/ or transformation of it biologically active component. Extraction of

pegaga juice is performed using industrial food processor and filtered by muslin-cloth.

The residue may contained some phenolic compounds and markedly decrease the

amount of this component in juice extract. Skrede and Wrolstad (2002) found that the

extensive loss of polyphenolic compounds occurred during processing single strength

juice. The industrial processing of pasteurized highbush blueberry recovered only 32%

of anthocyanin, whereas 18% remained in press-cake residue after pressing the pulp.

Similarly, Koo and Suhaila (2001) noticed that at high temperatures certain phenolics

decompose or combine with other plant components. Moreover, it was probably due to

the degradation of these compounds, as the best substrate for polyphenol oxidase (PPO),

for browning process.

The specific components contributed to total polyphenol content in pegaga are

not yet been identified clearly. However, the quantitave analysis by Thin Layer

Chromatography (TLC) demonstrated that pegaga (leaf, stolon and underground part)

contained various components of phenolic including flavonoids; apigenin (7.08 mg/g),

kaempferol (4.33 mg/g), quercetin (7.08 mg/g) and rutin (0.11mg/g) (Radzali, et al.,

2001). In almost similar investigation using HPLC system, Koo and Suhaila (2001)

reported that some locally consumed plants such as pegaga are found to be rich in

flavonoid content, which are including quercetin (423.5 mg/kg) and kaempherol (20.5

mg/kg). Flavonoid compounds such as kaempferol, quercetin, luteolin, mycertin and

80

catechin were contributed to the antioxidative activities in plant materials (Bors and

Saran, 1987). Component of phenolic antioxidants in herbs include catechins in tea

extract (Yen & Chen, 1995; Wang, et al., 2000), curcumin in C. longa (Ruby, et al.,

1995) and quercetin in Polygonum hydropiper (Haraguchi, et al., 1992).

0 200 400 600 800 1000 1200 1400 1600

Total polyphenol (mg/100ml)

F

A

B

C

CM1

CM2

Pega

ga d

rink

sam

ple

Ferulic acid eqv. Gallic acid eqv.

a

Figure 4.1: Total phenolic compounds (as ferulic acid and gallic acid

equivalents) of different sample of pegaga drink (n=3). Key: F (fresh sample); A

(65 C/15 min); B (80 C/5 min); C (canned); CM1 (commercial sample -Loo Ent.); CM2

(commercial sample-HPA). Values with same letter (a,b,c) are not significantly different

(P>0.05) between samples.

As previously investigated, Velioglu et al. (1998) reported that phenolic

compounds are responsible for the antioxidative activity in selected vegetables, fruits

and grains. In a similar finding, Yen and Chen (1995) noticed that tea leaf exhibited

marked antioxidative activity as it contain significant amount of polyphenols. Nicoli et

al. (1999), also found that the antioxidative effectiveness in most plant materials is

d

d

e

c

b

81

reported to be mostly due to phenolic compounds. The antioxidant potential of phenolic

compound is identified through the stability of the aroxy radical formed in the structure

of the compounds itself. According to Pokorny, et al., (2001b), in antioxidant activity,

the mechanism of protection from oxidative insults of each compound is very specific.

The role of flavonoids as antioxidant properties were reported in many research finding

(Cuvelier, et al., 1994; Bors and Saran, 1987; Kikuzaki and Nakatani, 1993).

Flavonoids, which are present in pegaga extract, are known as primary antioxidants and

acts as free radical acceptors and chain reaction breakers.

4.5 Ascorbic acid content

Commercially, ascorbic acid is fortified in food products as food supplement.

The reduction of ascorbic acid as a consequence of food processing procedures is

frequently discussed. In this study, the possible correlation of ascorbic acid in

antioxidant activity of pegaga drink and the retention of ascorbic acid as one of

important nutrient in pegaga was evaluated.

Figure 4.2 shows the ascorbic acid content in different samples of pegaga drink.

The amount of ascorbic acid was reduced significantly after heat treatment. Unheated

samples contained the highest amount of ascorbic acid tasted (4.23mg/100ml), followed

by heat sample at 65ºC/15 minutes and 80ºC/5 minutes (1.76mg/100ml each) and the

lowest concentration was observed in canned drink (0.7mg/100ml). The commercial

pegaga drink (CM1) contains much higher ascorbic acid (2.11 mg/100ml) than CM2

(1.41mg/100ml), however its amount was found to be lower than unheated or fresh

sample. The amount of ascorbic acid in fresh drink, however is significantly lower than

those determined from guava juice (80.1mg/100g), passion juice (39.1 mg/100g) and

lemon juice (10.5 mg/100g) but almost similar to G. schomburgkiana juice (4.6

mg/100g) (Suntornsuk, et al., 2002). The residual ascorbic acid content in heat-treated

82

drinks, was lower than the unheated product. This observation is in agreement with the

reported by Mahanom, et al. (1999), that the loss of ascorbic acid in dried herbal tea,

dried at 50ºC for 9 hours and 70ºC for 5 hours, is about 75.60% and 34.19%,

respectively. In addition, the concentration of ascorbic acid in tomato puree and tomato-

oil samples was reduced to 46% and 55%, subjected to heat treatment at 95ºC for 30 min

(Nicoli, et al., 1997b). Freeze-dried of guava juice and emblic myrobolan juice also

cause the decrease amount of ascorbic acid up to 41.4% and 20.4%, respectively

(Suntornsuk, et al., 2002)

00.5

11.5

22.5

33.5

44.5

Con

cent

ratio

n of

asc

orbi

c ac

id

(mg/

100m

l)

F A B C CM1 CM2

Sample

Figure 4.2: Ascorbic acid content of different sample of pegaga drink (n=3).

Key: F (fresh sample); A (65 C/15 min); B (80 C/5 min); C (canned); CM1

(commercial sample -Loo Ent.); CM2 (commercial sample-HPA). Values with same

letter (a,b,c) are not significantly different (P>0.05) between samples.

As expected, heat treatment dramatically reduced (41.6-83.45%) the ascorbic

acid content in all heat-treated pegaga drink samples. The losses of ascorbic acid

maybe attributed to the thermal treatment applied (Yang and Atallah, 1985). Ascorbic

a

c c

e

b

d

83

acid is easily destroyed by oxidation, especially at higher temperature and during

washing, processing and storage. Generally, the effect of temperature on ascorbic acid

content is far more severe than the effect of heating duration. Since ascorbic acid is

soluble in water, it is readily lost via leaching from cut or bruised surfaces of raw

material, however in processed foods the most significant losses results from chemical

degradation (Tannebaum, 1985). Transformation of ascorbic acid to diketoglutanic acid

due to reaction with air, light and metal ions may also contribute to the losses

encountered (Harris, 1975; Addo, 1981). Ascorbic acid can be degraded by active

oxygen and by reactions initiated by transition metals. As an antioxidant, it is removes

oxygen in systems and gets oxidized to dehydroascorbic acid. Besides, the antioxidant

behavior also enhances the loss of ascorbic acid (Jadhav, et al., 1996).

4.6 Antioxidant activity

4.6.1 Antioxidant activity in linoleic acid system (FTC Assay)

The positive and negative effect of heat treatment of foods on their antioxidative

activities was previously reported. Since pegaga was found to have antioxidant activiy,

the present of antioxidant compounds in fresh and heat-treated pegaga drink may delay

oxidation of linoleic acid and exhibited the antioxidative activity. The Ferric

thiocyanate assay was used to evaluate the antioxidant activity of pegaga drink, only at

primary state of oxidation.

The individual antioxidant activity of samples or the effect of heat treatments on

the peroxidation of linoleic acid is shown in figure 4.3. Each sample of herbal pegaga

drink showed a low absorbance values at 500 nm, which indicated high level of

antioxidant activity. The exposure of food components to high temperatures can cause

negative change not only to nutritional quality, but also their antioxidant activity.

84

Generally, the oxidative activity of linoleic acid is markedly inhibited by any samples of

pegaga drink compared to control assay. The results showed that the level of antioxidant

activity is reduced when the temperature is increased. Fresh sample of pegaga exhibited

much higher (P<0.05) antioxidant activity than heat-treated samples. The % of

inhibition of peroxidation is 72.98% for fresh and 53.73, 64.80% and 69.88% for sample

C, B and A respectively (Figure 4.3). The lipid peroxidation inhibitory activity of all

pegaga drink samples significantly lower than raw pegaga leaves (98.2%) as previously

reported by Vimala, et al. (2003). However, the antioxidant activity of fresh pegaga

drink was comparable to those reported for oolong tea and higher than green tea. Yen &

Chen (1995) in their investigation indicated that oolong tea and green tea exhibited

73.6% and 40% inhibition of linoleic acid peroxidation, respectively. Our finding is also

similar to the work of Duh and Yen (1997), who reported that the addition of herbal

extracts significantly increased the inhibition the linoleic acid peroxidation. In terms of

mechanism, it is prolongs the induction period by the lowering rate of accumulation of

oxidative products.

All pegaga drink samples exhibited higher activity than natural antioxidant such

as -tocopherol and ascorbic acid but lower than synthetic antioxidant, butylated

hydroxytoulene (BHT) at concentration of 200ppm. In agreement with our result, earlier

studies by Abdul Hamid, et al. (2002) revealed that the activity of pegaga evaluated

from similar method, is significantly lower than BHT. However, the antioxidant activity

of -tocopherol at concentration of 300ppm and above is not significantly different from

that exhibited by leaves and roots extract of pegaga. Observation of antioxidant activity

under Ferric Thiocynate (FTC) assay, done by Mohd Zin et al. (2002) showed that ethyl

acetate extract of mengkudu exhibited significant activity, which are comparable to that

of both -tocopherol and BHT.

In the previous studies the changes of antioxidant activity in relation to

processing and storage was carried out in different food system. In most cases, the

exposure of natural antioxidant to high processing temperatures caused to the decreased

85

in that particular important component. The reduction of the natural antioxidants could

be due to evaporation and transformation of the food component during processing that

could have pro-oxidant activity (Pokorny et al., 2001b; Nicoli et al., 1999). A brownish

colour was disserved in heat-treated drink, as a result of Millard reactions or degradation

of chlorophyll pigment. The reduction of antioxidant activity in heat-treated samples

can be attributed to the formation of compounds with pro-oxidants properties during

processing. Namiki and Hayashi (1983) reported that highly reactive radicals having

pro-oxidant properties might be formed in early stages of the Millard reactions, which

the formation of both pro-oxidant and antioxidant properties are always depend upon the

intensity and the duration of heat treatment (Nicoli, et al., 1999).

40.00% 50.00% 60.00% 70.00% 80.00% 90.00%

% inhibition of linoleic acid peroxidation

FreshABC

CM1CM2

Vit. EVit.CBHT

Pega

ga d

rink

and

stan

dard

sa

mpl

e

Figure 4.3: % Inhibition of peroxidation as mean (n=3) in pegaga drinks and standard

sample. Key: F (fresh sample); A (65 C/15 min); B (80 C/5 min); C (canned); CM1

(commercial sample -Loo Ent.); CM2 (commercial sample-HPA); Vit.E ( -tocopherol),

Vit.C (ascorbic acid) and BHT (Butylated hydroxy toluene). Values with same letter are

not significantly different at P=0.05

a

bc

d

ef

fg

h

86

The antioxidant capacity of herb extract is composed of a mixture of

antioxidants, which generally include phenolics, carotenoids and tocopherol. The

antioxidant content and profile varied greatly when it was exposed to different

environment and processes. As a result, the antioxidant activity of herbs in different

processing treatment may differ considerably from one to another.

It clearly that drink samples under study showed the decreased of % inhibition

with increasing of processing temperature, in agreement with the results reported by

Abdul Hamid et al. (2002), who studied the characterization of antioxidative activities of

various extracts of pegaga. They noted that the antioxidant activity of pegaga extract

was stable up to 50ºC of incubation temperature and reduced significantly at 70 to 90ºC.

4.6.2 Antioxidant activity by Ferric reducing ability of plasma (FRAP assay)

The results of antioxidant activity from linoleic acid peroxidation was compared

with FRAP value. Figure 4.4 demonstrates the FRAP values of pegaga drink as a

consequence of processing procedures. The FRAP value was interpolated from a

standard calibration curve with the linear regression was y = 7.387e-5x + 0.002. Heat

processing studied showed negative effects thus resulted in a decrease in the antioxidant

potential of the pegaga drink. The greatest FRAP value was observed in fresh sample

(860 µmol/liter) followed by A (65 C/15 minutes), B (80 C/5minutes) and C (canned).

FRAP values of heat-treated samples are in the range of 404 - 740 µmol/litre. Two

commercial samples (CM1 and CM2) showed the appreciable amount of antioxidant

activity, which were able to reduce about 620 µmol/litre and 370 µmol/litre of Fe (III),

respectively. The antioxidant activity, however, is significantly lower than value

reported by Gardner, et al., (2000), who observed that the ability of vegetable and

orange juice to reduce Fe(III) are approximately 1.2mM and 6mM, respectively. These

results indicated a similar trend as to the FTC assay. Tsai, et al. (2002) found that the

87

FRAP activity of roselle extract and green tea was 2 mmol/litre and 8 mmol/litre,

respectively. The FRAP value was obtained from 1 g of roselle and green tea, extracted

in 300ml water at 100 C for 3 min. Results showed that, the level of antioxidant activity

in pegaga drink is slightly lower than green tea and roselle extract. Our results differ

from previous report on green tea beverages. The optimum activity was obtained when

it was prepared at high infusion temperature with long infusion time. The tea beverages

prepared at 20-70 C of infusion temperature was significantly lower than at the infusion

temperature of 90 C Lingley-Evans (2000). However, the antioxidant compounds in

black tea is ideally extracted at 70-90 C in 1-2 minutes infusion time.

Commercial antioxidant at 200 ppm (vitamin E) showed the highest FRAP value

(569.37 µmol/litre) followed by synthetic antioxidant, specifically BHT (543.75

µmol/litre), at the same concentration. The FRAP value of vitamin C was 398.36

µmol/litre. In contrast with FTC assay, BHT exhibited lower antioxidant activity than

fresh and heat-treated pegaga drink. Our result is similar to the previous studies, which

reported that the water extract of Chrysanthemum and Roselle exhibited a greater

reducing power than 200 ppm of -tocopherol and BHA (Duh and Yen, 1997).

After heat treatments, the antioxidant capacity were reduced by 14 – 53.59%

evaluated by FRAP assay. The polyphenols, which are present in the pegaga drink, can

be destroyed or transformed into other phytochemicals during heat treatment and

processing. Transformation of existing structure (Kikuzaki and Nakatani, 1993),

oxidation of phenolic compounds during processing steps and interaction of phenolic

antioxidant with other food components (Nicoli, et al., 1999) may also explain the

reduction in the value of antioxidant activity in pegaga drink. Results also indicated that

the heating period did not significantly affect the antioxidant properties in pegaga drink.

Sample A with prolong heating period (15 min) was still shows higher antioxidant

activity compared to sample B and Sample C. Skorikova and Lyashenko (1972)

previously mentioned a negative correlation between the heating period and the phenol

content of apple and pear juices. However, the browning as well as the antioxidant

88

activity of the tomato samples increased with the increase in heating time Nicoli, et al.

(1997b). Nicoli et al. (1997a) also reported that the antioxidant activity did not

increased linearly with the increasing of roasting time of coffee brews.

0 200 400 600 800 1000

FRAP value (µmol/L)

FreshABC

CM1CM2Vit. EVit.CBHT

Pega

ga d

rink

and

stan

dard

sam

ple

Figure 4.4: FRAP activity as mean (n=3) in different thermal processing of pegaga

drinks. Key: F (fresh sample); A (65 C/15 min); B (80 C/5 min); C (canned); CM1

(commercial sample-Loo Ent.); CM2 (commercial sample-HPA). Values with same

letter are not significantly different at P=0.05

Although the antioxidant activity of pegaga drink was reduced as a consequence

to heat processing, the antioxidant activity of the samples were still considerably high as

they exhibited more than 50% inhibition of linoleic acid peroxidation. The trend is

almost similar to the antioxidant activity determined by FRAP assay. The high capacity

of antioxidant in heat-treated drink is probably due to development of new component

having antioxidant properties. Processing steps such ageing, oxygenation and heat

treatments can promote progressive polymerisation of phenols to form brown coloured

macromolecular products, which are expected to possess the same antioxidant activity of

the original phenols (Manzocco, et al., 1999). Wang, et al. (1996), also observed that

ab

c

d

d

e

f

f

g

89

heat-processed tomato juice and grape juice had a much higher antioxidant activity than

fresh products, however the mechanism for the increase in activity is not clear. Thermal

treatment is responsible to induce the increase in the amount of phenolic antioxidant,

particularly anthocyanin and total cinnamates (Scalzo, et al., 2004). Results obtained

from previous studies also noted that the increased in antioxidant activity of plant foods

during prolonged heat treatments is because of the formation of Millard reactions

product (Nicoli et al., 1999). Monzocco, et al., 1999 found that the increase of chain-

breaking activity in Marsala-type wine is related to the development of non-enzymatic

browning (MRPs). Nicoli et al. (1997a) was reported a similar result in their

investigation on the antioxidant properties of coffee brew in relation to the roasting

degree. Millard products, especially melanoidins, can also bind iron and copper ions

into inactive macromolecular complexes (Pokorny, 2001b). In our research, the relation

of antioxidant activity with the formation of browning during processing of pegaga drink

was not investigated specifically.

Other factors such the synergism with other food components or chelating agents

particularly citric acid could be attributed to the appreciable level of antioxidant activity

in heat-treated samples. It has been reported that most natural antioxidative compounds

often work synergically with each other to provide a broad spectrum of antioxidant

activtiy that creates an effective defense system against free radical attack (Lu and Foo,

1995).

The antioxidant activity varies considerably from one heating temperature to

another. The antioxidant capacities of two commercial pegaga drinks were not similar

to fresh sample and heat-treated samples used in this study. Commercial processing step

is thought to be responsible for the reduction of antioxidant activity. The varieties of

pegaga used in commercial products could also contributed to the variation. Pegaga

drink contains multiple-components in its formulation including citric acid, sugar and

preservative, and undergoes a series of processes such as heating, pasteurization and

90

concentration. Under these conditions, reaction between components can occur and

affect the antioxidant property.

4.6.3 Correlation of FTC assay and FRAP assay

The effect of thermal processing of pegaga drink on antioxidant capacity was

assessed by measuring the amount of peroxide in initial stages of lipid oxidation (FTC

method) and their ability to reduce Fe (III) (FRAP assay).

Figure 4.5 shows a linear correlation of FTC against FRAP assay. The two

assays are strongly correlated (r=0.93) at p=0.05. Since results from both methods were

significantly associated, any one of two models may be a useful tool for evaluating the

antioxidant capacity of pegaga drink. However different results were obtained when the

antioxidant activity of BHT, ascorbic acid and vitamin E were measured. We found that

BHT compound, which strongly inhibited peroxidation of linoleic acid, did not showed

high antioxidant potential via FRAP assay.

91

FTC (Absorbance at 500nm)

FR

AP

(Abs

orba

nce

at 5

93nm

)

0.05

0.07

0.09

0.11

0.13

0.15

0.30 0.36 0.42 0.48 0.54 0.60

Figure 4.5: Correlation of FRAP and FTC measurement of antioxidant activity in

pegaga drink. The correlation between the two assays is highly significant (r=0.93,

P<0.05).

There was no significant correlation observed between two assays of

antioxidative activity measurement of pegaga drink, synthetic antioxidant and natural

antioxidant (r=0.5385, p=0.136) based on a linear regression y=0.0464x + 35.615 as

shown in figure 4.6. This suggests that any of these methods may demonstrate the

antioxidant capacity through different mechanism. Furthermore, the differences in

antioxidative activities observed from FTC and FRAP assay could also be ascribed to

several factors, including antioxidative mechanisms exhibited by the compounds, the

structures of the different antioxidant properties such as phenolic compounds and

probably due to the synergistic effects of the different compounds that present in the

sample (Zainol, et al., 2003). The finding is in accordance with their report that the

antioxidative activity of pegaga extract show different pattern measured by FTC and

Thiobarbituric acid (TBA) assays. In other experiment, both FRAP and Electron Spin

Resonance Spectroscopy (ESR) assays gave comparable results that were strongly

92

correlated (r=0.96) at P<0.001 (Gardner, et al., 2000). ESR was previously used to

measure the ability of antioxidant properties to donate a hydrogen atom or electron to

synthetic free radical potassium nitrosodisulphonate (Fremy’s salt). FRAP assay also

gave an accurate measurement of antioxidant capacity in Roselle extract (Tsai, et al.,

2001) and fruit juices (Gardner, et al., 2000).

FRAP values (umol/L)

FTC

(% in

hibi

tion

of li

nole

ic p

erox

idat

ion)

40

45

50

55

60

65

70

75

80

300 400 500 600 700 800 900

Figure 4.6: Regression of FRAP assay against FTC measurement of antioxidant

activity of pegaga drink, BHT, vitamin E and vitamin C (r= 0.5385, p=0.136)

4.7 Antioxidant activity of phenolic compounds and ascorbic acid.

Both phenolic compounds and ascorbic acid are mainly recognized for their

valuable sources of antioxidant in fruits and vegetables. The previous studies on herbs

showed that the role of phenolic compounds as antioxidant is more significant compared

to ascorbic acid. Calculated coefficients of correlation between total polyphenol and

93

antioxidative activity of various pegaga drink sample are shown in figure 4.7. A

correlation was found based on a linear regression, y = 1.197x – 264.26 with y =

antioxidant activity (FRAP assay) and x = phenolic compound expressed as gallic acid

equivalent. The antioxidative activity of pegaga drink towards FRAP assay was

significantly correlated (r=0.8078, p<0.05) with their phenolic compounds. However,

there was low correlation (r=0.6185) obtained between the total phenolics content and %

inhibition of linoleic acid peroxidation in pegaga drink. The results reflect that the

activity of phenolic antioxidant was accounted the oxidation of linoleic acid at the

primary stages without considering the secondary state of oxidation. Thiobarbituric acid

assay (TBA) is used to measure the peroxide, which is gradually decomposed to lower

molecular compounds in secondary or advanced stages of oxidation process (Kikuzki

and Nakatani, 1993). Present finding also indicated that in FRAP assay, pegaga drink

with higher total phenolic contents were also superior in activity. In contrast, the

activity in the FTC assay shows that all the results were not influence by the total

phenolic content of extract. The differences may be also due to differences in

distribution pattern of phenolics or other antioxidants in the corresponding samples.

Dorman, et al., 2003 reported that results obtained from the different assay depend on

the chemical nature and structure of phenolic compounds present in the extracts.

It was established that the antioxidant capacity of pegaga is strongly correlated

(r2=0.90) with total phenolic content. At the same time it is suggested as major

antioxidant compounds of pegaga (Zainol, et al., 2003). However, the correlation

coefficient of total polyphenol and antioxidant activity in pegaga drink was significantly

lower than raw material. This result indicated that formation of heat-induced antioxidant

(Nicoli, et al., 1997) and synergist effect of secondary antioxidant (Lindsay, 1985) had

also contributed to antioxidant activity in herbal pegaga drink. However, previous study

on green tea extract reported that the Maillard reaction appeared not to be an important

factor for the browning of tea during processing and storage. The oxidation of phenolic

compounds was judge to be the key element in colour changes (Wang, et al., 2000).

94

Preliminary data on FRAP assay showed that the decrease in antioxidant activity

is mainly due to the decrease of phenolic compounds. Similar antioxidant activity has

been described for phenolic-rich beverages such as grape wines and teas (Frankel, et al.,

1995; Rice-Evans, et al., 1996). The decrease in phenolic total polyphenol is always

associated with significant decrease of antioxidant activity (Tsai, et al., 2002). The

finding also supported by Duh & Yen (1997), who noticed that the water extracts of

Chrysanthemum and Roselle possessed high contents of phenolic compounds and had

effective activities as radical scavengers. They also concluded that herbal water extracts

have effective activities as hydrogen donors and as primary antioxidants by reacting

with the lipid radical.

FRAP value (umol/L)

Tot

al p

olyp

heno

l con

tent

(gal

lic a

cid

eqv.

mg/

100m

l)

600

800

1000

1200

1400

1600

300 400 500 600 700 800 900

Figure 4.7: Correlation coefficient of antioxidant activity (FRAP assay) and total

polyphenol content

The correlation between antioxidant activity and their phenolic compounds was

successfully established in a few studies. For example, Lunder (1992) reported that

there was a good correlation between the antioxidant activity and the epigallocatechin

gallate (EGCg) content. In other study, Gardner, et al. (1997), noted that the antioxidant

95

potential in teas is ascribed to catechin-derivatives. The activity of phenolic antioxidants

seem to be related to their ability as hydrogen donors, which can converted the peroxy

radicals to more stable product (Rice-Evans, et al., 1995). The ability of the fruit juices

to reduce Fe (III) to Fe (II) was also closely related to their phenolic contents (Gardner,

et al., 2000) and it reflects the ability of many phenolic compounds to donate hydrogen

atoms from hydroxyl groups on their ring structures (Scott, 1997). Phenolic compounds

are able to form complexes with Fe3+ and generally the chelating ability of phenolics is

related to the high nucleophilic character of the aromatic rings. Husin, et al. (1997),

indicated that the flavanoids such as myricetin, quercetin and rhamnetin were scavengers

of hydroxyl radical and that the scavenging effect increased with increasing number of

hydroxyl groups substituted in the aromatic B-ring. In cases of pegaga, however,

specific phenolic components as well as mechanisms, which attributed to antioxidative

activities, are not identified yet. Piskula and Terao, (1998) reported that the potential of

dietary flavonoids has recently created an interest among scientist for treating many

diseases. The biological activity of flavonoids includes action against allergies,

inflammation, free radicals, hepatotoxins, microbes, ulcers, viruses and tumor (Marcia

Zimmerman, 2001). Flavonoids function as primary antioxidants, chelators and

superoxide anion scavengers (Rajalakshmi and Narasimhan, 1996) and it has been

established to act as free radical acceptors and chain reactions breakers (Larrauri, et al.,

1996). Cao, et al. (1996) also reported that it has much stronger antioxidant activities

against peroxy radicals than vitamin E, vitamin C and glutathione. The quercetin was

identified as the antioxidant property in Polygonum hydropiper, a medicinal herb

(Haraguchi, et al., 1992) and onion (Makris and Rossiter, 2001). This compound has

been effective in inhibiting copper-catalyzed oxidation. Similarly, it is clear that

quercetin and kaempferol exhibit a strong antioxidant capacity (Hertog and Hollman,

1996; Namiki, 1990; Larrauri, et al., 1996). Since quercetin and kaempferol are

appeared as part of major flavonoids components in pegaga (Radzali, et. al., 2001; Koo

and Suhaila, 2001), it is possible to explain that these constituents may also contribute in

the antioxidant capacity of pegaga drink. However, the heat processing treatment may

reduced quercetin and kaempferol content in a drink and resulted in opposite effect of

their contribution on total activity.

96

Ascorbic acid is one of the most effective antioxidants in fruits and vegetables

(Leong and Shui, 2002). As observe in table 4.3, the correlations of antioxidant activity

towards FRAP and FTC assays with ascorbic acid content are 0.8364 and 0.7461,

respectively. Initial results suggested that the source of antioxidant capacity of pegaga

drink and commercial pegaga drink sample might also from ascorbic acid. However, the

ascorbic acid was not present as a major component in fresh and heat-treated pegaga

drink. Thus, further investigation of actual contribution of ascorbic acid on antioxidant

activity is necessary.

Table 4.3: Correlation (r) of antioxidant activity with total polyphenol and ascorbic acid

content of the pegaga drink (a P<0.05, b P>0.05)

Total polyphenol Ascorbic acid

FRAP 0.8078a 0.8364b

FTC 0.6525b 0.7461b

The reducing rate in antioxidant activity is also associated with the reduction of

ascorbic acid content during heat processing of pegaga drink. Ascorbic acid was found

to be a good antioxidant property in most fruit juices. Gardner, et al. (2000) reported

that the contribution of ascorbic acid on the antioxidant capacity of orange, florida

orange and grapefruit were about 66%, 100% and 89%, respectively. However, in

pineapple and vegetable juice, it contributes less than 5% of antioxidant activity,

calculated from the ability of vitamin C to reduce Fremy’s radical. Total antioxidant

activity of blood orange juice was decreased in accordance with observed decrease of

ascorbic acid (Arena, 2001). Wang et al. (1996) calculate that the contribution of

vitamin C to total ORAC activity of fruits (including strawberry, orange, grape and

banana) was usually less than 15%. In terms of mechanism, ascorbic acid quenches

97

various activated oxygen spices and also reduces free radicals and primary antioxidant

radicals (Jadhav, et al., 1996).

Recently, many research works indicate that an increased intake of ascorbic acid

is associated with a reduce risk of chronic diseases such as cancer. The recommended

dietary allowance (RDA) for adult is 60 and 100 mg/day in United Stated and Malaysia,

respectively (Bender, 1993; Anon, 1990). However, the current RDA for ascorbic acid

is not sufficient for optimum prevention against chronic diseases. Therefore, Carr and

Frei (1999) suggested new RDA of 120mg/day for suitable action of ascorbic acid to

protect diseases. Since very low amount of ascorbic acid was observed in all pegaga

drink (0.7-4.23 mg/100ml) as compared to total polyphenol (730.27-1470.14 mg/100ml

gallic acid equivalent), its contribution to antioxidant activity was assumed not

significant or negligible.

4.8 The factors influence on antioxidant activity

During preparation of herbal drink, food additives are commonly added into

drink in order to improve the quality and for the shelf-life extension. Although some

tests indicated that food preservative (sodium metabisulphite) and citric acid may be

correlated with antioxidant activity in processed foods, further research on the specific

role of these particular components is required. To understand the contribution of food

additives on antioxidant activity in fresh sample of herbal pegaga drink, a study was

carried out on the effect of addition of sugar, citric acid and sodium metabisulphite.

98

4.8.1 Effect of citric acid on antioxidant activity

In food production, citric acid is commonly used as an acidulant and a cheltor in

many food products including fruit and vegetable beverages. Chelating agent arrest

oxidation by chain termination or serve as oxygen scavengers. Chelating agents are

valuable antioxidant synergists since they remove metal ions and that catalyze oxidation

(Lindsay, 1985). In addition, the role of citric acid as chelating agent is also referred to

synergist effect since it is greatly enhanced the action of phenolic antioxidant (Pokorny,

2001b). As organic acid, citric acid also provides an acidic environment in our food

systems that enhance the stability of primary antioxidant (Madhavi, et al., 1996).

Figure 4.8 illustrate the effect of addition of citric acid at various concentrations

on their ability to inhibit the linoleic acid peroxidation in pegaga drink. Increased

absorbance of sample and the reaction mixture indicated decreased % inhibition of

linoleic acid (Yen and Chen, 1995). The oxidative activity of linoleic acid was inhibited

by pegaga drink sample with addition of any concentration of citric acid. The increasing

amount of citric acid up to 0.2% (200ppm) added to pegaga drink result in the increase

of the antioxidant activity. The antioxidant activity was slightly reduced at concentration

of 0.3%.

99

00.050.1

0.150.2

0.250.3

0.350.4

0.45A

bsor

banc

e at

500

nm

Control 0% 0.10% 0.20% 0.30%

Concentration of citric acid

Figure 4.8: The effect of citric acid on the antioxidant activity (FTC assay) of

pegaga drink (n=3). Values with same letter are not significantly different (p>0.05)

In contrast, as shown in figure 4.9, no antioxidant activity (FRAP values) was

observed in pegaga drink after the addition of citric acid. The FRAP value dropped

significantly from absorbance value 0.032 nm in control sample to in the range of

–0.039 nm to –0.046 nm. It is noted that the increase of concentration of citric acid was

related to the reduce pH of drink. The pH of pegaga drink was reduced from 5.91nm

(without addition of citric acid) to 3.22nm, 2.91nm and 2.81nm for 0.1%, 0.2% and

0.3% of citric acid, respectively. These results suggested that the addition of citric acid

or low in pH significantly inhibited the FRAP values.

a bc

d

100

FRAPpH

Concentration of citric acid (%)

FRA

P va

lue

(Abs

orba

nce

at 5

93 n

m)

pH

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

-0.06

-0.04

-0.02

0.00

0.02

0.04

-0.05 0.05 0.15 0.25 0.35

Figure 4.9: The effect of citric acid on the antioxidant activity (FRAP assay) of pegaga

drink (n=3). Values with same letter are not significantly different (p>0.05)

The effectiveness of citric acid as chelating agent are depended on it

concentration and the food component involved. It is used with both primary and oxygen

scavengers at levels of 0.1-0.3% (Gardner, 1972). According to Lindsay (1985), citric

acid at concentration of 20-200ppm is effective synergists in all lipid system. Almost

similarly, citric acid chelates metal ions at levels of 0.005-0.2% in fat and oils (Dziezak,

1986). Citric acid also increases the effectiveness of TBHQ. A combination of a 0.02%

TBHQ and 0.01% citric acid increases the oxidative stability of olive oil from 2 hours to

58 hours (Sherwin, 1990).

Although citric acid enhances the inhibition of linoleic acid peroxidation (FTC

assay), the reduction in pH due to the addition of citric acid also shows negative effect

on the ability to reduce Fe (III) to Fe (II). Similar result was reported by Abdul Hamid,

et al. (2002), who noted that pegaga extracts exhibited optimum antioxidant activity at

pH 7 and the activity declined significantly at up and below this pH level.

a

b

bc

101

4.8.2 Effect of total soluble solid on antioxidant activity

Soluble solid content in food products can be increased by evaporation of

moisture level and via addition of sugar. Several studies have shown that high sugar

concentration increases the stability of some phenolic compound by lowering its water

activity (Wrolstad, et al., 1990). The possible effect of soluble solid content on

antioxidant activity of pegaga drink was investigated.

The total soluble solid (TSS) in pegaga drink was increased by the addition of

sugar at different concentration. To evaluate the effect of TSS on antioxidant activity in

pegaga drink, the level of TSS was increased from 1 Brix to 15 Brix. Figure 4.10

shows the antioxidant activity of pegaga drink at different level of total soluble solid

(TSS). The antioxidant activity of pegaga drink strictly depended on total soluble solid

(TSS), which high absorbance value of FRAP assay was observed at 15 Brix (0.057

nm) followed by 10 Brix (0.050 nm), 5 Brix (0.044 nm) and control sample (0.032

nm). A slightly contrast, the antioxidant activity was gradually increased at 5 Brix and

10 Brix by FTC assay, but declined thereafter with the addition of sugar up 15 Brix

(figure 4.11). Pegaga drink at concentration of 15 Brix, however shows the lowest

antioxidant activity compared to control sample (1 Brix) and other samples tasted.

According to Takeoka, et al. (2001), the increase in total soluble solid level up to 25-30

Brix appeared to influence the loss of antioxidant property in tomatoes such lycopene

content. They reported that the longer processing time, required to achieve the desired

final solid levels, might be associated with increased losses of lycopene. In our study,

the role of total soluble solid on overall antioxidant activity was still unclear. However,

it was because of different rates in chemical oxidation of phenolic compounds, which

are depending on some intrinsic food variables and it processing condition such as water

activity (aw) (Nicoli, et al., 1999).

102

0

0.01

0.02

0.03

0.04

0.05

0.06

FRAP Values (Absorbance at

593 nm)

1 Brix 5 Brix 10 Brix 15 Brix

Total soluble solid (oBrix)

Figure 4.10: The effect of total soluble solid on the antioxidant activity (FRAP assay)

of pegaga drink (n=3). Values with same letter are not significantly different (p>0.05).

The increase in total soluble solid through the addition of sugar also reduced the

aw value in pegaga drink. The relationship between food products stability and water

activity was previously investigated (Tannenbaum, et al., 1985; Karel and Yong, 1981;

Labuza, 1985). The rate of chemical reactions such as lipid oxidation and degradation

of vitamin C, generally increased as water is added up to a higher aw value with

maximum rates typically occur in the range of intermediate moisture foods (0.7-0.9 aw)

(Fennema, 1985). Karel and Young (1981) have suggested that the present of free water

may accelerate oxidation by increasing the solubility of oxygen and by macromolecules

to swell, thereby exposing more reactions.

d

cb

a

103

00.05

0.10.15

0.20.25

0.30.35

0.40.45

Absorbance at 500nm

Control 1 Brix 5 Brix 10 Brix 15 Brix

Total soluble solid (Brix)

Figure 4.11: The effect of total soluble solid on the antioxidant activity (FTC assay) of

pegaga drink (n=3). Values with same letter are not significantly different at p>0.05.

4.8.3 Effect of sodium metabisulphite

Sulphites are widely used in food and beverages as food preservatives. They

serve as secondary antioxidant and have been demonstrated to be capable of controlling

food quality through prevention of browning, reduction in discoloration of pigments and

protection against microbial spoilage (Lindley, 1998).

The effect of various concentration of sodium metabisulfite on antioxidant

activity of pegaga drink is shown in Figure 4.12, 4.13 and 4.14. As observe, the addition

of sodium metabisulphite contributed to the retention most of the antioxidant activity in

pegaga drink. The antioxidant activity of pegaga drink was increased with increasing

concentration of sodium metabisulphite. The ability of sample with sodium

metabisulphite to reduce Fe(III) to Fe (II) was strongly increased from absorbance value

cb

a a

104

0.032 nm to 0.062 nm, 0.097 nm, 0.109 nm and 0.122nm at 200ppm, 250ppm, 300ppm

and 350ppm, respectively.

Similar results were observed in FTC assay that the % inhibition of linoleic

peroxidation was also increased accordingly. The sample of pegaga drink markedly

inhibited the oxidation of linoleic acid with the addition of sodium metabisulphite. The

% inhibition of linoleic acid oxidation was increased about 22.68% at a concentration of

350ppm sodium metabisulphite compared to control. Manzocco, et al. (2001) reported

that the addition of SO2 contributed to the retention most of the original chain breaking

activity of the dried apple cubes. In similar finding, Wang, et al. (1996) observed that

commercial tomato and grape juice had much higher antioxidant activity than fresh

materials. The high antioxidant activity was also found in the commercial wine and

juice sample, partially due to the presence of food preservatives such as sodium

metabisulphite and vitamin C, which is commonly, added to commercial food products

(Tsai, et al., 2002).

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

FRAP Values (Absorbance at

593 nm)

0 200 250 300 350

Concentration of sodium metabisulphite (ppm)

Figure 4.12: The effect of sodium metabisulphite on the antioxidant activity

(FRAP assay) of pegaga drink (n=3). Values with same letter are not significantly

different (p>0.05).

ab

c

d

e

105

Sulphites are highly effective in preventing browning in fruits and vegetables,

however they have weak antioxidant properties and are used as oxygen scavengers.

Sulphites inhibit numerous enzymes including polyphenol oxidase, lipoxygenase and

ascorbic oxidase. It also prevents the oxidation of essential oils and carotenoids

(Madhavi, et al., 1996). The mechanism of action of sulfites in preventing browning

reaction involved several actions including direct inhibition of the enzyme, interact with

intermediates of reaction and prevent their participation in reactions leading to the

formation of brown pigments, or act as reducing agents promoting the formation of

phenols from quinones (Taylor, et al., 1986). Although the antioxidant activity of

pegaga drink increased after the addition of sodium metabisulphite, their used is

restricted mainly because of reports of adverse allergic reaction that is attributed to the

consumption of over limit of sulphites in food products.

Sodium metabisulphite (ppm) Concentration of

FRA

P va

lues

(A

bsor

banc

e at

593

nm

)

0.02

0.04

0.06

0.08

0.10

0.12

0.14

-50 0 50 100 150 200 250 300 350 400

Figure 4.13: Correlation coefficient of antioxidant activity (FRAP assay) and

concentration of sodium metabisulphite (n=5, p<0.05, r=0.9653).

106

00.050.1

0.150.2

0.250.3

0.350.4

0.45A

bsor

banc

e at

500

nm

Control 0 200 250 300 350Concentration of sodium metabisulphite (ppm)

Figure 4.14: The effect of sodium metabisulphite on the antioxidant activity (FTC

assay) of pegaga drink (n=3). Values with same letter are not significantly different

(p>0.05).

4.9 Triterpene glycosides

Pegaga is consumed not only as vegetable or used in medicinal purposes but also

in food preparations. Recently, increasing attention had been paid to the present

phytochemicals in herbal products. In nutritional aspect, there is increase evident that

beside macro and micro-nutrients, foods also contain a great number of compounds,

which may exhibit a protective action (Nicoli, et al., 1999). Most of the industrial food

preparations are believed to be responsible for the significant loss of bioactive

constituents of plant materials. However, in some cases treatments and processing

resulted in the enhancement of certain properties. The present study elaborates on the

effect of heat treatment during preparation of herbal drink on phytochemicals

composition of pegaga, particularly madecassoside, asiaticoside, asiatic acid and

madecassic acid.

abc

de

107

4.9.1 Isocratic HPLC Assay

The isocratic HPLC assay for qualitative and quantitative determination of

triterpene glycoside in pegaga drink was developed. Gradient HPLC is widely used to

get the separation of four active compounds at single run. In this study, the assessment

was done in isocratic HPLC with the Waters 2487 Dual Absorbance Detection using

various types of mobile phase at different concentrations.

Preliminary study was carried out to choose the best combination of methanol-

water that commonly used as mobile phase for analysis of saponins including triterpene

glycosides using High Peformance Liquid Chromatography (HPLC) (Court, et al., 1996;

Inamdar, et al., 1996; Verma, et al., 1999). Beside, a few different variables including

eluent strength, column and flow-rate were studied in order to accomplish optimum

separation of four active components of pegaga. The separation of active constituents in

pegaga was performed at the room temperature (Morganti, et al., 1999; Burnouf-

Radosevich and Delfel, 1986) with using methanol-water as a mobile phase in isocratic

HPLC system. Due to difference in polarity of the triperpene acids and its glycosides,

different concentrations of methanol (in the range of 10-90%) were used to get the better

eluent. From a few series of experiment it was observed that no peak of both triterpene

acids and its glycosides were detected at very low concentration of methanol including

20:80 and 10:90 of methanol:water. The optimum separation for madecassoside and

asiaticoside were obtained at ratio of 80:20 methanol:water after 7.87 and 8.53 minutes

(tR), respectively. The concentration of 90% methanol was observed to be excellent in

triterpene acids separation. Similarly, in isocratic HPLC assessment, Inamdar, et al.

(1999) reported that the two triterpene acids were separated by using high concentration

of methanol or acetonitrile but their glycosides needs low concentration of methanol or

acetonitrile. The chromatographic separation was peformed with a Genesis C18, flow

rate at 0.4ml/min and attenuation of 1 AUSF. The chromatograms corresponding to the

standard of asiaticoside and madecassoside are shown in figure 4.16 and 4.17.

108

Figure 4.15: HPLC-Chromatogram for standard madecassoside (tr=7.87 min)

Figure 4.16: HPLC-chromatogram for standard asiaticoside (Rt = 8.53)

109

Concentration of standard solution (mg/ml)

Are

a(1E

3 m

V.s)

0

500

1000

1500

2000

2500

3000

3500

4000

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45

Figure 4.17: Calibration curve for madecassoside (area vs concentration of standard

madecassoside)

In order to get a linear plot, the concentrations of standard solution were

prepared in the range of 0.05 – 0.4 mg/ml. The correlation coefficients of standard

calibration curves of both components were closed to 1 (Figure 4.18 and 4.19) with the

equation y = 8675.9x + 94.036 for madecassoside and y = 6767.1x + 110.94 for

asiaticoside that y equal to area and x indicates the concentration of madecassoside and

asiaticoside

y = 8675.9x + 94.036

110

Concentration of standard solution (mg/ml)

Are

a (1

E3 m

V.s)

200

800

1400

2000

2600

3200

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45

Figure 4.18: Calibration curve for asiaticoside (area vs concentration of standard

asiaticoside)

The peak for madecassic acid and asiatic acid were identified by using methanol-

water at concentration of 90:10 as mobile phase. The retention times (tR) of madecassic

acid and asiatic acid were 9.11 and 11.51, respectively. Figure 4.20 and 4.21 represent

the standard peak for madecassic acid and asiatic acid.

111

Figure 4.19: HPLC-chromatogram for standard madecassic acid (Rt=9.11)

The standard calibration curves for madecassic acid and asiatic acid were linear

over the range of 0.025-0.4 mg/ml and 0.05-0.4 mg/ml with correlation coefficients (r2)

equal to 0.9996 and 0.9999, respectively (Figure 4.22 and 4.23). The typical calibration

curves were given by the regression equation y = 14125x + 75.092 and y = 31621x +

1.2049, where y indicates the peak area and x represents the concentration of madecassic

acid and asiatic acid (mg/ml). The combination of 80:20 methanol:water was not

satisfactory for the analysis of triterpene acids due to difference in polarity, which the

compounds were not eluted out using existing mobile phase. Table 4.4 summarized the

results of HPLC analysis.

112

Figure 4.20: HPLC-chromatogram for standard asiatic acid (Rt=11.51)

Concentration of standard solution (mg/ml)

Are

a (1

E3 m

V.s)

0

1000

2000

3000

4000

5000

6000

7000

0.0 0.1 0.2 0.3 0.4

Figure 4.21: Calibration curve for madecassic acid (area vs concentration of standard

madecassic acid)

y = 14125x + 75.092

113

Concentration of standard solution (mg/ml)

Are

a (1

E3 m

V.s)

0

2000

4000

6000

8000

10000

12000

14000

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45

Figure 4.22: Calibration curve for asiatic acid (area vs concentration of asiatic acid)

A study on triterpene glycosides was done using different extraction methods,

which were water based and methanol extract from insoluble material of pegaga drink.

The extraction of triterpene glycoside was very poor in water based as compared to

methanol extract. None of the samples showed the presence of all four bioactive

components when water based samples were injected on HPLC while extraction with

methanol resulted in a higher quantity of triterpene glycosides in pegaga drink sample.

Similarly, a maximum recovery of asiaticoside (97%) was achieved by using methanol

as an extracting solvent while the extraction was very poor with ethanol and water

(Verma, et al., 1999). Methanol and aqueous methanol are effectively use as an

extraction solvent of triterpene glycosides in pegaga (Inamdar, et al., 1996; Verma, et

al., 1999)

y = 31621x + 1.2049

114

Table 4.4: The results of HPLC analysis

Compound Calibration

curve

L (mg/ml) Retention

time (tR)

(minute)

Correlation

Coefficient

(r2)

Madecassoside y=8675.9x+94.036 0.05 – 0.4 7.87 0.9989

Asiaticoside y=6767.1x+110.94 0.05 – 0.4 8.53 0.9973

Madecassic acid y=1415x+75.092 0.025 – 0.4 9.11 0.9996

Asiatic acid y=31621x+1.2049 0.05 – 0.4 11.51 0.9999

4.9.2 Quantitative determination of triterpene glycosides in pegaga drink

Herbal drink such as tea is widely consumed due to its desirable taste as well as

their antixidative, antimicrobial and anticarcinogenic properties (Osawa and Namiki,

1981). At the same time, there is now an increased interest in herbal drink or tea from

local plant. Herbal drink from pegaga was developed for similar purposes.

Traditionally, pegaga is commonly used as herbal tea or herbal drink for it cooling effect

especially among the Chinese. The commercial production of pegaga into processed

food or value-added products increased the market potential and usage. This assessment

was conducted to evaluate the triterpene glycosides content before and after heat

processing treatment of pegaga drink.

Table 4.5 illustrates the results of individual component of triterpene glycosides

(mg/100ml) content in pegaga drinks and commercial products. The total amount of

triterpene glycoside in every sample was tabulated. The average contents of

madecassoside in fresh drink were 12.2-22.1% higher than those in the corresponding

heat-treated samples. The level of this component dropped from 3.12 mg/100ml (F) to

2.70mg/100ml (A) and 2.43 mg/100ml (B). The amount of madecassoside was first

declined at 65 C/15 minutes and 80 C/5 minutes, however, it slightly increased when

115

the temperature was further increased to 100 C as observed in sample C (2.74

mg/100ml). It could be assumed that the target constituent was extracted and dissolved

easily at high temperature during heating process of pegaga drink. Madecassoside

content of commercial sample (CM1) was almost similar with fresh or control sample

(2.93 mg/ml) and significantly higher than other commercial sample, CM2 (2.54

mg/100ml).

Asiaticoside content shows a different trend that its concentration in fresh drink

was significantly lower (3.92 mg/100 ml) than in sample A (4.32 mg/100ml). However,

was significantly higher than other heat-treated samples. The concentration of

asiaticoside was remarkably reduced to 8-22.5% when exposed to high temperature up

to 80 C as in sample B (3.61 mg/100ml) and sample C (3.03 mg/100ml). Therefore, it

may be concluded that the heat treatment at moderate temperature (65 C) is likely to

increase the ability of water (as a medium) to dissolve the asiaticoside. In accordance

with the report of Vongsangnak, et al., (2003), which obtained the maximal saponin

yield when the extraction temperature was controlled around 50 C. On the other

research, Pan, et al., (2002) noticed that the application of high temperature (20-50 C)

enhanced the extraction efficiency. This is a result of an increased in diffusivity of the

solvent into cells and at the same time it increased the ability of components to adsorb

from the cells.

116

Tab

le 4

.5: R

esul

ts fo

r trit

erpe

ne g

lyco

side

s ass

ay

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

___

Tri

terp

ene

glyc

osid

es c

onte

nt (m

g/10

0ml)

of m

etha

nol e

xtra

cts o

f peg

aga

drin

k

Sam

ple

Mad

ecas

sosi

de

+S D

Asi

atic

osid

e

+SD

Mad

ecas

sic

acid

+SD

Asi

atic

acid

+SD

Tri

terp

ene

glyc

osid

es

+SD

F3.

12+0

.10a

3.

91 +

0.10

b 2.

56 +

0.2

0c

2.45

+ 0

6a

12.0

4 +

0.14

a

A2.

70+

0.05

cd

4.32

+0.

05a

2.70

+0.

03bc

1.

03 +

0.09

c 10

.75

+0.

14b

B2.

43+

0.07

e 3.

61 +

0.0

7c

3.02

+ 0

.20a

b 1.

02 +

0.07

c 10

.08

+ 0.

17cb

C2.

74+

0.08

c 3.

03 +

0.0

5d

3.22

+ 0

.33a

0.

97 +

0.0

5c

9.96

+ 0

.68c

CM

12.

93+

0.13

b 2.

60 +

0.3

0e

2.79

+ 0

.10b

c 1.

86 +

0.2

8b

10.1

8+ 0

.46b

CM

22.

54+

0.09

de

3.10

+ 0

.14d

1.

37 +

0.10

d+

1.0

5 +

0.09

c 8.

06+

0.32

d

Mea

ns w

ith th

e sa

me

lette

r (a,

b, c

) in

each

col

umn

are

not s

igni

fican

tly d

iffer

ent a

t p=0

.05

117

The amount of asiaticoside in all samples, however, significantly lower than

obtained in methanol extract of oven-dried pegaga at 30-50 C (36mg/100g dry weight),

previously reported by Verma (1999). After heat treatment, the quantity of madecassic

acid is not significantly different over a range of temperature of 0 C (control) to 100 C

(C). However, data showed that the lowest amount of this particular compound was

observed in fresh drink followed by A, B and C, in which the concentrations were 2.56,

2.70, 3.02 and 3.22 mg/100ml, respectively. The madecassic acid content in commercial

samples, CM1 and CM2 were 2.79 and 1.37 mg/100ml, respectively.

Asiatic acid was not stable at the higher temperature and this resulted in a

decreasing amount in all heat-treated sample. The concentration of asiatic acid varies in

the range of 2.45-0.97mg/100 ml. Asiatic acid content dropped to 10.7%, 16.3 % and

17.3% in A, B and canned sample, respectively. Pasteurization processed at

65 C/15min (A) to 100 C (C) resulted in a significant change of asiatic acid content in

pegaga drink. Again, the asiatic acid content of commercial sample, CM1 (10.18

mg/ml) was significantly higher than CM2 (8.06 mg/ml).

The total amount of triterpene glycosides in individual sample was recorded.

The overall result shows a different trend from individual assessment. Still heat

treatment has a great influence on the concentration of total triterpene glycosides. Heat

treatment at 65 C/15 minutes, 80 C/5 minutes and up to 100 C in canned drink caused

significant changes in the amount of total triterpene glycosides. The degree of reduction

were in the order of: F (12.04 mg/100ml) > A (10.75 mg/100ml) >B (10.08 mg/100ml)

>C (9.96 mg/100ml). The changes in phytochemical contents occurred due to the

chemical degradation and conversions of some thermolabile asiaticoside and

madecassoside to another components during heat treatments. Similar results were

reported in ginsenosides during steaming process of P. ginseng (Ren and Chen, 1999).

The levels of four active constituents of both commercial samples are always lower than

observed in fresh and heat-treated sample. CM1 and CM2 contained about 10.18 and

8.06 mg/100ml of total triterpene glycosides, respectively. The variations of triterpene

118

glycosides content in different samples occur due to various factors such as species,

geographical source, cultivation, harvest, storage, as well as preparation method of herb.

It can be seen that the increase in heating temperature decreases the

concentration of madecassoside (65 C and up to 80 C), asiaticoside (80 C and up to

100 C) and asiatic acid under all heating conditions. It could be explained that the used

of heat may also slightly reduced the concentration of saponins (Court, et al., 1996).

According to Choi, et al. (1982), a little thermal degradation of saponin occurred during

microwave extraction at 80 C. The effect of heating treatment on active constituents

such as triterpene glycosides in pegaga has not been investigated in previous reports.

However, the effect of steaming process on Panax notoginseng containing saponins

investigated as reported by Lau, et al., (2003). This information might be useful to

relate the chemical stability and characteristics of other saponin components such

asiaticoside, madecassoside and its triterpene acids. For example, the notoginsenoside

R1, ginsenoside Rg1, Re, Rb1, Rc and Rd was degraded after exposure at high

temprature during steaming process. The amount was significantly declined upon

prolong steaming duration.

There are no comparative studies on the qualitative and quantitative analysis of

triterpene glycosides of raw and processed food products from pegaga. Some reports

described only the quantitative determination of pharmaceutical products with different

extraction method and HPLC conditions (Schaneberg, et al., 2003; Morganti, et al.,

1999; Guenther and Wagner, 1996; Inamdar, et al., 1996). Generally, the concentration

of asiaticoside, madecassic acid and asiatic acid in pegaga drink significantly lower than

obtained in centellase tablet formulations containing pegaga extract. This commercial

tablet contains about 13.2 mg asiaticoside, 10.1 mg madecassic acid and 4.03 mg asiatic

acid (Inamdar, et al., 1996).

119

Kartnig (1998) noted that the pegaga extract contains not less than 2% triterpene

ester glycosides including asiaticoside and madecassoside, it is in a ranged of 1-8%. In

terms of triterpenoid fraction in pegaga drink excluding commercial samples,

asiaticoside accounted the highest percentage (30.4-40.2%) followed by madecassoside

(24.1-27.5%), madecassic acid (21.3-32.3%) and asiatic acid (9.7-20.1%). The trend

was almost similar to quantitative evaluation of individual constituents in the plant

extract that previously investigated by Inamdar, et al. (1996). According to Brinkhaus

(2000), the extracts and total triterpenoid fraction of pegaga in pharmacological studies

consists of asiatic acid (30%), madecassic acid (30%) and asiaticoside (40%). No

madecassoside content has been recorded.

The total triterpenoid fraction of commercial formulation of Centellase tablet

contained about 48.3% (13.2mg) asiaticoside, 37% (10.1mg) madecassic acid and 14.7%

(4.13mg) asiatic acid. However, no madecassoside was presence in this commercial

tablet (Inamdar, 1996). The amount is depended on it formulation and the

pharmaceutical preparation.

120

0

10

20

30

40

50

60T

rite

rpen

e gl

ycos

ides

sco

nten

t (%

)

F A B C CM1 CM2

Pegaga drink sample

MadecassosideAsiaticosideMadecassic acidAsiatic acid

Figure 4.23: Triterpenoid fraction (%) of pegaga extract from drink samples

Daily consumption of 60mg-120mg of standardized extracts of pegaga

containing up to 100% total triterpenoids is suggested in modern herbal medicine

(Murry, 1995; WHO, 1999). The used about 60 mg/day or 120 mg/day of pegaga

extract is effective in the treatment of venous insufficiency (Pointel, et al., 1997). It has

further been proposed that oral application of total triterpenoid fraction of Centella

asiatica (TTFCA) with dosage of 60 mg/day for about 10 weeks is suggested for venous

hypertension treatment (Belcero, et al., 1990). No significant side effects are

experienced with internal or topical used of pegaga except for person who allergic to this

herb (Murray, 1995; Danese, et al., 1994). It may be possible to suggest that the

consumption of 500 ml, accounted for 60.2 mg of total triterpene glycosides, in once or

twice a day of fresh pegaga drink is good enough for our health benefits. On the other

hand, consumption of 600 ml or more heat-treated herbal drink containing about 59.76 –

64.56 mg triterpene glycosides daily could also contribute appreciable amount of active

constituents to the body.

121

Rush, et al. (1993) reported that asiaticoside is converted in vivo to asiatic acid

by hydrolytic cleavage of the sugar moiety. Similarly, Grimaldi, et al. (1990) explained

that asiaticoside is transformed into asiatic acid in vivo through metabolic interaction.

They also suggested that the therapeutic effects of asiaticoside might be mediated

through conversion to asiatic acid. Since the actual absorption of these phytochemicals

on our body is still unclear, a further investigation is needed to prove their significant

role on pharmacological activity and toxicological effect through the consumption of

pegaga as herbal drink.

4.10 Antioxidant activity of asiaticoside

Shukla, et al., (1999a) reported the antioxidant effect of asiaticoside. They

reported that topical application of 0.2% asiaticoside solution twice daily for 7 days to

skin wounds shows an increased in both enzymatic and non-enzymatic antioxidant

activity namely superoxide dismutase (35%), catalase (67%), glutathione peroxidase

(49%), vitamin E (77%) and ascorbic acid (36%) in newly formed tissue. It also results

in several fold decrease in lipid peroxide levels (60%) as measured in terms of their

thiobarbituric acid reactive substance (TBARS). Jayasharee, et al. (2003) also reported

that oral treatment of extract of Centella asiatica for 14 days significantly increased anti-

oxidant enzymes like superoxide dismutase, catalase and glutathione peroxidase.

Results showed that asiaticoside was associated (r=0.63471 and r=0.879 towards FRAP

and FTC, respectively) with antioxidant activity of pegaga drink. However, the

concentration of asiaticoside in pegaga drink was relatively low (3.03-4.32 mg/100ml)

as compared to the amount required for antioxidant effect (0.2g/100ml). Shukla, et al.,

(1999) also reported that lower concentrations of asiaticoside (0.05% and 0.1%) were

found to have no significant effect on wound healing activity. Results obtained from the

study assumed that the presence of very small amount of asiaticoside in pegaga drink

indicates a relatively low or negligible contribution on total antioxidant activity.

122

CHAPTER 5

CONCLUSION AND RECOMMENDATION

5.1 Conclusion

Fresh pegaga drink (F) contained about 1470.14 mg/100ml of total polyphenol

(GAE equivalent), which was significantly higher than pasteurized sample at

65 C/15 minutes (903.23 mg/100ml) and 80 C/5 minutes (805.54 mg/100ml);

and canned pegaga drink (730.27 mg/100ml).

The degradation of ascorbic acid occurred at a higher rate in canned pegaga

drink, followed by pasteurization at 80 C/5 minutes and 65 C/15 minutes. The

concentration in fresh pegaga drink was significantly higher, which is about

4.23mg/100ml.

Antioxidant assay results revealed that the control sample (F) of pegaga drink

exhibited much higher (P<0.05) antioxidant activity than heat-treated samples.

The FRAP values of 860 µmol/litre was obtained from untreated or fresh sample

(F) and the activity from 404 to 740 µmol/litre were observed in heat-treated

drinks. The % inhibition of peroxidation was 72% for fresh sample (F) and in

the ranged of 26-56% for heat-treated samples. The reduction of natural

occurring antioxidants in pegaga drink could be due to the transformation of the

123

food component during processing into compound that possessed pro-oxidant

property.

The two assays (FRAP and FTC) were strongly correlated (r=0.93) at p=0.05.

However, very low correlation was obtained (r=0.54) when antioxidative activity

of pegaga drink, synthetic antioxidant (BHT) and natural antioxidant (ascorbic

acid and vitamin E) were taken into account.

Initial results suggested that total polyphenol is a major contributor to the

antioxidant activity of herbal pegaga drink, which significantly correlated

(r=0.8071) at p<0.05 towards FRAP assay.

The analysis of individual component of triterpene glycosides shows a different

trend. The concentration of madecassoside in fresh sample (F) was 12.2-22.1%

higher than heat-treated drink. Canning process retains higher level of

madecassoside as compared to pasteurization at 65 C/15 minutes (A) and 80 C/5

minutes (B).

The maximum amount of asiaticoside (4.32 mg/100ml) was observed during

processing at 65 C/15 minutes (A). The amount in fresh sample was

3.92mg/100ml. This result suggested that the heat processing at moderate

temperature (65 C) and longer heating period (15minutes) enhanced the

extraction efficiency of asiaticoside.

The average content of madecassic acid was higher in canned drink (3.22

mg/100ml) followed by B (3.02mg/100ml), A (2.70 mg/100ml) and fresh sample

(2.56 mg/100ml).

The heat processing of pegaga drink resulted lower amount of asiatic acid, where

the asiatic acid content varies in the range of 2.45-0.97 mg/100ml. The non-

thermally treated drink or fresh sample (F) contained higher amount of total

124

triterpene glycosides.followed by sample A, B and C. The change in these

components is due to chemical degradation and conversions of some

thermolabile asiaticoside and madecassoside to another components.

5.2 Recommendations and further works

Changes in antioxidant activities and other constituents in pegaga drink are

already demonstrated. However, the specific components, structures and

mechanism that involved in antioxidant activity should also be studied in detail.

The future research works should be focuses on flavonoid content such as

quercetin and kaempferol and their contribution to antioxidant activity in pegaga

drink.

Previous works by Vimala, et al. (2003) reported that pegaga leaves extract

contain a high antioxidant activity towards superoxide free radical scavenging

activity (SS) and radical scavenging activity (DPPH). The similar assessment

should be carried out for pegaga drink in order to evaluate its ability to reduce

the excess free radical and to determine the level of prevention of tissue and cells

damage. Scavenging of DPPH radical determines the antioxidant potential of the

test sample, which shows its effectiveness, prevention, interception and repair

mechanism against injury in biological system.

Further research should be oriented to the optimisation of antioxidant activity

and triterpene glycosides content in herbal pegaga drink in order to understand

the factors controlling the retention of these phytochemicals. Beside, the data on

the potential interactions of natural bioactive constituents with other food

components during industrial processing and home preparation of food and

beverages is very limited. Therefore, the optimum retention of these

phytochemicals under various processing parameters and their interactions with

125

other food components should also be studied in future. Furthermore, their

stability under different parameters such as storage conditions, packaging, light,

water activity, degree of oxidation and High Temperature Short Time (HTST)

processing technology need also be evaluated in future.

On the other hand, consumers believe that herbal pegaga products that were

assumed rich in antioxidants and triterpene glycosides may afford a degree of

protection against free radical damage and higher in pharmacological activity.

The data on their adsorption in blood stream, pharmacological benefit and

toxicity over the range of studies of still remain unknown and further information

should be provided.

126

REFERENCES

Abdul Hamid, A., Md. Shah, Z., Muse, R. and Mohamed, S. (2002). Characterisation of antioxidant activities of various extracts of Centella asiatica (L) Urban. FoodChemistry. 77: 465-469

Abdurahman O. Musaiger, Mousa A. Ahmed and Maddur V. Rao (1998). Chemical composition of some traditional dishes of Oman. Food Chemistry. 76 (1/2): 17-22

Addo, A.A. (1981). Ascorbic acid retention of stored dehydrated Nigerian vegetables. Nutrition Report International. 24(4): 769-775

Akinyele, I.O., Keshinro, O.O. and Akinnawo, O.O. (1990). Nutrient Losses During and After Processing of Pineapples & Oranges. Food Chemistry. 37: 181-188

Ames, B.M. (1983). Dietary carcinogens and anticarcinogens : oxygen radicals and degeneratives diseases. Science. 221: 1256-1263

Anese M. dan Nicoli M.C. (2001). Optimising Phytochemical Release By Process Technology” In: Dfannhauser, W., Fenwick, G.R. and Khokhar, S. Eds. Biologically-active phytochemicals in food. Cambridge, U.K.: Bookcraft Ltd. 455-470

Annison, G. and D.L. Topping , D.L. (1994). Nutritional role of resistant starch: chemical structure vs physiological function. Annual Reviews of Nutrition 14: 297–320.

Anon (1980). Recommended dietary allowances. Committee on dietary allowance. Washington, DC, USA: Food and Nutrition Board (NAS/NRC).

Anon (1990). Malaysia Food Act 1983 and Food Regulation 1985. Kuala Lumpur: MDC Sdn.Bhd. 191

Business Time (2000). Content in Herbal Market Must Rise. Business Time. 25.04.2000. Kuala Lumpur

127

AOAC (1980). AOAC Official Method of Analysis. 13th ed. Arlington: VA, Association of Official Analytical Chemists, Inc.

AOAC (1984). AOAC Official Method of Analysis. 14th ed. Arlington: VA, Association of Official Analytical Chemists, Inc.

Arena E., Fallico, B. dan Maccarone, E. (2001). Evaluation of antioxidant capacity of blood orange juices as influences by constituents, concentration process and storage. Food Chemistry. 74(4): 423-427

Asp, N.G. (1995). Classification and methodology of food carbohydrates as related to nutritional effects. American Journal of Clinical Nutrition .61: 930S - 937S .

Babu,T.D., Kuttan, G. and Padikkala, J. (1995). Cycotoxic and anti-tumor properties of certain taxa of Umbelliferae with special reference to Centella asiatica (L.) Urban. Journal of Ethnopharmacology. 48(11): 53-57

Bao, B. and Chang, K.C. (1994). Carrot juice colour, carotenoids and nonstachy polysaccharides as affected by processing condition. J. Food Science. 59:1155-1158

Barlow, S.M. (1990). Toxicological aspects of antioxidants used as food additives. In: Hudson, B.J.F. Ed. Food Antioxidant. London: Elsvier. 253-307

Basaga, H., Acikel, F. and Tekkaya, C. (1997). Antioxidative and free radical scavenging properties of rosmery extract. Lebensm.-Wiss.u.-Technol. 31: 694-698

Belcaro, G.V., Grimaldi, R. and Guidi, G. (1990). Improvement of Capillary Permeability in Patients with Venous Hypertension after Treatment with TTFCA. Angiology. 4: 533-540

Bengtsson, B.l. (1969). Nutritional effects of food processing. J. Food technol. 4: 141-145

Bender, A.E. (1987). Development in Food Preservation-4. Cambridge: Elsevier Applied Science. 1.

Bender, D.A. (1993). Micronutrients: the vitamins and minerals. An introduction of nutrition and methabolism. 2nd ed. London: UCL Press.

Benzie, I.F.F. dan Strain J.J. (1996). The Ferric Reducing Ability of Plasm (FRAP) as Measure of ‘Antioxidant Power’ : The Frap Assay. Analytical Biochemistry. 239:70-76

128

Beveridge, T., Franz, K.Y. and Harrison, J.E. (1986). Clarified natural apple juice: production and storage stability of juice and concentrate. Journal of Food Science. 51(433): 411-414

Birch, G.G., Bointon, B.M., Rolfe, E.J. and Selman, J.D. (1974). Quality changes related to vitamin C in fruit juice and vegetables processing. In: Birch, G.G. and Parker, K. Eds. Vitamin C. London: Applied Science. 40

Bloch, A. and Thompson, C.A. (1995). Positition of the American Diet Association: Phytochemicals and Functional Food. J. Am. Diet Assoc. 95: 493

Boiteau, P. and Ratsimamanga, A.R. (1956). Asiaticoside extracted from Centellaasiatica, its theraputic uses in the healing of experimental or refactory wounds, leprosy, skin tuberculosis and lupus. Therapie. 11: 125-149

Bolin, H.R. and Stafford, A.E. (1974). Effect of processing and storage on provitamin A and vitamin C in apricots. J. Food Science. 39: 1034-1036

Bonte, F., Dumas, M., Chaudagne, C. and Meybeck, A. (1994). Influence of Asiatic acid, Madecassic Acid and Asiatcoside on Human Collagen I Synthesis. PlantaMedica. 60: 133-135

Bors, W. and Saran, M. (1987). Radical scavenging by flavonoid antioxidants. FreeRadical Res. Commun. 2: 289-294.

Buedo, A.P., Elustondo, M.P. and Urbicain, M.J. (2000). Non-enzymatic browning of peach juice concentrate during storage. Innovative Food Science & Emerging Technologies. 1(4):. 255-260

Brinkhaus, B., Lindner, M., Schuppan, D. and Hahn, E.G. (2000). Chemical, pharmacological and clinical profile of the East Asian medical plant Centellaasiatica: Review Article. Phytomedicine. 7(5): 427-448

Burnouf-Radosevich, M. and Delfel, N.E. (1986). High-performance liquid chromatography of triterpene saponins. J. Chromatography A. 368: 433-438.

Cabritta, L., Fossen, T. and Andersen, O.M. (2000). Colour and stability of six common anthocyanidin 3-glucoside in aqueous solution. Food Chemistry. 68: 101-107

Cao, G., Sofic, E. and Prior, R.L. (1996). Antioxidant capacity of tea and common vegetables. J. Agric. Food Chem. 44: 3426-3431

Carabasa-Giribert, M. and Ibraz-Ribas, A. (2000). Kinetics of colour development in aqueous glucose systems at high temperatures. Journal of Food Engineering. 44(3): 181-189

Caragay, A.B. (1992). Cancer-preventive foods and ingredients. Food Technol. 4: 65-68

129

Carr,A.C. and Frei, B. (1999). Toward a new recommended dietary allowance for vitamin C based on antioxidant and health effect in human. American Journal og Clinical Nutrition. 69: 1086-1107

Castellani, C., Marai, A. and Vacchi, P. (1981). The Centella asiatica. Bolletin chimica farmacia. 120: 570-605

Cerutti, P.A. (1985). “Prooxidant states and tumor promotion”. Science. 227. 375-381

Chaterjee, T.K., Chakraborty, A. and Pathak, M. (1992). Effect of Plant Extract Centella asiatica (Linn.) on cold resistant Stress Ulcer in Rats. Ind. J. Exp. Biol.30: 889-891

Cheftel, J.C., Cuq, J. and Lorient, D. (1985). Amino acids, peptides and proteins. In: Fennema, O.R. Ed.. Food Cehmistry. 2nd ed. New York: Marcel Dekker Inc. 319.

Che Rahani, Z. (1998). Teknologi Pemprosesan Minuman Buah-buahan. Nota Kursus Pemprosesan Hasilan Buah-buahan Tropika. Johor Bahru: MARDI 1-13

Chen, Y.J., Dai, Y.S., Chen, B.F., Chang, A., Chen, H.C., Lin, Y.C., Chang, K.H., Lai, Y.L., Chung, C.H. and Lai, Y.J. (1999). The effect of tetrandrine and extracts of Centella asiatica on acute radiation dermatitis in rats. Biol Pharm Bull. 22(7): 703-706

Cheng, C.L. and Koo, M.W.L. (2000). Effect of Centella asiatica on ethanol induced gastric mucosal lesions in rats. Life Science. 67(21): 2647-2653

Choi, J.H., Kim, D.H., Sung, W.J. and Oh, S.K. (1982). Kinetic studies on the thermal degradation of ginsenosides in ginseng extract. Hanguk Sikp’um Kwahakhoe Chi. 14: 197-202

Chuah, E.C. (1984). Principle of Thermal Processing. Maklumat Teknologi Makanan. ISSN 0127-4821. June 1984. Kuala Lumpur. MARDI. 1-2

Clegg, K.M. (1966). Citric acid and the browning of solutions containing ascorbic acid. Journal of the Science of Food and Agriculture. 17(12): 546

Cook, N.C. and Samman, S. (1996). Flavonoids – chemistry, merabolism, cardioprotective effect and dietary sources. Nutritional Biochemistry. 77: 66-76

Cornwell, C.J. and Woodstad, R.E. (1981). Causes of browning inpear juice concentrate during storage. Journal of Food Science. 46: 515-518

130

Court, W.A., Hendel, J.G. and Elmi, J. (1996). Reversed-phase high peformance liquid chromatography determinations of ginsenoside of Panax quinquefolium. J.Chromatography A. 755: 11

Crozier, A., Micheal, E.J.L., McDonald, M.S., Black, C. (1997). Quantitative analysis of the flavonoid content of commercial tomatoes, onion, lettuce and celery. J.Agric. Food Chem. 45. 590-595

Cuvelier, M.E., Berset, C. and Richard, H. (1994). Antioxidant constituents in sage (Salvia officinalis). J. Agric. Food Chem.42: 665-669

Danese, P., Carnevali, C., Bertazzoni, M.G. (1994). Allergic contact dermatitis due to Centalla asiatica extract. Contact Dermatitis. 31: 201

Dawes, H.M. and Keene, J.B. (1999). Phenolic composition of kiwi fruit juice. J. Agric. Food Chemistry. 47(6): 2398-2403

De Lucia, C., Sertie, J.A.A., Camargo, E.A. and Panizza, S. (1997). Pharmacological and toxicological studies on Centella asiatica extract. Fitoterapia. 68: 413-416

Desrosier, N.W. and Desrosier, J.N. (1977). The Technology of Food Preservation. Westport, Connecticut: AVI Publishing Company, Inc.

Diplock, A.T. (1994). Antioxidant nutrient and diseases prevention: an overview. American Journal of Clinical Nutrition. 53(1): 189s-193s

Donovan, J.L., Meyer, A.S. and Waterhouse, A.L. (1998). Phenolic composition and Antioxidant activity of Prunes and Prune Juice (Prunus domestica). J. Agric. Food Chemistry. 46: 1247-1252

Dorman, H.J.D., Peltoketo, A., Hiltunen, R. and Tikkanen, M.J. (2003). Characterisation of the antioxidant properties of de-odourised aqeous extracts from selected Lamiaceac herbs. Food Chemistry. 83(2): 255-262

Duh, P. and Yen. G. (1997). Antioxidative activity of three herbal water extracts. FoodChemistry. 60(4): 639-645

Duke, J.A. (1992). Handook of phytochemical constituents of GRAS herbs and economic plants. Boca Raton, FL: CRC Press.

Dziezak, J.D. (1986). PreservativeSystems in Food, Antioxidatives and Anti Microbial Agents. Food Technol. 40: 94-131

Eichner, K. (1981). Antioxidative effect of Millard reaction intermediates. Progress in Food Nutrition and Science. 5: 441-451

131

Elkins, E.R. (1979). Nutrien Content of Raw & Canned Green Beans, Peaches & Sweet Potatoes. Food Technol. 66-79

Erdman Jr, J.W. (1979). Effect of Preparation and Service of Food and Nutrient Value. Food Technol. 62-65

Ewald, C., Fjelkner-Modig, S., Johnsson, K., Sjoholm, I. And Akesson, B. (1999). Effect of processing on major flavonoids in processed onion, green bean and peas. Food Chemistry. 64: 231-235

Faridah A.F. (1998). The commercialization of Local Medicinal Herb in Skin Care and Toiletries Products. In: Nair M.N.B. and Nathan G. Eds. Medicinal Plants: CURE for 21st Century (Biodiversity, Conservation and Utilization of Medicinal Plants. Selangor: UPM. 130-132

Favell, D.J. (1998) A comparison of the vitamin C content of fresh and frozen vegetables. Food Chemistry. 621: 59-64

Fennema, O.R. (1985). Water and Ice. In: Fennema, O.R. Ed. Food Cehmistry. 2nd ed. New York: Marcel Dekker Inc. 23-67.

Fezah, O., Radzali, M. Marziah, M., Johari,R. and Mohd. A.S. (2000). Polyphenol and Salicyclic Acid Levels in Fresh and Air-dried Powder of Centella asiatica, L. (Urban). Proceeding of the 16th National Seminar on Natural Products. Selangor: MARDI. 107-110

Francis, F.J. (1985). Pigments and other colorants. In: Fennema, O.R. Ed. FoodChemistry. 2nd ed. New York: Marcel Dekker Inc. 546-582

Frankel, E.N. (1993). In search of better methods to evaluate natural antioxidants and oxidative stability in food lipids. Trends in Food Science & Technology. 4: 220-225

Frankel, E.N., Waterhouse, A.L. and Teissedre, P.L. (1995). Principal phenolic phytochemicals in selected California wines and their antioxidant activity in inhibiting oxidation of human low-density proteins. J. of Agric. Food Chemistry. 43: 890-894

Frankel, E.N. and Meyer, A.S. (2000). The problem of using one dimensional method to evaluate multifunctional food and biological antioxidants. J. Sci. Food Agric. 80:1925-1941

Fransworth N.R., Bingel A..S., and Fond H.H.S. (1976). Oncogenic and tumor promoting spermatophytes and pteridophytes and their active principles. Cancer Treatment Report. 60(8): 1171-1214.

132

Gardner, P.T., White, T, A.C., McPhail, D.B. and Duthie, G.G. (2000). The relative contributions of vitamin C, carotenoids and phenolics to the antioxidant potential of fruit juices. Food chemistry. 68: 471-474

Gartner, C., Stahl, W. and Sies, H. (1997). Lycopene is more bioavailable from tomato paste than from fresh tomatoes. Am. J. Clin. Nutr. 66: 116-122

Gazzani, G., Papetti A., Massolini, G. and Daglia, M. (1998). Anti- and Prooxidant Activity of Soluble Components of Some Common Diet Vegetables and Effect of Thermal Treatment. J. Agric.Food Chem. 46: 4118-4122

Gil-Izquierdo, A., Gil M. I., Conesa, A.M. and Ferreres, F. (2001). Effect of storage temperatures on Vitamin C and phenolic content of artichoke (Cynara scolymus L.) heads. Innovative Food Science & Emerging Technologies. 2(3): 199-202

Gil-Izquierdo, A., Gil M. I., and Ferreres, F. (2002). Effect of processing techniques at industrial scale on orange juice antioxidant and beneficial health compounds. J.Agric. Food Chem. 50(18): 5107-5114

Godoy, H.T. and Rodriquez-Amaya, D.B. (1987). Changes in individual carotenoids on processing and storage mango (Magnifera indica) slices and puree. Int. J. Food Sci. Technol. 22:451-460.

Goh, S.H., Chuah, C.H., Mok, J.S.L. and Soepadmo (1985). Malaysian Medicinal Plants for the Treatment of Cardiovascular Diseases. Kuala Lumpur: Academe Art and Printing Services Sdn. Bhd. 77-78.

Gordon, M. (1990). The mechanism of antioxidation action in vitro. In: Hudson, B.J.F. Ed. Food Antioxidants. London: Elsevier. 1-18

Gordon, M. (2001). The development of axidative rancidity in foods. In: Pokorny, J., Yanishlieva, N. and Gordon, M. Eds. Antioxidants in food: practical applications. Cambridge, England: Woodhead Publishing Limited. 7-21

Goni, L.G., Manas, E. and Saura Calixto, F. (1996). Analysis of resistant starch; a method for foods and food products. Food Chemistry 56(4): 455–459.

Gunther, B. and Wagner, H. (1996). Quantitative determination of triterpene in extracts and phytopreparation of Centella asiatica (L.) Urban. Phytomedicine. 3: 59-65

Grimaldi, R., De Ponti, F.D., D’Angelo, L., Caravaggi, M., Lecchini, S. Frigo, G.M. and Crema, A. (1990). Pharmacokinetics of the total triterpenic fraction of Centellaasiatica after single and multiple asministrations to healthy volunteers. A new assay for asiatic acid. J Ethnopharmacol. 28(2): 235-241

Guseva, N.G., Starvoitova, M.N. and Mach, E.S. (1998). Madeccassol treatment of systemic and localized scleroderma. Ter Arkh. 70(5): 58-61

133

Halliwell, B., Aeschbach, R., Loliger, J. and Aruoma, O.I. (1995). “The characterization of antioxidants.” Food and Chemical Toxicology. 33(7). 601-617

Haraguchi, H., Hashimoto, K., and Yagi, A. (1992). Antioxidative substances in leaves of Polygonum hydropiper. J. Agric. Food Chem. 40: 1349

Harborne, J.B. (1998). Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis. 3rd ed. London: Champman and Hall.

Harris, R.S. (1975). General Discussion on the Stability of Nutrients. In: Harris, R.S. and Karmas, E. Eds. Nutritional Evaluation of Food Processing. 2nd ed. Westport: The AVI Publishing Co. Inc.

Hayase, F., Hirashima, S., Okamoto, G. and Kato, H. (1989). Scavenging of active oxygen by melanoidins. Agricultural and Biological Chemistry. 53: 3383-3385

Hemeda, H.M. and Klein, B.P. (1990). Effect of naturally occurring antioxidants on peroxidase activity of vegetables extracts. Journal of Food Science. 55: 184-185

Heinonen, I.M., Lehtonen, P.J. and Hopia, A.I. (1998). Antioxidant Activity of Berry and Fruit Wines and Liquors. J. Agric. Food Chem. 46: 25-31

Henrix, C.M. and Redd, J.B. (1995). Chemistry and technology of citrus juices and by-products. In: Ashurst, P.R. Ed. Production and packaging of non-carbonated fruit juice and fruit beverages. London: Blackie Academic Professional. 53-87

Hertog, M.G.L., Hollman, P.C.H. and Katan, M.B. (1992). Content of potentially anticarcinogenic flavonoids of 28 vegetables and 9 fruits commonly consumed in the Netherlands. J. Agric. Food Chemistry. 40: 2379-2383

Hertog, M.G.L. and Hollman, P.C.H. (1996). Potential health effects of dietary flavonol quercetin. European Journal of Clinical Nutrition. 50: 63-71

Hudson, B.J.F. (1998). Food Antioxidants. London: Elsvier.

Husin, S.R., Cillard, J. and Cillard, P. (1987). Hydroxyl radical scavenging activity of flavanoids. Phytochemistry. 26: 2489-2491.

Hussein, l. and El-Tohamy (1990). Vitamin A potency of carrot and spinach carotenes in human metabolic studies. Int. J. Vit. Nutr. Res. 60: 229-235

Hunter, K.J. and Fletcher, J.M. (2002). The antioxidant activity and composition of fresh, frozen, jarred and canned vegetables. Trends in Food Science & Technology. 10: 94-100

134

Hurt, H.D. (1979). Effect of Canning in the Nutritive Value of Vegetables. Food Technol. 62-65

Imark, C., Kneubuhl, M. dan Bodmer S. (2000). Occurance and activity of natural antioxidants in herbal spirits. Innovative Food Science & Emerging Technologies. 1(4): 239-243

Inamdar, P.K., Yeole, R.D., Ghogare, A.B. dan De Souza, N.J. (1996). Determination of biologically active constituents in Centella asiatica. J. of Chromatography A..742: 127-130

Indu Bala Jagnathan and Ng, LeeTiek (1999). Herbs: The Green Pharmacy of Malaysia. Kuala Lumpur: Vinpress Sdn. Bhd. 21-23

Jackman, R.l., Yada, R.Y., Tung, M.A. and Speers, R.A. (1987). Anthocyanins as food colorants – a review. J. Food Biochem. 11: 201-247

Jadhav, S.J., Nimbalkar, S.S., Kulkarni, A.D. and Madhavi, D.L. (1996). Lipid Oxidation in Biological and Food System. In: Madhavi, D.L., Deshpande, S.S. and Salunkhe, D.K. Eds. Food Antioxidants. London and New York: Elsevier Applied Science. 5-63.

Jayashree, G., Kurup Muraleedhara, G., Sudarslal, S. and Jacob, V.B. (2003). Anti-oxidant activity of Centella asiatica on lyphoma-bering mice Fitoterapia. 74(5):431-436

Jeffery, B., Harbone, FRS., Baxter, H., Moss, G.P. Eds (1999). Phytochemical Dictionary. A Handbook of Bioactive Compounds from Plants. 2nd ed. UK: Taylor & Francis Ltd. 802

Johnson, J.R., Braddock, R.J. and Chen, C.S. (1995). Kinetic of ascorbic acids loss and nonenzymatic browning in orangeserum:experimental rate constants. Journal of Food Science. 60: 502-505

Julkunen-Tiitto, R. (1985). Phenolic constituents in the leaves of Northern Willows: methods for the analysis of certain phenolics. J. Agric. Food Chem. 33: 213-217.

Kaack, K and Austed, T. (1998). Interaction of vitamin C and flavanoids in elderberry (Sambucus nigra L.) during juice processing. Plant Foods for Human Nutrition.52: 187-198

Kaanane, A., Kane, D. and Labuza, T.P. (1988). Time and Temperature Effect on Stability of Moroccan Processed Orange Juice during Storage. Journal of Food Science. 53(5): 1470-1473

135

Kabasakalis, V., Siopidou, D. and Moshatou, E. (2000). Ascorbic acid content of commercial fruit juices and its rate of loss upon storage. Food Chemistry. 70: 325-328

Kartnig, T. (1988). Clinical Application of Centella asiatica (L.) Urb. In: Cracker, L.E. and Simon J.E. Eds. Herbs, Spices and Medicinal Plants: Recent Advance in Botany, Horticulture and Pharmacology. Pheonix: Oryx Press. 145-178

Khal, R. dan Hilderbrant, A.G. (1986). Methodology for studying antioxidant activity and mechanism of action of antioxidant. Food Chem.Toxicol. 24: 1007-1014

Kikugava, K., Kunugi, A. and Kurechi, T. (1990). Chemistry and Implications of Degradation of Phenolic Antioxidants In: Hudson, B.J.F., Ed. Food Antioxidants.London and New York: Elsevier Applied Science. 65-98.

Kikuzaki, H. & Nakatani, N. (1993). Antioxidant Effects of Some Ginger Constituents. Journal of Food Science. 58(6): 1407-1410

Klurfeld, D.M. (1992). Dietary fiber-mediated mechanisms in carcinogenesis. CancerRes. 52(7): 2055-2059.

Koleva, I.I., Van Beek, T.A., Linssen, J.P.H, de Groot A. dan Evstatieva L.N. (2002). Screening of Plant Extract for Antioxidant Activity: a Comparative Study on Three Testing Methods. Phytochemical Analysis.13: 8-17

Koo, H.M. and Suhaila Mohamed (2001). Flavonoid (Myricetin, Quercetin, Kaempherol, Luteolin, and Apigenin.) Content of Edible Tropical Plants. JournalAgriculture Food Chemistry. 49(6): 3106-3112

Labuza, T.P. (1985). An Integrated Approach To Food Chemistry. In: Fennema, O.R. Ed.. Food Cehmistry. 2nd ed. New York: Marcel Dekker Inc. 913-938

Langley-Evans, S.C. (2000). Antioxidant potential of green and black tea determined using the ferric reducing power (FRAP) assay. International Journal of food Science and Nutrition. 51: 181-188

Larson, R.A. (1988). The antioxidants of higher plants. Phytochemistry. 27(4):. 969-978

Larrauri, J.A., Ruperez, P., Bravo, L. and Saura-Calixto, F. (1996). High dietary fibre powders from orange and lime peels: associated polyphenols and antioxidant capacity. Food Chemistry. 29(8): 757-762

Lathrop, P.J. and Leung, H.K. (1980). Rates of Ascorbic Acid Degradation During Thermal Processing of Canned Pea. Journal of Food Science. 45: 152-153

136

Lau, A., Woo, S. and Koh, H. (2003). Analysis of saponin in raw and steamed Panaxnotoginseng using high-performance liquid chromatography with diode array detection. Journal of Chromatography A, 1011: 77-87

Lea, A.G.H. and Arnold, G.M. (1978). The Phenolics of Ciders: Bitterness and Astringency. J. Sci. Food Agric. 29: 478

Lea, A. G. H. (1991). Apple juice. In: Hick, D. Ed. Production of non-carbonated fruit juice and fruit beverages. Glassgow: Blackie. 182-225.

Lea, A.G.H. (1992). Flavour, colour and stability of fruit products: the effect of polyphenols. In: Hemingway, R.W., Laks, P.E. Eds. Plant polyphenols. New York: Plenum Press. 827-847

Lindley, M.G. (1998). The impact of food processing on antioxidants in vegetable oil, fruits and vegetable. Trends in Food Science & Technology. 9(8-9): 336-340

Lindsay, R.C. (1985). Food Additives. In: Fennema, O.R. Ed. Food Cehmistry. 2nd ed. New York: Marcel Dekker Inc. 629-688

Ling, A.P.K., Marziah, M. and Tan, S.E. (2000). Triterpenoids Distribution in Whole Plant and Callus Cultures of Centella asiatica Accessions. Proceeding of the 16th

National Seminar on Natural Products. Selangor: MARDI. 165-168

Lingnert, H. and Waller, G.R. (1983). Stability of antioxidants formed during histidine and glucose by Millard reactions. Journal of Agricultural and Food Chemistry. 31: 27-30

Lolinger, J. (1991). The use of antioxidant in foods. In: Arouma, O.I. and Halliwell, B, Eds. Free Radical and Food Adhves. London: Taylor and Francis. 121-150

Lu, F. and Foo, L.Y. (1995). Phenolic antioxidant component of evening primrose. In: Ong, A.S.H., Niki, E. and Packer, L. Eds. Nutritional, lipids, health and diseases. Champaign: American Oil Chemists Society Press.

Luh, B.S. (1980). Tropical Fruit Beverages. In: Nelson P.E. and Tressler, D.K. 3rd ed. Fruit and Vegetables Juice Processing. Westport, Connecticut: The AVI Publishing, Co. Inc. 344-435

Lunder, T.l. (1992). Catechins of green tea: antioxidant activity. In: Huang, M.T., Ho, C.T. and Lee, C.Y. Eds. Phenolic Compounds in Food and their Effects on Health: Antioxidant and Cancer Prevention. Washington: American Chemical Society. 114-120

Mallet, J.F., Cerrati, C., Ucciani, E, Gamisans, J. and Gruber, M. (1994). Antioxidant activity of plant leaves in relation to their alpha-tocopherol content. Foodchemistry. 49: 61-65

137

Madhavi, P.L., Deshpande, S.S and Salunkhe, D.K. Eds (1996a) Food Antioxidant: Technological, toxicological and health perspectives. New York: Marcel Dekker, Inc. 1-4

Madhavi, P.L., Singhal, R.S. and Kulkarni, P.R. (1996b). Technological Aspects of Food Antioxidants In: Madhavi, P.L., Deshpande, S.S and Salunkhe, D.K., Eds. Food Antioxidant: Technological, toxicological and health perspectives. New York: Marcel Dekker, Inc. 159-266

Madsen, H.L. and Bertelsen, G. (1995). Spices as antioxidants. Trends Food Sci. Technol. 6: 271-277

Mahanom, H., Azizah, A.H. and Dzulkifly, M.H. (1999). Effect of different drying methods on concentrations of several phytochemicals in herbal preparation of 8 medicinal plants leaves. Mal. J. Nutr. 5: 47-54

Majchrzak, D., Mitter, S. and Elmadfa, I. (2004). The effect of ascorbic acid on total antioxidant activity of black and green tea. Food Chemistry. 88(3): 447-451

Mak, P.P., Ingham, B.H. and Ingham, S.C. (2001). Validation of Apple Cider Pasteurization Treatments against Escherichia coli, Salmonella and Listeria Monocytogenes. Journal of Food Protection. 64(11): 1679-1689

Makris, D.P. dan Rossiter, J.T. (2001). Domestic Processing on Onion Bulbs (Alliumcepa) and Asparagus Spears (Asparagus officinalis): Effect on Flavanol Content and Antioxidant Status. J. Agric. Food Chem. 49: 3216-3222

Manzocco, L., Anese, M. and Nicoli, M.C. (1998). Antioxidant properties of tea extract as affected by processing. Lebensm.-Wiss. u. Technol. 31: 694-698

Manzocco, L., Mastrocola, D. and Nicoli, M.C. (1999). Chain-breaking and oxygen scavenging properties of wine as affected by some technological procedures.Food Research International. 31(9): 673-678

Manzocoo, L., Calligaris, S., Mastrocola, D., Nicoli, M.C. and Lerici, C.R. (2000). Review of non-enzymatic and antioxidant capacity in processed foods. Trends in Food Science & Technology. 11(9-10): 340-346

Marin, F.R., Martinez, M, Urbie Salgo, T., Castillo, . and Frutos, M.J. (2002). Changes in nutraceutical composition of lemon juices according to different industrial extraction systems. Food Chemistry. 28: 319-324

138

Mazzotta, A.S. (2001). Thermal Inactivation of Stationary-Phase and Acid-Adapted

Escherichia coli, Salmonella and Listeria Monocytogenes in Fruit Juices. Journal

of Food Protection. 64(3): 315-32

Mehrlich, F.P. and Felton, G.E. (1971). Pineapple Juicee. In: Tressler, D.K. and Joslyn, M.A. Fruit and Vegetable Juice Processing Technology. 2nd ed. Westport, Connecticut: The AVI Publishing Co. Inc. 185

Meiners, C.R., Derise, N.L., lai, H.C., Crews, S.J., Ritchey, S.J. and Murphy, E.W. (1976). The content of nine mineral eleme ts in raw and cooked mature dry legumes. J. Agric. Food Chem. 24: 1126-1130

Meng, Z.M. and Zheng, Y.N. (1988). Determination of asiaticoside contained in sanjinplan. Zhonggtuo yaoke daxue xuebao. 19: 205-206

Miki, N. and Akatsu, K. (1971). Stability of Tomato Juice of Lycopene from Inner and Outer Part of the Flash. Nip. J. Food Science & Technology. 18: 309-312

Miller, N.J., Diplock, A.T. dan Rice-Evans C.A. (1995). Evaluation of the Total Antioxidant Activity as a Marker of the Deterioration of Apple Juice on Storage. J. Agric. Food Chem. 43: 1794-1801

Min, Z., Chunli, L. and Ping, C. (2004). Effect of processing conditions of the green-leafy vegetable juice enriched with selenium on its quality stability. Journal of Food Engineering. 62(4): 393-398

MOH (2002). Pharmaceutical Service Division Annual Report 2002. Petaling Jaya, Selangor: Ministry of Health Malaysia.

MOH (2004). “Berita Ubat-ubatan.” Newaletter of The Drug Control Autority Malaysia. Petaling Jaya: National Pharmaceutical Control Bureau, Ministry of Health Malaysia. Mac 2004. 23(1): 8-112

Mohamad Faisal, A.F. (2000). Current Scenario of Malaysian Herbal / Natural Product Industry. J. Trop. Med. Plants. 1: 36-42

Mohd Zin, Z., Abdul Hamid, A. dan Osman, A. (2001). Evaluation of the Antioxidant Activity of Exracts from Mengkudu (Morinda citrifolia) Root, Fruit and Leaf. Proceedings of Conference on Functional Food- Latest Development. Putra Jaya: UPM. 139-146

Monnier, L., Pham, T.C., Aguiree, L., Orsetti, A. and Mirouze, J. (1978). Influence of indigestible fibers on glucose tolerance. Diabetes Care. 1: 83-88

139

Morales,F.J. and Jimenez-Perez (2001). Free radical scavenging capacity of Millard reaction products as related to colour and fluorescence. Food Chemistry. 72(1):119-125

Morganti, P., Fionda, A., Elia,U. dan Tiberi, L. (1999). Extraction and analysis of cosmetic active ingredients from an anti-cellulitis transdermal delivery system by high-performance liquid chromatography. Journal of Chromatographic Science. 37(2): 51-55

Muhammed Idris M.A., Noraini H. and Ng L.T. (1999). Medicinal plants: trade and investment prospects in Malaysia. In: Ali A.M., Shaari, K. and Zakaria Z., Eds. Phytochemicals and biopharmaceutins from the Malaysian rain forest. Malaysia: FRIM. 21-30.

Moyer, J.C. and Aitken, H.C. (1971). Apple Juice. In: Tressler, D.K. and Joslyn, M.A. Fruit and Vegetable Juice Processing Technology. 2nd ed. Westport, Connecticut: The AVI Publishing Co. Inc. 186-233

Murray, M.T. (1995). The Healing Power of Herbs. Rocklin, CA: Prima Publishing. 173-183.

Namiki, M. and Hayashi, T. (1983). The Millard Reaction in Foods and Nutrition. Washinton D.C: American Chemical Society. 21

Namiki, M. (1990). Antioxidants/antimutagens in food Critical Review of Food Science and Nutrition. 29: 273-300

Nergiz, C. and Otles, S. (1993). Chemical composition of Nigella sativa L. seeds. Food Chemistry. 48: 259-261

Newall, C.A., Anderson, L.A. and Philipson, J.D. (1996). Herbal Medicine- A Guide for Health-care Profesionals. London: The Pharmaceutical Press. 296

Nicoli, M.C., Anese, M., Manzocco, L. and Lerici, C.R. (1997a). Antioxidant Properties of Coffee Brews in Relation to the Roasting Degree. Lebensm.-Wiss. U.-Technol. 30: 292-297

Nicoli, M.C., Anese, M. dan Parpinel, M. T., Franceschi, S. and Lerici, C.R. (1997b). Loss and/or formation of antioxidants during food processing and storage. Cancer Letters. 114: 1-4

Nicoli, M.C., Anese, M. dan Parpinel, M. (1999). Influence of processing on the antioxidant properties of fruit and vegetables. Trends in Food Science & Technology. 10(3): 94-100

Niwa, Y. and Miyachi, Y. (1986). Antioxidant action of natural health products and Chinese herbs.” Inflammation.10: 79-91

140

Osawa, T. and Namiki, M. (1981). A novel type of antioxidant isolated from leaf wax of Eucalyptus leaves. Agric. and Biol. Chem. 45(3): 735-739

Padula, M. and Rodriquez-Amaya, D.B. (1987). Changes in individual carotenoids and vitamin C on processing and storage of guava juice (Magnifera indica) slices and puree. Int. J. Food Sci. Technol. 22:451-460.

Pan, X.J., Niu,G.G. and Liu,H.Z. Comparison of microwave-assisted extraction and conventional extraction techniques from the extraction of tanshinones from Salvia miltiorrhiza bunge. Biochemical Engineering Journal. 12: 71-77

Pearson, D. (1976). Chemical Analysis of Foods. 7th ed. Edinburg London and New York: Churchill Livingstone. 14-16.

Pederson, C.S. (1980). Vegetable Juice. In: Nelson, P.E. and Tressler, D.K. eds. Fruitand Vegetable Juice Processing Technology 3th ed. Westport, Connecticut: The AVI Publishing Co. Inc. 573-596

Peleg, H., Naim, M., Rouseff, R.L. and Zehavi, U. (1991). Distribution of bound and free phenolic acids in oranges (Citrus sinensis) and grapefruits (Citrus paradisi).Journal of the Science of Food & Agriculture. 57: 417-426

Perry, C.M. (1998). Medicinal plants of East and Southeast Asia: attributed properties and uses. Mass, USA: MIT Press.

Piga, A., Del Caro, A., Pinna, I. And Agabbio, M. (2003). Changes in ascorbic acid, polyphenol content and antioxidant activity in minimally processed cactus pear fruits. 36(2): 257-263.

Pizzocaro, F., Torreggiani, D. and Gilardi, G. (1993). Inhibition of apple polyphenoloxidase (PPO) by ascorbic acid, citric acid and sodium chloride. Journal of food Processing and Preservation. 17: 21-30.

Pointel, J.P., Boccalon, M.D. Cloarec, M., Lederehat, M.D. and Joubert, M. (1987). Titrated Extract of Centella asiatica (TECA) in the Treatment of Venous Insufficiency of the Lower Limb. Angiology. 38: 46-50

Pokorny, J. (1987). Major factors affecting the autoxidation in lipids. In: Chan, H. Ed. Autoxidation of unsaturated lipids. London: Academic Press. 141-206

Pokorny, J., Yanishliera, N. and Gordon M. (Eds.) (2001a). Antioxidant in Food: Practical Application. Cambridge, England: Woodhead Publishing Ltd. 1-6

Pokorny, J. (2001b). Natural antioxidant functionality during food processing. In: Pokorny, J., Yanishliera, N. and Gordon M. Eds. Antioxidant in Food: Practical Application. Cambridge, England: Woodhead Publishing Ltd. 331-372

141

Potter, N.P. (1999). Food Science. 4th ed. Connecticut: The AVI Publishing Company Inc.

Prasad, N.N., Siddalingaswamy, M., Parameswariah, P.M., Radhakrishna, K., Rao, R.V., Viswanathan, K.R. and Santhanam, K. (2000). Proximate and mineral composition of some processed traditional and popular Indian dishes. FoodChemistry. 68(1): 87-94

Qi, S., Xie, J. and Li, T. (2000). Effect of asiaticoside on hypertrophic scars in a nude mice model. Chinese Journal of Burns. 16(1): 53-56

Ragazzi, E. and Veronesse, G. (1973). Quantitative analysis of phenolic compounds after thin-layer chromatographic separation. Journal of Chromatography. 77: 369-375

Rajalakshmi, D. and Narasimhan, S. (1996). Food Antioxidants: Sources and Methods of Evaluation. In: Madhavi, D.L., Deshpande, S.S. and Salunkhe, D.K. Eds. Food Antioxidants: Technological, Toxicological, and Health Perspectives. New York: Marcel Dekker, Inc. 65-157

Rash, E.R., Murry, G.R. and Graham, D.J. (1993). The comparative stedy-state bioavailability of the active ingredients of Madecassol. Eur J Drug Metab Pharmacokinet. 18(4): 323-326

Raspisarda, P., Tomaino, A., Lo Cascio., Bonina, F., De Pasquale, A. and Saija, A. (1999). Antioxidant effectiveness as influenced by phenolic content of fresh orange juice. Journal of Agricultural and Food Chemistry. 11: 4718-4723

Razali, M., Fezah, O., Marziah, M., Johari, R. Mohd. Aspollah, S. and Azizah, A. H. (2001). Qualitative and Quantitative Analyses of Flavonoids (Apigenin, Kaempferol, Quercetin and Rutin) from Centella asiatica (L) Urban. Proceedingof Conference on Functional Food –Latest Development. Putra Jaya: UPM. 187-191

Rice-Evans, C.A., Miller, N.J. and Paganga, G. (1996). Structure antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biology and Medicine. 20: 933-956

Roberto G., Baratta M. T., Deans S.G. dan Dorman H.J.D. (2000). Antioxidant and Antimocrobial Activity of Foeniculum vulgare and Crithmum maritimum Essential Oils. Planta Medica. 66: 687-693

Roig, M.G., Bello, J.F., Rivera, Z.S., and Kennedy, J.F. (1999). Studies on the occurrence of non-enzymatic browning during storage of citrus juice. FoodResearch International. 32(9): 609-619

142

Ruby, A.J., Kuttan, G., Babu, K.D., Rajasehnaran, K.N. and Kuttan, R. (1995). Anti-tumor and antioxidant activity of natural curcuminoids. Camcer Latters. 94: 79-83

Sairam, K., Rao, C.V. dan Goel, R.K.. (2001). Effect of Centella asiatica Linn onphysical and chemical factors induced gastric ulceration and secration in rats. Indian Joutnal of Experimental Biology. 39(2): 137-142

Sampson, J.H., Raman, A., Karlsen, G., Navsaria, H. dan Leigh, I.M. (2001). In vitro keratinocyte antiproliferant effect of Centella asiatica extract and triterpenoid saponins. Phytomedicine : International Journal of Phytotherapy and Phytopharmacolog. 8(3): 230-235

Santerre, C.R., Cash, J.N. and Vonnorman, D.J. (1988). Ascorbic acid / citric acid combination in the processing of frozen apple slices. Journal of Food Science.53: 1713-1716

Sapers, G. M. (1993). Browning in foods: control by sulfites, antioxidants, and other means. Food Technology. 68: 75-84

Scalzo, R. L., Iannoccari, T., Summa, C., Morelli, R. and Rapisarda, P. (2004). Effect of thermal treatment on antioxidant and antiradical activity of blood orange juice. Food Chemistry. 85: 41-47

Schaneberg, B.T., Mikell, J.R., Bedir, E. and Khan, I.A. (2003). An improved HPLC method for quantitative determination of six terpenes in Centella asiaticaextracts and commercial products. Pharmazie. 58(6): 381-384

Schuler, P. (1990). Natural antioxidant exploited commercially. In: Hudson, B.J. Ed. Food Antioxidants. London: Elsevier Applied Science. 99-170

Scott, G. (1977). Antioxidant in science, technology, medicine and nutrition. Chichester, England: Albion Publishing.

Sherwin, E.R. (1990). In: Branen, A.L., Davidson, P.M., and Salminen, S. Eds.. FoodAdditives. New York: Marcel Dekker. 139.

Shi, J., Maguer, M. L. and Bryan, M. (2002). Lycopene from tomato. In: Shi, J., Mazza, G. and Maguer, M.L. Eds. Functional Foods: Biochemical and Processing Aspects. Vol. 2. Boca Raton, Florida: CRC Press. 136-168.

Shui, G. dan Leong, L.P. (2002). Separation and determination of organic acids and phenolic compounds in fruit juices and drinks by high-performance liquid chromatography. J. of Chromatography A.. 977(1): 89-96

143

Shukla, A., Rasik, A.M., Jain, G.K., Shankar, R., Kulshrestha, D.K. dan Dhawan, B.N. (1999a). In vitro and in vivo wound healing activity of asiaticoside isolated from Centella asiatica. Journal of Ethnopharmacology. 65(1): 1-11

Shukla, A., Rasik, A.M. and Dhawan, B.N. (1999b). Asiaticoside-induced elevation of antioxidant levels in healing wounds. Phytother Res. 3(1): 50-54

Sing, B. and Rastogi, R.P. (1969). Reinvestigation of the Triterpenes of Centellaasiatica. Phytochemistry. 8: 917-921

Skrede, G., Wrolstad, R.E. and Durst, R.W. (2000). Changes in anthocyanins and polyphenolics during juice processing of highbush blueberries (Vaccinium corymbosum L.)” J. Food Sci. 65(2): 857-364

Skrede, G. and Wrolstad, R.E. (2002). Flavonoids from berries and grapes. In: John Shi, Mazza, G. and Marc le Maguer Eds. Functional Foods: Biochemical and Processing Aspects. 2nd ed. Boca Raton, Florida: CRC Press. 71-134

Skorikova, V. and Lyashenko, E.P. (1972). The effect of thermal processing on polyphenolic substances in apple and pear juice. Izvest. Vyss. Ucheb. Zaveb Pish.Tekhnol. 3: 80-82

Slinkard, K. and Singleton, V.L. (1977). Total phenol analysis: automation & comparison with manual methods. Am. J. Ecol Vitic. 28(1): 49-56

Spanos, G.A., Wrolstad, R.E. and Heatherbell, D.A. (1990). Influence of Processing and Storage on the Phenolic Composition of Apple Juice. J. Agric. Food Chem. 38:1572-1579

Suguna, L., Sivakumar., P. and Chandrakasan, G. (1996). Effect of Centella asiaticaextract on dermal wound healing in rats. Indian Journal of Experimental Biology. 34(12): 1208-1211

Sung, T.V., Lavaud, C., Porzel, A., Steglich., W. and Adam, G. (1991). Triterpenoids and their glycoside from the bark of Schefflera octophylla. Phytochemistry. 31(1): 22-231

Suntornsuk, L., Gritsanapun W., Nilkamhank,S. and Paochom, A. (2002). Quantitation of vitamin C content in herbal juice using direct titration. Journal of Pharmaceutical and Biomedical Analysis. 28: 849-855

Somchit, M.N., Halijah, H. dan Wan Kartini, W.H. (2002). Antiulcer effect of Centellaasiatica and Piper betle extracts: A comparative study. J. Trop Med. Plants. 3(1): 18-22

Tagi, K. (1987). Lipid perioxides and human diseases. Chemistry and Physics of Lipids. 45: 337-341

144

Takeoka, G.R., Dao, L., Flessa, S., Gillespie, D.M., Jewel, W.T., Huebner, B., Bertow, D. and Ebeler, S.E. (2001). Processing effects on lycopene content and antioxidant activity of tomatoes. J. Agric. Food Chem. 49: 3713-3717

Tannenbaum, S.R., Young, V.R., Archer, M.C. (1985). Vitamins and Minerals. In: Fennema, O.R. Ed.. Food Cehmistry. 2nd ed. New York: Marcel Dekker Inc. 477-544

Taylor, S.T., Higley, N.A. and Bush, R.K. (1986). Sulfates in foods: uses, analytical methods, residues, fate, exposive, assessment, metabolism, toxicity and hypersensitivity. Advances in Food Research. 30: 1.

Tee, E.S., Mohd Idris, N., Mohd Nasir, A. and Khatijah, I. (1997). Nutrient Composition of Malaysian Foods. 4th ed. Malaysian Food Composition Database Programme. Kuala Lumpur: Inst. Medical Research. 16

Teissedre, P.L., Frankel, A.L., Waterhouse, H.P. and German, J.B. (1996). Inhibition of in vitro human LDL oxidation by phenolic antioxidants from grapes and wines.” Journal of the Science of Food and Agriculture. 70. 55-61

Tiek, N.L. (1997). Pegaga (Centella asiatica) – More about its Healing Properties. FRIMin Focus. 2(2): 10-11

Tsai, P., McIntosh, J., Pearce, P., Camden, B. and Jordon, B.R. (2002). Anthocyanin and antioxidant capacity in Roselle (Hibiscus Sabdariffa) extract Food Research International. 35(4): 351-356.

Turton, S. (1993). Australian Journal of Medicinal Herbalism. New South Wales. 5(3): 57-61

Velioglu, Y.S. Mazza, G. Gao, L. and Oomach, B.D. (1998). Antioxidant activity and total phenolics of selected fruits, vegetables, and grain products. J. Agric. Food Chem. 46: 4113-4117

Veldhuis, M.K. (1971). Orange and Tangerine Juice. In: Tressler, D.K. and Joslyn, M.A. Fruit and Vegetable Juice Processing Technology. 2nd ed. Westport, Connecticut: The AVI Publishing Co. Inc. 31-91

Vimala, S. and Mohd Ilham Adenan (1999). Malaysian tropical forest medicinal plants: a source of natural antioxidants. J. of Tropical Forest Products. 5: 32-38

Vimala, S., Mohd Ilham Adenan, Abdull Rashih Ahmad and Rohana Shahdan (2003). Nature’s choice to wellness: antioxidant vegetables/ulam. Siri Alam dan Rimba No. 7. Kuala Lumpur: FRIM. 90-92

145

Vishu Rao, G., Shivakumar, H.G. and Parthasarathi, G. (1996). Influence of Aqueous Extract of Centella asiatica (Brahmi) on Experimental Wounds in Albino Rats. Indian Journal of Pharmacology. 28: 249-253

Vogel, H.G., De Souza, N., D’ Sa, A. (1990). Effect of Terpenoids Isolated from Centella asiatica on Granuloma Tissues. Acta Therapeutica. 16: 285-298.

Vongsangnak, W., Gua, J., Chauvatcharin, S. and Zhong, JJ. (2003). Towards efficient extraction of notoginseng saponins from cultured cells of Panax notoginseng.Biochemical Engineering Journal. 18(2): 115-120

Verma, R.K., Bharatariya, K.G., Gupta, M.M. and Sushil Kumar. (1999). Reverse-phase High Peformance Liquid Chromatography of Asiaticoside in Centella asiatica.” Phytochemical Analysis. 10: 191-193

Wang, H,, Cao, G. dan Prior, R.L. (1996). Total Antioxidant Capacity of Fruits. J. Agric. Food Chem. 44: 701-705

Wang, L., Kim, D. and Lee C.Y. (2000). Effect of Heat Processing and Storage on Flavonols and Sensory Qualities of Green Tea Beverage. J. Agric. Food Chem.48: 4227-4232

Whister, R.L. and Daniel, J.R (1985). Carbohydrate. In: Fennema, O.R. Ed.. FoodChemistry. 2nd ed. New York: Marcel Dekker Inc. 74.

WHO (1998). Medicinal Plants in South Pacific. Manila: WHO Regional Office for the Western Pacific. 42-43

WHO (1999). WHO Monograph on Selected Medicinal Plants. Geneva: World Health Organisation. 77-85

Wiart, C. (2000). Medicinal Plants of South East Asia. Malaysia: Pelanduk Publication.

Wong, P.K., Salmah, Y., Hasanah M.G. Che Man, Y. (2001). Effect of Different Processing Methods on Anthocyanin Pigments and Ascobic Acid Contents in Roselle (Hibiscus Sabdariffa) Juice. Proceeding of Conference on Functional Food – Latest Development. Putra Jaya: UPM. 162-167

Wrolstad, R.E., Skrede, G., Lea, P. and Enersen, G. (1990). Influence of sugar on antocyanin pigment stability in frozen strawberries. J. Food Sci. 55(4): 1064-1072

Yang, C.S.T. and Attalah, W.A. (1985). Effect of Four Drying Methods on the Quality of Intermediate Moisture Lowbush Blueberries. Journal of Food Science. 50:1233-1237

146

Yanishlieva-Maslarove, N.V. (2001). Inhibiting oxidation. In: Pokorny, J., Yanishliera, N. and Gordon M. Eds. Antioxidant in Food: Practical Application. Cambridge England: Woodhead Publishing Ltd. Abington. 331-372

Yen G. dan Chen H. (1995). Antioxidant Activity of various Tea Extracts in Relation to Their Antimutagenicity. J. Agric. Food Chem. 43: 27-32

Yumi Yuhanis. (2001). Extraction of Oleoresin and Antioxidant activity of Curcumin from Curcuma Longa. UTM: Master thesis.

Yuting, C., Rongliang, J., Zhongjian, J. and Young, J. (1990). Flavonoids as superoxide scavengers and antioxidants. Free Radical Biology and Medicine. 9: 145-150

Yusuf, N., Fadzillah, N.M., Daud, S.K. dan Marziah, M. (2000). Antioxidative constituents of centella asiatica. Proceeding of the 16th National Seminar on Natural Products. Mines: Selangor. 91-94

Zainol, M.K., Abd Hamid, A., Yusof, S. and Muse, R. (2003). Antioxidative activity and total phenolic compounds of leaf, root and petiole of four Centella asiatica accessions of (L) Urban. Food Chemistry. 81(4): 575-581

Zakaria, M. dan Mohd, M.A. (1994). Traditional Malaysia Medicinal Plants. Kuala Lumpur: Fajar Bakti Sdn Bhd.

Zheng, W. dan Wang, S.Y. (2001). Antioxidant activity and phenolic compounds in selected herbs. J. Agric. Food Chem. 49(11): 5165-5170

Zielinski, H., Kozlowska, H. and Lewczuk, B. (2001). Bioactive compounds in the cereal grains before and after hydrothermal processing Innovative Food Science & Emerging Technologies. 2:159-169.

147

APPENDIX A1

Figure 1: HPLC-Chromatogram of methanol extract of triterpene acid (Fresh sample)

Figure 2: HPLC-Chromatogram of methanol extract of triterpene acid (Sample A)

148

APPENDIX A2

Figure 3: HPLC-Chromatogram of methanol extract of triterpene acid (Sample B)

Figure 4: HPLC-Chromatogram of methanol extract of triterpene acid (Sample C)

149

APPENDIX A3

Figure 5: HPLC-Chromatogram of methanol extract of triterpene acid (Sample CM1)

Figure 6: HPLC-Chromatogram of methanol extract of triterpene acid (Sample CM2)

150

APPENDIX B1

Figure 7: HPLC-Chromatogram of methanol extract of glycosides (Fresh)

Figure 8: HPLC-Chromatogram of methanol extract of glycosides (Sample A)

151

APPENDIX B2

Figure 9: HPLC-Chromatogram of methanol extract of glycosides (Sample B)

Figure 10: HPLC-Chromatogram of methanol extract of glycosides (Sample C)

152

APPENDIX B3

Figure 11: HPLC-Chromatogram of methanol extract of glycosides (Sample CM1)

Figure 12: HPLC-Chromatogram of methanol extract of glycosides (Sample CM2)

153

APPENDIX C

Figure 13: HPLC-Chromatogram of water extract of glycosides (Fresh sample)

Figure 14: HPLC-Chromatogram of water extract of triterpene acid (Fresh sample)

154

APP

END

IX D

Figu

re 1

5: C

alib

ratio

n cu

rve

of st

anda

rd F

eSO

4.7H

20 (R

2 =0.9

9)

Con

cent

ratio

n of

Fe(

II) u

mol

/L

Absorbance at 593nm

-0.01

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

0.11

0.12 -200

0200

400

600

800

1000

1200

1400

y=7.

387E

-5x

+ 0.

002

155

APP

END

IX E

Figu

re 1

6: S

tand

ard

calib

ratio

n cu

rve

of g

allic

aci

d (r

2 =0.9

9)

Con

cen

trat

ion

of

gall

ic a

cid

(m

ol/L

)

Absorbance at 750nm

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5 0.

00.

20.

40.

60.

81.

01.

2