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 65qC/15 minutes, 80qC/5minutes and pasteurization at 80qC/5minutes
followed by canning and boiling at 100qC/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 65qC/15 minit, 80qC/5
minit dan pempasturan pada 80qC/5 minit diikuti dengan pengetinan dan pendidihan
pada 100qC/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 - E-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
qBrix - 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%), E-

carotene (2649 Pg), 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 93qC 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 85qC (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 71.1qC to 100qC.
On the other hand, Chuah (1984) reported that the process of pasteurization usually
consists of a process whereby the food is heated to temperature 60-90qC 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 60qC 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 80qC for 6 minutes on antioxidant
changes compared to non-thermally treated juice. After pasteurization at 80qC 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 qC for 1 to 5 minutes still
 6

maintained the sensorial quality of products (Che Rahani, 1998). The carrot juice heated
at 82qC for 5 minutes retained 57% of D-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 80qC 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 87qC 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 82qC for 5 minutes, followed by canning and immersed in boiling water
(100qC) 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 (90qC) and with long infusion
time. However, Langley-Evans (2000) suggested that the black tea is ideally prepared
between 70-90qC 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 (D-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 D-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; 65qC/15 minutes (A), 80qC/5minutes (B) and
canned process (heat at 80qC/5minutes followed by canning and boiling
at 100qC/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 90qC 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. E-carotene and ascorbic acid, known to have
antioxidative activities, are present at appreciable concentration (2649 Pg and 48.5mg,
respectively) in fresh pegaga. E-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

% Nutrient composition of edible portions (E.P),
E.P per 100g sample
Proximate composition*

Kcl g g g g g g
Energy Water Protein Fat CHO Fiber Ash
37 87.7 2.0 0.2 6.7 1.6 1.8
Indian
Pennywort 44 Vitamin**
(pegaga);
Hydrocotyle Pg Pg Pg mg mg mg mg
asiatica Retinol Carotene RE B1 B2 Niacin C
0 2649 442 0.09 0.19 0.1 48.5

Mineral**

mg mg mg mg mg
Ca P Fe Na 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 Pg retinol + (Pg 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 R5
Asiatic acid -H -H -CH3 -CH3 H

Asiaticoside -H -ȕ-D-glc-(6-1)- ȕ-D-glc- -CH3 -CH3 H

(4-1)--L-rha
Madecassic acid -OH -H -CH3 -CH3 H

Madecassoside -OH -ȕ-D-glc-(6-1)- ȕ-D-glc- -CH3 -CH3 H

(4-1)--L-rha

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 Pg/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 Trolox-C, Anoxomer,
Antioxidant Ethoxyquin
Oxygen Scavengers Sulfites, Ascorbic acid,
Ascorbyl palmitate
Secondary / Synergistic Chelating Agents EDTA, Polyphosphate,
Antioxidant Citric acid, Lecithin,
Tartaric acid
Secondary antioxidant Thiodipropionic acid,
Distearyl ester
Miscellaneous antioxidants Amino acid, Spices
extract, Flavonoids,
Vitamin A, E-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 50qC. 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 25-30qBrix 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), E-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 88qC for 1
minutes, 100qC for 12 seconds and 121qC 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 71.1qC for 15 sec
is equivalent in bacterial destruction to batch method at 62.8qC 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 71.1qC 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 68.1qC for 14 seconds and 71.1qC 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
116qC or 121qC and sometime in steam at temperatures of about 140qC (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

63qC for 30 minutes (Potter, 1986). The high acid (below pH 4.2) beverages could also
be process at the temperature as low as 60qC for about 10-20 minutes (Chuah, 1984).
According to Pederson (1980), the heating treatment at the temperature as low as 71.1qC
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
KILLING
TIME pH 5 to pH 7
(min)
10

pH 4.5
1.0
pH 3.5

0.1
99 110 121

TEMPERATURE (qC)
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 qC for 1 to 5 minutes (Che Rahani, 1998), but the
orange juice was thermally treated at slightly low temperature, 80qC for 6 minutes
(Scalzo, 2004). For the heat processing of guava drink and carrot juice, the
pasteurization at 82qC 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
87.8qC 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
78-93qC 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 77-82.2qC 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 90qC. 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 60qC, 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 87qC 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 87.8qC, 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, E-D-
glycosides hydrolyzed less rapidly than D-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 110-132qC 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 100qC, 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 50qC for 9 hour (75.60%)
compared to freeze drying (21.13%) (Mahanom, et al.,1999). Mild (75qC for 30 sec)
and standard pasteurization (95qC 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
(80qC/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 75qC and
storage for 15 weeks at 40qC, 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 50qC
yields the highest amount of total phenolics in the sample. Increasing the temperature
above 60qC, 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
150qC. The duration of heating below 100qC, 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 10qC to 23qC (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 50qC for 9 hours and 70qC 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 (97qC for 85 seconds
and 97qC 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 (100qC 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-
90qC 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
75qC and 95qC 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 75qC. However, the activity
was decreased from 150.1 mg to 143.7 mg after standard pasteurization at 95qC 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 E–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 102qC for 10, 20 and 30 minutes on antioxidant activity of
vegetable juice based on E-carotene bleaching test. When prepared at 2qC 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 102qC for 10 minutes exhibited higher antioxidant activity.
The antioxidant activity (based on E-carotene bleaching test) also increased with the
increasing of heating time and heating temperature. For example, the antioxidant
activity of carrot juice at 25qC 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

12
T3
11
Relative antioxidant activity

10

8
T2
7

5
T1
4

Heating time

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 65qC/15 minutes, 80qC/5 minutes
and in canning process (heat at 80qC/5minutes, canned and followed by boiling at
100qC/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 15q 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 5-15qBrix (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; 65qC/15 minutes, 80qC/5 minutes and in
canning process (heat at 80qC/5minutes, canned and followed by boiling at 100qC/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 4qC.
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) (90żC/1min)

Fresh sample (F) Heat treatment

65żC/15 min (A) 80żC/5 min (B) Canning process (C)

(heat at 80żC/5 min, canned,
boiled at 100żC/10 min)

Packed in LDPE bottle

Analysis
Figure 3.1: Flowchart of the preparation of pegaga drink
Samples Fresh sample Heat-treated samples Commercial sample
(Sample F) (Sample A, B and C) (Sample CM1 and CM2)

Assessments Physico-chemical Antioxidant

Triper activity Triterpene glycosides
characteristics content

pH, Total acidity, Colour index L*, a* Ferric thiocyanate assay Asiaticoside content,
and b* values, Total soluble solid, (FTC) and Ferric Reducing Madecassoside content, Asiatic
Proximate composition, Microelement, Ability of Plasma (FRAP) acid content and Madecassoside
Total polyphenol and Ascorbic acid assay content
content

Contribution of total polyphenol Correlation of FRAP and FTC Contribution of asiaticoside on

Output and ascorbic acid on antioxidant measurement of antioxidant activity antioxidant activity
activity using correlation (correlation coefficient, r)
coefficient, r

Figure 3.2: Experimental layout

 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
90żC/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 4qC.

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
105qC 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 550qC 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 420qC. 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 – 60qC

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 70-80qC. 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
105qC. 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 105qC for 1 hour. The crucible with residue was weighed (W1) and
ignited in muffle furnace at 450qC 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
70-80qC. 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 Pm 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 4qC 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 37qC. 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 40qC.

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. D-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
5qBrix, 10qBrix and 15qBrix. Fresh drink (1qBrix) 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 Ȝ 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 Pl 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-Pm membrane filter and 10 Pl 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 1.0qBrix (fresh) and 11.2-11.8qBrix (heat-treated drink), which is almost
similar to orange juice (9-15 qBrix) and apple juice (11-14qBrix). The total soluble solid
in CM2 was significantly lower (7.6qBrix) than CM1 (12.6qBrix) and heat-treated drink.
For heat-treated samples (65qC/15 minutes, 80qC/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 (65qC/15 minutes, 80qC/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 65qC/15 minutes shows higher development of browning followed by canned
and pasteurization at 80qC/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 TA L* a* b*
(Brix) (%) +sd +sd +sd
F (Fresh) 5.93 1.0 3.85 24.43+0.21c 2.83+0.04a 4.88+0.06b
AA (65qC/15min) 3.72 11.2 14.37 27.84+0.31ab 2.17+0.08b 6.88+0.18a
B (80qC/5min) 3.79 11.8 14.72 28.86+0.48a 2.09+0.08bc 6.03+0.18ab
C (Canned) 3.72 11.2 14.38 27.04+2.57ab 2.67+0.24a 6.56+2.27a
CM1 3.89 12.6 7.71 26.90+0.77ab 2.22+0.16b 4.80+0.21b
CM2 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 100qC, about 50% of total polyphenol in pegaga drink remain. The loss of total
polyphenol content under heat processing treatment at 65qC for 15 minutes and 80qC 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).

d
CM2
b
Pegaga drink sample

CM1
e
C

B d

A c
a
F

0 200 400 600 800 1000 1200 1400 1600

Total polyphenol (mg/100ml)

Ferulic acid eqv. Gallic acid eqv.

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
(65qC/15 min); B (80qC/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
 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)

4.5 a
Concentration of ascorbic acid

4
3.5
3
(mg/100ml)

2.5 b
2 c c
d
1.5
e
1
0.5
0
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 (65qC/15 min); B (80qC/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
 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 D-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 D-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 D-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).

BHT a
Vit.C h
Pegaga drink and standard

Vit. E g
CM2 f
e
sample

CM1
f
C
d
B
c
A
b
Fresh
40.00% 50.00% 60.00% 70.00% 80.00% 90.00%
% inhibition of linoleic acid peroxidation

Figure 4.3: % Inhibition of peroxidation as mean (n=3) in pegaga drinks and standard
sample. Key: F (fresh sample); A (65qC/15 min); B (80qC/5 min); C (canned); CM1
(commercial sample -Loo Ent.); CM2 (commercial sample-HPA); Vit.E (D-tocopherol),
Vit.C (ascorbic acid) and BHT (Butylated hydroxy toluene). Values with same letter are
not significantly different at P=0.05
 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 (65qC/15 minutes), B (80qC/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 100qC 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 20-70qC of infusion temperature was significantly lower than at the infusion
temperature of 90qC Lingley-Evans (2000). However, the antioxidant compounds in
black tea is ideally extracted at 70-90qC 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 D-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.

e
Pegaga drink and standard

BHT
Vit.C f
d
Vit. E
g
CM2
sample

CM1 c
f
C
d
B
A b
Fresh a

0 200 400 600 800 1000

FRAP value (µmol/L)

Figure 4.4: FRAP activity as mean (n=3) in different thermal processing of pegaga
drinks. Key: F (fresh sample); A (65qC/15 min); B (80qC/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
 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

0.15
FRAP (Absorbance at 593nm)

0.13

0.11

0.09

0.07

0.05
0.30 0.36 0.42 0.48 0.54 0.60
FTC (Absorbance at 500nm)

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).

80
FTC (% inhibition of linoleic peroxidation)

75

70

65

60

55

50

45

40
300 400 500 600 700 800 900
FRAP values (umol/L)

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.

1600
(gallic acid eqv. mg/100ml)
Total polyphenol content

1400

1200

1000

800

600
300 400 500 600 700 800 900
FRAP value (umol/L)

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

a
FRAP 0.8078 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

0.45
0.4 d
Absorbance at 500nm

0.35
0.3
c
0.25
0.2 a b
0.15
0.1
0.05
0
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.
 100

0.04 6.5
FRAP value (Absorbance at 593 nm) a
6.0
0.02
5.5

0.00 5.0
4.5

pH
-0.02 4.0
b 3.5
-0.04 c
b 3.0
FRAP
-0.06 2.5
-0.05 0.05 0.15 0.25 0.35 pH
Concentration of citric acid (%)

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.
 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 1qBrix to 15qBrix. 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 15qBrix (0.057
nm) followed by 10q Brix (0.050 nm), 5qBrix (0.044 nm) and control sample (0.032
nm). A slightly contrast, the antioxidant activity was gradually increased at 5qBrix and
10qBrix by FTC assay, but declined thereafter with the addition of sugar up 15qBrix
(figure 4.11). Pegaga drink at concentration of 15qBrix, however shows the lowest
antioxidant activity compared to control sample (1qBrix) 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

a
0.06
b
0.05 c

0.04 d
FRAP Values
(Absorbance at 0.03
593 nm)
0.02

0.01

0
1 Brix 5 Brix 10 Brix 15 Brix
o
Total soluble solid ( Brix)

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.
 103

0.45 c
0.4 b
0.35 a a

0.3
Absorbance at 0.25
500nm 0.2
0.15
0.1
0.05
0
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
 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).

0.14 a
b
0.12 c
0.1
FRAP Values 0.08 d
(Absorbance at
593 nm) 0.06
e
0.04
0.02
0
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).
 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.

0.14

0.12
(Absorbance at 593 nm)

0.10
FRAP values

0.08

0.06

0.04

0.02
-50 0 50 100 150 200 250 300 350 400
Concentration of
Sodium metabisulphite (ppm)

Figure 4.13: Correlation coefficient of antioxidant activity (FRAP assay) and

concentration of sodium metabisulphite (n=5, p<0.05, r=0.9653).
 106

0.45
0.4 e
0.35 d
c
Absorbance at 500 nm

0.3 b a
0.25
0.2
0.15
0.1
0.05
0
Control 0 200 250 300 350
Concentration 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.
 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 O 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

4000

3500

3000
Area(1E3 mV.s)

2500
y = 8675.9x + 94.036
2000

1500

1000

500

0
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45
Concentration of standard solution (mg/ml)

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
 110

3200

2600
Area (1E3 mV.s)

2000

1400

800

200
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45
Concentration of standard solution (mg/ml)

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)

7000

6000

5000
Area (1E3 mV.s)

4000 y = 14125x + 75.092

3000

2000

1000

0
0.0 0.1 0.2 0.3 0.4
Concentration of standard solution (mg/ml)

Figure 4.21: Calibration curve for madecassic acid (area vs concentration of standard
madecassic acid)
 113

14000

12000

10000
Area (1E3 mV.s)

y = 31621x + 1.2049
8000

6000

4000

2000

0
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45
Concentration of standard solution (mg/ml)

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)
 114

Table 4.4: The results of HPLC analysis

Compound Calibration L (mg/ml) Retention Correlation
curve time (tR) Coefficient
(minute) (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 65qC/15 minutes and 80qC/5 minutes, however, it slightly increased when
 115

the temperature was further increased to 100qC 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 80qC 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 (65qC) 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 50qC. On the other
research, Pan, et al., (2002) noticed that the application of high temperature (20-50qC)
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

Table 4.5: Results for triterpene glycosides assay

___________________________________________________________________________________________________
Triterpene glycosides content (mg/100ml) of methanol extracts of pegaga drink
Sample Madecassoside Asiaticoside Madecassic Asiatic Triterpene glycosides
+S D +SD acid +SD acid + SD
+SD
F 3.12 +0.10a 3.91 + 0.10b 2.56 + 0.20c 2.45 + 06a 12.04 + 0.14a
A 2.70 + 0.05cd 4.32 + 0.05a 2.70 + 0.03bc 1.03 + 0.09c 10.75 + 0.14b
B 2.43 + 0.07e 3.61 + 0.07c 3.02 + 0.20ab 1.02 + 0.07c 10.08 + 0.17cb
C 2.74 + 0.08c 3.03 + 0.05d 3.22 + 0.33a 0.97 + 0.05c 9.96 + 0.68c
CM1 2.93 + 0.13b 2.60 + 0.30e 2.79 + 0.10bc 1.86 + 0.28b 10.18+ 0.46b
CM2 2.54 + 0.09de 3.10 + 0.14d 1.37 + 0.10d + 1.05 + 0.09c 8.06+ 0.32d

Means with the same letter (a, b, c) in each column are not significantly different at p=0.05
 117

The amount of asiaticoside in all samples, however, significantly lower than

obtained in methanol extract of oven-dried pegaga at 30-50qC (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 0qC (control) to 100qC
(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
65qC/15min (A) to 100qC (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 65qC/15 minutes, 80qC/5 minutes and up to 100qC 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 (65qC and up to 80qC), asiaticoside (80qC and up to
100qC) 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 80qC. 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

60

50

40
Triterpene glycosides s

Madecassoside
30
content (%)

Asiaticoside
20 Madecassic acid
Asiatic acid
10

0
F A B C CM1 CM2
Pegaga drink sample

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

x Fresh pegaga drink (F) contained about 1470.14 mg/100ml of total polyphenol
(GAE equivalent), which was significantly higher than pasteurized sample at
65qC/15 minutes (903.23 mg/100ml) and 80qC/5 minutes (805.54 mg/100ml);
and canned pegaga drink (730.27 mg/100ml).

x The degradation of ascorbic acid occurred at a higher rate in canned pegaga

drink, followed by pasteurization at 80qC/5 minutes and 65qC/15 minutes. The
concentration in fresh pegaga drink was significantly higher, which is about
4.23mg/100ml.

x 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.

x 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.

x 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.

x 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 65qC/15 minutes (A) and 80qC/5
minutes (B).

x The maximum amount of asiaticoside (4.32 mg/100ml) was observed during

processing at 65qC/15 minutes (A). The amount in fresh sample was
3.92mg/100ml. This result suggested that the heat processing at moderate
temperature (65qC) and longer heating period (15minutes) enhanced the
extraction efficiency of asiaticoside.

x 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).

x 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

x 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.

x 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.

x 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.

x 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. Food
Chemistry. 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 Centella

asiatica, 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. Planta
Medica. 60: 133-135

Bors, W. and Saran, M. (1987). Radical scavenging by flavonoid antioxidants. Free

Radical 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 Centella
asiatica: 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. Food
Chemistry. 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. Food
Chemistry. 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 Centella
asiatica 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. Cancer

Res. 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. Journal
Agriculture 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 Panax
notoginseng 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. Food
chemistry. 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 (Allium
cepa) 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. Fruit
and 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. Food
Chemistry. 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. Proceeding
of 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. Food
Research 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 on
physical 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 asiatica
extracts 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.. Food
Additives. 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 Centella

asiatica. 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 asiatica

extract 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 Centella
asiatica 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. FRIM
in 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.. Food
Chemistry. 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

APPENDIX D

0.12
0.11
0.10
0.09
0.08
0.07 y=7.387E-5x + 0.002
0.06
0.05
0.04
0.03

Absorbance at 593nm
0.02
0.01
0.00
-0.01
-200 0 200 400 600 800 1000 1200 1400

Concentration of Fe(II) umol/L

Figure 15: Calibration curve of standard FeSO4.7H20 (R2=0.99)

 155

APPENDIX E

4.5

4.0

3.5

3.0

2.5

2.0

1.5

Absorbance at 750nm
1.0

0.5

0.0
0.0 0.2 0.4 0.6 0.8 1.0 1.2

Concentration of gallic acid (mol/L)

Figure 16: Standard calibration curve of gallic acid (r2=0.99)