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 Date

: : :

………………………………… SANIAH BTE KORMIN …………………………………

iii

ACKNOWLEDGEMENT

First and foremost, thanks to God Almighty for the guidance and help in giving me the strength to complete this thesis. I would also like to take this opportunity to express my utmost gratitude to my supervisor, Prof. Dr. Mohd Roji Sarmidi for his valuable guidance and advice throughout this thesis study. Appreciation is also to Pn. Faridah Husin, Research officer, Food Technology Center, MARDI Serdang, for her kindness in supporting this study. I would like to express my sincere appreciation to research assistants in MARDI Johor Bahru for their help during the various laboratory tasks. A word of thanks also goes to all personnel and technicians in Chemical Engineering Pilot Plant, UTM due to their full support in my research experiments especially to En. Abdul Rahim Abd. Rahman and En Muhammad Subri Abd. Rahman. Finally, I am also forever indebted to my lovely husband, Mohd Azli Sairan for his continuous encouragement and many sacrifices.

iv

ABSTRACT

The health benefit of herbal pegaga drink, which is associated with triterpene glycosides content and antioxidant activity attract a lot of interest from the public and food and herbal industries. The works carried in this research investigated the effect of heat processing at 65 C/15 minutes, 80 C/5minutes and pasteurization at 80 C/5minutes followed by canning and boiling at 100 C/10 minutes on these phytochemicals and compared to untreated herbal pegaga drink or fresh sample. The results revealed that the untreated pegaga drink exhibited much higher (P<0.05) antioxidant activity than the heat-treated samples. The Ferric Reducing Ability of Plasm (FRAP) values was 860 µmol/litre for the untreated sample and in the range of 404 - 740 µmol/litre for heattreated 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. phytochemicals are good sources of antioxidant. These

v

ABSTRAK

Faedah kesihatan bagi minuman herba pegaga yang dikaitkan dengan kehadiran triterpena glikosida dan aktiviti pengantioksidan telah menarik minat yang tinggi daripada orang awam dan pengusaha industri herba serta makanan. Kajian ini dijalankan bagi menyiasat kesan proses pemanasan pada suhu 65 C/15 minit, 80 C/5 minit dan pempasturan pada 80 C/5 minit diikuti dengan pengetinan dan pendidihan pada 100 C/10 minit ke atas perubahan fitokimia tersebut dan dibandingkan dengan minuman tanpa rawatan atau sampel segar. Keputusan menunjukkan minuman pegaga tanpa rawatan menghasilkan aktiviti pengantioksidan yang lebih tinggi (P<0.05) berbanding sampel yang dipanaskan. Nilai ‘Ferric Reducing Ability of Plasma’ (FRAP) adalah 860 µmol/liter bagi sampel tanpa rawatan dan dalam julat 404 - 740 µmol/liter untuk sampel yang dipanaskan. Minuman tanpa rawatan merencat 72% pengoksidaan asid linoleik dan peratus perencatan bagi sampel yang dipanaskan adalah di antara 2656%. 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 ACKNOWLEDGEMENT ABSTRACT ABSTRAK TABLE OF CONTENTS LIST OF PLATE LIST OF TABLES LIST OF FIGURES LIST OF SYMBOLS LIST OF APPENDICES

ii iii iv v x xi xii xiii xv xviii

1

INTRODUCTION 1.1 1.2 Objective Scopes

1 9 9 11 11 12

2

LITERATURE RIVIEW 2.1 2.2 Medicinal Plants in Malaysia Herbal Products in Food Industries

vii 2.3 Plant Material (Centella asiatica) 2.3.1 2.3.2 2.3.3 2.4 2.4.1 2.5 Plant Description Medicinal Applications Bioactive Constituents in Pegaga Proximate Composition and Nutritive Values of Pegaga Triterpene Glysoside (Asiaticoside, Madecassoside, Asiatic acid, Madecassic acid) 2.5.1 2.5.2 2.5.3 2.5.4 Chemical Structure of Triterpene Glycosides Health-Promoting Effect of Triterpene Glycosides Antioxidative Activity of Triterpene Glycosides Methods for Assessing Triterpene Glycosides 2.5.4.1 2.5.4.2 2.6 Ascorbic acid 2.6.1 2.7 The Contribution of Ascorbic acid in Antioxidant Activity Polyphenol 2.7.1 2.7.2 2.8 Phenolic Compounds in Pegaga The Contribution of Phenolic compounds in Antioxidant Activity Antioxidant activity 2.8.1 2.8.2 2.8.3 Antioxidant activity in Herbs Antioxidant Activity of Pegaga The Role of Synergistic or Secondary Antioxidants 2.8.3.1 Effect of citric acid 32 32 2.8.3.2 Effect of sulphites 28 30 30 31 25 26 26 Extraction HPLC Analysis 21 21 22 22 24 20 18 20 18 12 12 13 14 15 16

Nutrient Composition

viii 2.8.4 2.8.5 2.8.6 2.8.7 Effect of enzymatic oxidation on antioxidant activity Effect of concentration and sugar content The Mechanism of Antioxidant Activity Assesment of Antioxidant Activity 2.8.7.1 Ferric Reducing Ability of Plasma (FRAP) 2.8.7.2 2.9 2.9.1 2.9.2 2.9.3 2.9.4 Ferric Thiocyanate (FTC) 37 38 42 44 45 48 49 50 52 54 54 56 56 56 59 59 Heat processing of Food and Beverages The retention of nutrient and phytochemical during processing of foods Effect of food processing on nutrient composition Effect of heat processing on natural antioxidant Effect of heat processing on antioxidant activity 2.9.4.1 Development of pro-oxidant during heat processing 2.9.4.2 Development of heat-induced antioxidant 2.10 3 Effect of heat processing on triterpene glycosides 34 35 36 37 33

MATERIAL AND METHODS 3.1 3.2 Introduction Material and Sample Preparation 3.2.1 3.2.2 3.2.3 3.3 Juice Extraction Preparation of Pegaga Drink Commercial Pegaga Drink Sample

Experimentals and Analytical Methods

ix 3.3.1 Physico-chemical Characteristics 3.3.1.1 Colour Index 3.3.1.2 Total Soluble Solid and pH 3.3.1.3 3.3.2 3.3.2.1 Total Acidity Moisture Proximate and Micronutrient Analysis 3.3.2.2 Ash 3.3.2.3 Protein 3.3.2.4 3.3.2.6 3.3.2.7 3.3.4 3.3.5 Fat Carbohydrate and Energy Microelement 3.3.2.5 Fibre 59 59 60 60 59 60 60 61 62 62 63 63 64 65 66 66 66 67 68 69 70 70 71 74 77 81 83

3.3.3 Ascorbic Acid Assay Total Polyphenol Assay Antioxidant Assay 3.3.5.1 Ferric Reducing Ability of Plasm (FRAP) Assay 3.3.5.2 Ferric Thiocyanate Method (FTC) 3.3.6 3.3.7 3.4 4 Study on Factors Influence to the Antioxidant Determination of Triterpene Glycosides Activity of Pegaga Drink Statistical Analysis

RESULTS AND DISCUSSION 4.1 4.2 4.3 4.4 4.5 4.6 Introduction Physico-chemical Characteristics of Pegaga Drink Nutrient Composition Total Polyphenol Ascorbic Acid Content Antioxidant Activity

x 4.6.1 Antioxidant Activity in Linoleic Acid System (FTC Assay) 4.6.2 4.6.3 4.7 4.8 Antioxidant Activity by Ferric Reducing Ability of Plasma (FRAP Assay) Correlation of FTC Assay and FRAP Assay 90 92 97 98 101 103 106 107 114 121 122 122 122 124 126 147 – 155 Antioxidant Activity of Phenolic Compounds and Ascorbic Acid The Factors Influence on Antioxidant Activity 4.8.1 Effect of Citric Acid on Antioxidant Activity 4.8.2 4.8.3 4.9 Effect of Total Soluble Solid on Antioxidant Activity Effect of Sodium Metabisulphite Triterpene Glycosides 4.9.1 Isocratic HPLC Assay 4.9.2 4.10 5 Quantitative Determination of Triterpene Glycosides in Pegaga Drink Antioxidant Activity of Asiaticoside 86 83

CONCLUSION AND RECOMMENDATION 5.1 5.2 5.2 Introduction Conclusion Recommendations and further works

REFERENCES Appendices

xi

LIST OF PLATE

PLATE 1 Pegaga (Centella asiatica)

TITLE

PAGE 13

xii

LIST OF TABLES

TABLE 2.1 2.2 4.1 4.2 4.3 4.4 4.5

TITLE Nutritional composition of pegaga Classification of food antioxidant Physico-chemical characteristic of pegaga drink The nutritional value and trace element of pegaga drink Correlation (r) of antioxidant activity with total polyphenol and ascorbic acid content of the pegaga drink Results of HPLC analysis Results for triterpene glycosides assay

PAGE 17 29 72 76 96 114 116

xiii

LIST OF FIGURES

FIGURE 2.1 2.2 2.3 2.4 2.5

TITLE Structure of triterpene glycosides The group of saponin glycosides Structure of ascorbic acid Influence of pH of heating medium on heat resistence of spores Changes in overall antioxidant activity due to development of different stages Millard reaction at different temperatures

PAGE 19 19 23 41 52

3.1 3.2 4.1 4.2 4.3 4.4 4.5 4.6

Flowchart of the preparation of pegaga drink Experimental layout Total phenolic compounds (as ferrulic and gallic acid equivalent) of different sample of pegaga drink Ascorbic acid content of different sample of pegaga drink % inhibition of peroxidation as mean (n=3) in pegaga drink and standard sample FRAP activity as mean (n=3) in different thermal processing of pegaga drink Correlation of FRAP and FTC measurement of antioxidant activity in pegaga drink Regression of FRAP assay against FTC measurement of antioxidant activity of pegaga drink, BHT, vitamin E and vitamin C

57 58 80 82 85 88 91 92

4.7

Correlation coefficient of antioxidant activity (FRAP assay)

94

xiv and total polyphenol content 4.8 4.9 4.10 4.11 4.12 4.13 4.14 4.15 4.16 4.17 4.18 4.19 4.20 4.21 4.22 4.23 The effect of citric acid on the antioxidant activity (FTC assay) of pegaga drink. The effect of citric acid on antioxidant activity (FRAP assay) of pegaga drink. The effect of total soluble solid on the antioxidant activity (FRAP assay) of pegaga drink The effect of total soluble solid on the antioxidant activity of pegaga drink The effect of sodium metabisulphite on the antioxidant activity (FRAP assay) of pegaga drink Correlation coefficient of antioxidant activity and concentration of sodium metabisulphite The effect of sodium metabisulphite on inhibition of linoleic acid peroxidation of pegaga drink HPLC-Chromatogram for standard madecassoside HPLC-Chromatogram for standard asiaticoside Calibration curve for madecassoside Calibration curve for asiaticoside HPLC-Chromatogram for madecassic acid HPLC-Chromatogram for asiatic acid Calibration curve for madecassis acid Calibration curve for asiatic acid Triterpenoid fraction (%) of pegaga extract from drink samples 108 108 109 110 111 112 112 113 120 106 105 104 103 102 100 99

xvi O2 H2O2 OH. LDL CHO HTST RP PPO DPPH SS TBHQ FDA TBARS ORAC BCBT ABTS CMC TSS TA HCL GAE TPTZ UV HCL NaOH K2S04 EDTA DMRT SAS CIE
-

Superoxide radical Hydrogen peroxide Hydroxyl radical Low debsity lipoprotein Carbohydrate High temperature short time Reverse phase Polyphenol oxidase Radical scavenging activity Superoxide free radical scavenging activity tert-butylhydroquinone Food Drug and Administration Thiobarbituric acid reactive species Oxygen radical absorbance capacity -carotene bleaching test 2.2’, azino-bis(3-ethyl-benz-thiozoline-6-sulfonic acid) Carboxy methylcellulose Total soluble solid Total acidity Hydrochloric acid Gallic acid equivalent Trypyridyl-s-triazine Ultraviolet-visible Hydrochloric acid Ferum sulfate Sodium hydroxide Pottasium sulfate Ethylenediamine tetra-acetic acid Duncan’s multiple range test Statististical Analysis System Commision Internationale de L’Eclairage

-

Fe2SO4.7H2O -

-

xv

LIST OF SYMBOLS Rt L r2 L* a* b* ppm rpm HPLC GAE TSS TA Brix NEB RDA TLC FTC FRAP TBA BHT BHA MRPs ESR SO2 SD ROS Retention time Linearity Correlation coefficient Colour index for lightness Colour index for redness Colour index for yellowness part per million rotation per minute High Performance Liquid Chromatography Gallic acid equivalent (mg/100ml) Total soluble solid Total acidity Unit for total soluble solid Non-enzymatic browning Recommended Daily Allowance Thin Layer Chromatography Ferric Thiocyanate Ferric Reducing Ability of Plasma Thiobarbituric acid Butylated hydroxytoulene Butylated hydroxy anisole Maillard Reaction Products Electron Spin Resonance Spectroscopy Sodium dioxide Standard deviation Reactive oxygen species

xvii EGCg RE B1 B2 E.P Vitamin C Ca Fe Na K epigallocatechin gallate Total vitamin A activity Vitamin B1 (Thiamine) Vitamin B2 (Riboflavin) Edible portion Ascorbic acid Calcium Iron Sodium Pottasium

xviii

LIST OF APPENDICES

APPENDIX A1 A2 A3 B1 B2 B3 C D E

TITLE HPLC-Chromatogram of methanol extract of triterpene acid (Fresh sample and Sample A) HPLC-Chromatogram of methanol extract of triterpene acid (Sample B and Sample C) HPLC-Chromatogram of methanol extract of triterpene acid (Commercial sample CM1 and CM2) HPLC-Chromatogram of methanol extract of glycosides (Fresh sample and Sample A) HPLC-Chromatogram of methanol extract of glycosides (Sample B and Sample C) HPLC-Chromatogram of methanol extract of glycosides (Commercial sample CM1 and CM2) HPLC-Chromatogram of water extract of triterpene acid and Glycosides for fresh sample Calibration curve of standard FeSO4.7H20 Standard calibration curve of gallic acid (GAE)

PAGE 147 148 149 150 151 152 153 154 155

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. asiaticoside and antioxidant property. The pharmacological activity is attributed to its phytochemical constituents such as

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 oxidationreduction reactions (Roberto, et al., 2000). It is known that BHT and BHA retard lipid oxidation, however, due to increasing consumer awareness of health aspect, their used is slowly replaced by alternative antioxidants, which are without toxic effect. Recently, there is growing interest in the used of natural antioxidant in food products. Natural antioxidants are perceived as safe, less toxic and beneficial for human health, however it is very expensive and not widely commercialized. Sources of natural antioxidants are spices and herbs, and such materials have been used throughout history for flavouring and preservative agent (Kikuzaki and Nakatani, 1993).

High concentrations of phytochemical in plant extracts are associated with strong antioxidant activity. Ascorbic acid and phenolic compounds including vitamins, pigments and flavonoids have been identified to be responsible for antioxidant properties in most plants, for example anthocyanin in Roselle extract (Tsai, et al., 2002), hydroycinnamic acid in blood orange juice (Arena, et al., 2001) and catechins in tea extract (Kikuzaki and Nakatani, 1993). Polyphenols belong to a heterogeneous class of compounds with great variety of effects. These compounds are reported to quench oxygen-derived free radicals by donating a hydrogen atom or an electron to the free radical (Yuting, et al., 1990). The antioxidant effect of polyhenols has been reported in many in vitro studies including human low-density lipoprotein (LDL) and liposomes (Teissedre, et al., 1996). The relationship between antioxidant activity with ascorbic acid content and phenolic compounds has recently been discussed in many research works (Gil-Izquierdo, et al., 2002; Arena, et al., 2001; Gil-Izquierdo, et al., 2001; Dawes and Keene, 1999). The flovonols quercetin was identified as the antioxidant

4 property in Polygonum hydropiper, a medicinal herb (Haraguchi, et al., 1992) and onion (Makris and Rossiter, 2001). The antioxidant activity of orange juice, pineapple juice and many fruit juices are found to be associated with the concentration of ascorbic acid (Gardner, et al., 2000). On the other hand, ascorbic acid is widely used as an antioxidant in many food products, including processed fruits, vegetables, meat, fish, soft drinks and beverages (Madhavi, et al., 1996b).

Nutritionally, pegaga contains appreciable level of asiaticoside (1-8%), carotene (2649

-

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

(Brinkhaus, et al., 2000; Tee, et al., 1997; Fezah, et al., 2000). These compounds play an important role on promoting human health through their antioxidant activity (Velioglu, et al., 1998; Gil-Izquierdo et al., 2001; Jeniffer, et al., 1998; Gazzani, et al., 1998). Abdul Hamid, et al. (2002), determined that various extracts from different parts of pegaga exhibit antioxidant activity. Phenolic compounds were found out to be the major contributor of antioxidant properties (Zainol, et al., 2003). Since quercetin and kaempferol also appeared as part of major flavonoids components in pegaga (Radzali, et. al., 2001; Koo and Suhaila, 2001), it is possible that these constituents may contributed in the antioxidant capacity of pegaga drink. However, the specific phenolic components that involves in antioxidant activity of pegaga are not clearly identified. In other study, Shukla, et al. (1999a) investigated the role of asiaticoside as antioxidant property in wound healing activity. Asiaticoside derived from pegaga has been attributed to increase the antioxidant levels at an initial stage of healing. Beside, carotenoid and ascorbate peroxidase are also present as antioxidative constituents in this herb (Yusuf, et al., 2000). In fact, recent traditional applications indicated that a high intake of pegaga is associated with the reduced risk of a number of chronic diseases (Brinkhaus, et al., 2000).

Fruits and vegetable products are often subjected to heat treatments in order to preserve their quality and prevent the microbial growth. The most important commercial method of juice and drink preservation is pasteurization. This method is

5 based on time and temperature relationship (Moyer and Aitken, 1971). The standard pasteurization process destroys harmful bacteria and deactivates detrimental enzymes without adversely affecting the taste, quality and the nutritional value (Nagy and Shaw, 1970). Although, High Temperature Short Time (HTST) processing treatment or flash pasteurization retained most quality and nutrient in processed foods, but the cost of the equipments is high.

The traditional pasteurization processes or known as batch pasteurization often heat the juice or drink for longer periods of time, at slower heat-up rates, using considerably higher temperatures. Most of the vat or batch pasteurization of acidified beverages applied at below 93 C in order to maintain the sensory quality and to reduce the nutrient loss. For example, the mango puree heated under batch process in steamjacketed kettle until reaches 85 C (Luh, 1970).

The most important factor determining the minimum thermal process is the pH of the product (Noraini, 1984). According to Pederson (1980), for highly acid drink and juice (the pH is lower than pH 4.2) would normally be processed at 71.1 C to 100 C. On the other hand, Chuah (1984) reported that the process of pasteurization usually consists of a process whereby the food is heated to temperature 60-90 C either to destroy the nonsporing pathogens or to prolong the shelf-life of the food, usually but not conjunction with some added preservatives which prevent the spores of microorganisms from germination. High temperature heat processes are unnecessary for acid juices because the heated spores of spore-forming bacteria are unable to germinate at pH 4.2 or lower (Pederson, 1980). The heat treatment of beverages held at 60 C for 10-20 minutes is also recommended for the acidic products (Chuah, 1984). Scalzo (2004) studied the effect of thermal treatments of blood orange juice at 80 C for 6 minutes on antioxidant changes compared to non-thermally treated juice. After pasteurization at 80 C for 6 minutes, the inhibition DPPH (%) was reduced from 49.1% (unheated juice) to 43.2%. The processing of pineapple and “asam jawa” drink at 85 to 90 C for 1 to 5 minutes still

6 maintained the sensorial quality of products (Che Rahani, 1998). The carrot juice heated at 82 C for 5 minutes retained 57% of -carotene (Bao and Chang, 1994). The heating temperature for canned fruit and vegetables beverage is depended on the microbial level of the raw materials, the acidity of the products, the size of the can and the thermal conductivity of the product. Canned mango puree was heated in open steam jacketed kettle to 80 C for 10minutes. After hot-filling, the sealed cans were immersed in boiling water for another 20 minutes (Godoy and Rodriguez-Amaya, 1987). In other processing practice, the guava juice was heated to 87 C for 5 minutes, hot filled and sealed cans pasteurized in boiling water for 30 minutes. (Padula and Rodriguez-Amaya, 1987). The authors found that carotene content was maintained after heating at these processing condition. In other report, Che Rahani (1998) recommended the heat processing of guava drink at 82 C for 5 minutes, followed by canning and immersed in boiling water (100 C) for another 10 minutes.

One of the issues in plant material processing is on the effect of processing method on the phytochemical profile of the products. According to Nicoli, et al. (1999), the health benefit of plant material is dependent on their processing methods. Food processing procedures are generally believed to be responsible for the depletion of natural antioxidant and at the same time it is expected to have a lower health protecting capacity than fresh produce. Gazzani, et al., (1998) reported that processing steps significantly influenced the antioxidant activity of plant materials. This is due to the loss of antioxidant or the formation of compounds with pro oxidant action may lower their antioxidant capacity. The naturally occurring antioxidant such as ascorbic acid and phenolic compounds are generally degraded under thermal treatment (Mahanom, et al., 1999; Makris and Rossiter, 2001; Fezah, et. al., 2000). Thermal treatment also responsible for the reduction of antioxidant activity in processed products (Hunter and Fletcher; 2002; Takeoka, et al., 2001). Pro oxidant compounds that formed in early stage of Millard reactions significantly decreased the antioxidant activity (Nicoli, et al., 1999).

7 Thermal treatments are also frequently used in the extraction of phytochemicals substances from fruits and vegetables (Gazzani, et al., 1998). Some antioxidant substances are well extracted during preparation of herbs extract at high temperatures. For example, the maximum antioxidant capacity from in vitro studied is associated with the drinking of green tea prepared at high temperatures (90 C) and with long infusion time. However, Langley-Evans (2000) suggested that the black tea is ideally prepared between 70-90 C with infusion times not exceeding 1-2 min for maximum antioxidant recovery. According to Scalzo, et al., (2004), thermal treatment generally induced and increased the extractability of the phenolic substances of orange juice, such as anthocyanins and total cinnamates. The presence of intermediate oxidation state of polyphenol is also reported to exert a higher antioxidant activity (Manzocco, et al., 1998). On the other hand, alterations to the structure of existing antioxidants, as well as the formation of novel antioxidant components may enhance the initial antioxidant status (Gazzani, et al., 1998; Nicoli, et al., 1997b; Nicoli, et al., 1999). Heat treatment accelerates the oxidation reactions responsible for the formation of compounds with pro oxidant properties and compounds having antioxidant activity. Example of such reaction is Maillard reaction products. The brown-coloured Maillard reaction products formed in advanced stage of non-enzymatic browning reaction have clearly shown to improve antioxidant activity in vitro. Complex relations between these variables are generally obtained in multicomponent and in formulated foods (Manzocco, et al., 2000). Thus, the heat processing treatment could caused negative effect as well as enhanced their antioxidant activities on the herbalproducts.

The antioxidant potential of herbs dependent on many factors involved in it preparations. The right choice of processing parameters of herbal products may help to retain their phytochemicals content. In most cases, temperature control, minimizing oxygen content and protection from light can help to ensure maximum retention of antioxiants (Lindley, 1998). On the other hand, the eventual processing damage can be minimized by the addition or enrichment of the product with natural antioxidants and/or reconstituted with secondary antioxidants. According to Lindley (1998), the addition of

8 free radical chain breakers ( -tocopherol), reducing agents and oxygen scavengers (ascorbic acid), chelating agents (citric acid) and ‘secondary’ antioxidant (carotenoids) may be able to stabilize and prevented oxidation damage in fruits and vegetables. Pokorny (2000) reported that modification of a recipe during preparation of food and ready meals improved the stability against oxidation especially with the addition of spices. Recent studies also indicated that the addition of sulphur dioxide (S02) or sodium metabisulphite and vitamin C during processing of commercial food products balanced the depletion of natural antioxidant (Tsai, et al., 2002; Majchrzak, et al., 2004). The presence of metabisulphite has been demonstrated to control the spoilage and promote the retention of the natural antioxidant. Sulphites were successfully used to prevent the non-enzymatic browning in food and vegetables (Sapers, 1993), reduction in decoloration of pigments, changes in texture and loss of nutritional quality (Lindley, 1998). Other food additives such as citric acid generally enhanced the antioxidant activity via synergist effect with natural antioxidant like -tocopherol. Citric acid was also used as metal chelators to inhibit oxidative reactions (Madhavi, et al., 1996). Citric acid is widely used as acidulant and preservatives in food system. The high levels of total soluble solid usually help to stabilize or reduce the deterioration rate of food products. For example, high sugar concentrations are effectively to protect the degradation of anthocyanin (Wrolstad, et al., 1990), the strong antioxidant compound in Roselle (Tsai, et al., 2002) and berry fruits (Skrede, et al., 2000). The effect of sugar concentration is most likely due to lower in water activity (Skede and Wrolstad, 2002).

The impact of food processing and handling on nutrients such as vitamins and minerals are well established. However, the stability and the fate of phytochemicals in processed food have not been investigated to similar extent. It is always believe that phytochemical from pegaga are depleted by processing, particularly where thermal treatments are employed. The level of antioxidant activity and the presence of significant concentration of triterpene glycoside in pegaga are of interest to the herbal industry. However, the effect of processing parameters on both antioxidant activity and triterpene glycoside contents of products from pegaga is yet to be investigated

9 thoroughly. Since triterpene glycosides such as madecassoside, asiaticoside, madecassic acid and asiatic acid have been reported to contribute to the pharmacological activities, it is important to study the effect of processing treatment of pegaga on the fate of these components.

1.1

Objective

The main objective of the study was to investigate the effect of heat processing on the antioxidant activity and triterpene glycosides content of herbal pegaga drink

1.2

Scope

In order to achieve the objective, the scopes of the study are identified as follows: 1. The herbal pegaga drink was prepared under three different heat processing conditions; 65 C/15 minutes (A), 80 C/5minutes (B) and canned process (heat at 80 C/5minutes followed by canning and boiling at 100 C/10 minutes (C)). The unheated pegaga drink known as fresh sample (F) and two commercial samples, CM1with no thermal treatment and CM2, which heat processed at 90 C for 1 minutes were used as comparison. All pegaga drink samples (F, A, B, C, CM1 and CM2) were used for further assessment. 2. The physico-chemical characteristics of pegaga drink samples (F, A,B, C, CM1 and CM2) including pH, total acidity, total soluble solid, colour, proximate analysis, total polyphenol and ascorbic acid content was

10 studied. These assessments provide the basic data or information of characteristics of sample studied. 3. The level of antioxidant activity in pegaga drinks prepared under different heat processing conditions was assessed using two antioxidant assays namely Ferric thiocyanate (FTC) method and Ferric reducing ability of plasm (FRAP) methods. 4. The effect of addition of sodium metabisulphite and citric acid, and total soluble solid of fresh herbal pegaga drink on antioxidant activities were evaluated. The contribution of total polyphenol and ascorbic acid on antioxidant activity was also evaluated. 5. The concentration of four components of triterpene glycosides in pegaga drinks; including asiatic acid, madecassic asid, asiaticoside and madecassoside were examined. The contribution of asiaticoside on antioxidant activity of herbal pegaga drinks was also evaluated.

11

CHAPTER 2

LITERATURE REVIEW

2.1

Medicinal plants in Malaysia

Recently, there has been a worldwide interest towards the application of natural products in the health care. There are 80% of world’s populations who are dependent on the natural products for health care (Muhammad Idris, et.al, 1999).

In Peninsular Malaysia, there are about 1,230 plant species with medicinal value have been recorded (Latif, 1983). In 2002 alone, Ministry of Health received about 22,493 applications for registration of herbal medicinal products. There are 10,758 of traditional medicinal products that were registered until december 2002 and 146 premises were licensed (MOH, 2002).

Malaysian herbal remedies such as tongkat ali putih (Eurycoma longifolia), pegaga (Centella asiatica), hempedu bumi (Andographis paniculata) and limau purut (Citrus hystrix) have been recognised to provide health benefit. Their beneficial active components have a potential to be developed into commercial products (Mohamad Faisal, 2000).

12 2.2 Herbal Products in Food Industries

The global market for herbal products is estimated to be worth US$80 billion in 2000, and is expected to increase to US$200 billion in 2008 and US$5 trillion in 2050. It is estimated that from about RM2 billion Malaysian herbal markets in 1999, only RM 100 million was locally produced while the reminder was imported (Business time, 2000). The herbal/natural product industry is considered to be one of the most dynamic sectors with annual growth estimated at 20% a year (Mohamad Faisal, 2000).

Generally, Small and Medium Industries (SMI’s) contributed most of the production of herbal food products in the market, however, they are low in technical know how and managerial skills. Herbal food products attract a lot of interest from food producer due to the changes in market trends. To date, no data has been reported on the market value of herbal food products in Malaysia.

2.3

Plant Material (Centella asiatica)

2.3.1

Plant Description

Pegaga or Centella asiatica (L.) Urban is a genus of the plant family Apiaceae (Umbelliferare). Medicinal herb, which has a mildly bitter taste also commonly known as Hydrocotyle asiatica L., Indian Pennywort or Hydrocotyle asiatique in france (Ling, et. al., 2000). Other names of pegaga include ‘Luci Gong Gen’ or ‘Tung Chain’ in China, ‘Vallarai’ for tamil nadu in India and ‘Daun Kaki Kuda’ in Indonesia (Perry, 1980; Goh, et al., 1995).

13 Pegaga can be found easily in moist habitats or wet swampy area through out India, Malaysia, Madagascar, China, Southern United State Amerika and Middle Africa (Brinkhaus, et al., 1996; WHO, 1998; Perry, 1980).

Pegaga is a perennial creeping plant with cup-shaped of leaves, glabrous stems and rooting at nodes. The leaves are thin, soft and green in colour. The whole plant including leaves, stem and root are consumed as ‘ulam’ and therapeutic agents (Brinkhaus, 2000; Indu Bala & Ng, 2000).

Plate 2.1: Pegaga (Centella asiatica)

2.3.2

Medicinal Applications

Pegaga is used for medicinal purposes since prehistoric time (Kartnig, 1988) and it is used to treat a wide range of indications especially against gastrointestinal diseases, gastric ulcer, indigestion, gastritis and inflammantory diseases of the liver (Brinkhaus, 2000; WHO, 1998).

14 Pegaga based products are available in the form of powder, infusions, soluble and extract of fresh and dried plant, in both conventional and homeopathic preparation. It is also prepared in the form of ointments and creams (Brinkhaus, 2000). Madecassol (asiaticoside) in tablet, ointment and powdered form was used as anti-inflammatory (Chen, et al., 1999) and autoimmune (Guseva, et al., 1998). In terms of cosmetic application, it is used to promote skin regeneration and stimulate biosynthesis of collagen through the formation of lipids and proteins. Pegaga extract is reported to be effective on scar treatment (Faridah, 1998; Brinkhaus, et al., 2000).

2.3.3 Bioactive constituents in pegaga

The chemical constituents of pegaga are classified into main groups including essential oil, flavone derivatives, triterpenic steroids, triterpenic acids and triterpenic acid sugar ester or saponin (Brainkhaus, et al., 2000). Pegaga also contains various important constituents for clinical and pharmaceutical uses (Bonte, et.al., 1994). Chemicals that were previously investigated from pegaga are brahmic acid, brahminoside, brahmoside, centellic acid, centelloside, hydrocotyline, isobrahmic 3acid, glucosylkaempferol, 3-glucosyl-quercetin, indocentelloside,

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 -carotene and ascorbic acid, known to have accounted for 391 mg, 171mg and 32 mg per 100g, respectively. Pegaga is not a good source of protein, carbohydrate and fat.

antioxidative activities, are present at appreciable concentration (2649 g and 48.5mg, respectively) in fresh pegaga. -carotene and carotenoids can act as antioxidant and are Besides the more effective quenchers in singlet oxygen. In terms of mechanism, they are preventing the formation of hydroperoxides (Rajalakshmi and Narasimhan, 1996). popular phytochemical constituents in pegaga, these particular compounds also

17 contributed to the positive health. The nutrition composition in pegaga is shown in table 2.1.

Table 2.1: Nutritional composition of pegaga % E.P Nutrient composition of edible portions (E.P), per 100g sample Proximate composition*
Kcl Energy g Water g Protein g Fat g CHO g Fiber g Ash

37 Indian Pennywort (pegaga); Hydrocotyle asiatica 44

87.7

2.0

0.2

6.7

1.6

1.8

Vitamin**
g Retinol g Carotene g RE mg B1 mg B2 mg Niacin mg C

0

2649

442

0.09

0.19

0.1

48.5

Mineral**
mg Ca mg P mg Fe mg Na mg K -

171

32

5.6

21

391

Sources: Note:

* Nutrient Composition of Malaysian Foods (Tee et.al., 1997) ** Nutrient Composition of Malaysian Foods (Tee et.al., 1988) RE (Total Vitamin A activity) is expressed as retinol equivalents and calculated as g retinol + ( g carotene/6)

18 2 .5 Triterpene Glycosides (Asiaticoside, Medacosside, Asiatic acid and Madecassic acid)

The bioactive constituent of therapeutic interest in pegaga is pentacyclic triterpenoid group known as asiaticoside (De Lucia, et al., 1997) or saponin -containing triterpene acids and their sugar esters, the most important being: asiatic acid, madecassic acid and the three asiaticosides, asiaticoside, asiaticoside A and asiaticoside B (Sing and Rastogi, 1969; Brinkhaus, et al., 2000). Pegaga contains not less than 2% triterpene ester glycosides, asiaticoside and madecassoside (Kartnig, 1988). et.al. 1992). Asiaticoside and asiatic acid were also reported to be found naturally in Schefflera octophylla (Sung,

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 R3 R5

HO

HO HOH2C R1

OR2

Saponins Asiatic acid Asiaticoside Madecassic acid Madecassoside

R1 -H -H -OH -OH

R2 -H - -D-glc-(6-1)- -D-glc(4-1)--L-rha -H - -D-glc-(6-1)- -D-glc(4-1)--L-rha

R3 -CH3 -CH3 -CH3 -CH3

R4 -CH3 -CH3 -CH3 -CH3

R5 H H H H

Figure 2.1: Structure of triterpene glycoside: asiatic acid, asiaticoside, madecassic acid, and madecassoside (Brinkhaus, et al., 2000) Saponin

Glycone Sugar Neutral saponins Steroids

Aglycone Sapogenin Acid saponins 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. storage (Bolin and Stafford, 1974). Sulfur dioxide treatment can also affect the ascorbic acid losses during processing, as well as during

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, In other quercetin, luteolin, and mycertin are low molecular weight polyphenolic compounds that are widely distributed in vegetables and fruits.(Hertog, et al., 1992). investigations, Bors and Saran (1987) reported that many flavonoids such as kaempherol, quercetin, luteolin, mycertin, eridictyol, and catechin have been shown to have antioxidant activities.

The biologically active phenolic compounds containing one or more aromatic rings are found naturally in plant foods, where they provide much of flavour, colour and texture. Phenolic compositions are also observed to be responsible in taste such as bitterness and astringency (Lea and Arnold, 1978).

26 2.7.1 Phenolic compounds in pegaga

Total polyphenol was determined in all parts of pegaga including leaves, stem and root and it shows the highest in the leaves for about 0.23 g/mg dried methanol extract. The high concentration of polyphenol is thought to be responsible for the antiinflammatory 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. activities of pegaga. They also suggested that phenolic compounds are the major contributors to the antioxidative

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 Phenolic antioxidant, consuming foods and beverages rich in phenolic content is related with reduced incidences of heart disease (Muhammad Idris, et al., 1999). particularly flavanoids, exhibit a wide range of biological effects including antiinflammatory, 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 (YanishlievaMaslarova, 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 Primary antioxidant Group / Mechanism of action Compounds Phenols ‘Hindered’ Phenols Miscellaneous Antioxidant Oxygen Scavengers Secondary / Synergistic Chelating Agents Antioxidant Secondary antioxidant Miscellaneous antioxidants Gallates, Hydroquinone BHT, BHA, TBHQ Primary Trolox-C, Ethoxyquin Sulfites, Ascorbic acid, Ascorbyl palmitate EDTA, Citric Polyphosphate, acid, Lecithin, acid, Spices -carotene, Anoxomer,

Tartaric acid Thiodipropionic Distearyl ester Amino extract, Vitamin A, Tea extract Sources: Rajalakshmi and Narasimhan (1996). acid, Flavonoids,

30 2.8.1 Antioxidant activity in herbs

In traditional application, tea, herbs, fruit, vegetables and spices have been widely used as major source of antioxidant (Cao, et.al.,1996; Rajalakshmi and Narasimhan , 1996; Madsen & Bertelsen, 1995; Velioglu, 1998; Wang, et.al., 1996). Most of tropical herbs are rich with antioxidant activities, for example Morinda citrifolia, cucuma longa, zingiber officinale and lemon grass. There are wide range of components identified as antioxidant compound in herbs. Several studies have been made concerning relationships between the antioxidant activity and curcumin in C. longa (Ruby, et al., 1995), carnosic acid in sage and rosemary (Cavelier, et al., 1994), quercetin in Polygonum hydropiper (Haraguchi, et al., 1992), catechin in tea herb (Wang, et al., 2000), vitamin E in green-leafy vegetables (Mallet, et al., 1994), total polyphenol in Chrysanthemum morifolium and Hordeum vulgare (Duh and Yen 1997), flavonoid (Makris dan Rossiter, 2001; Catarina, et al., 1999) and anthocyanin in roselle (Tsai, et al., 2002). Phenolic components also appear to be major contributors to the antioxidant potential of tea herbs and non-citrus juice (Wang, et al., 2000; Miller et al., 1997).

2.8.2

Antioxidant activity of pegaga

Pegaga is well known to have a high antioxidant activity (Abdul Hamid, et al., 2001). It has been established that the presence of polyphenol in pegaga extract is contribute its antioxidative efficiency activity with the correlation of r2=0.9 (Zainol, et al., 2003). The specific component of phenolic that contributed to antioxidant activity in this herb is not reported clearly. Vimala, et al., (2003) reported that pegaga leaves were found to have very high antioxidant activity in three different pathway including superoxide free radical scavenging activity (86.4%), inhibition of linoleic acid peroxidation (98.2%) and radical scavenging activity, DPPH (92.7%). The consumption

31 of pegaga was useful to protect the cells from oxidative damage, to destroy excess free radicals and keep the oxidative stress state in balance. Shukla, et al., 1999 investigated the role of asiaticoside as antioxidant properties in wound healing activity. Asiaticoside derived from pegaga has been attributed to increase the antioxidant levels at an initial stage of healing. Yusuf, et al., (2000) also observed the antioxidative axtivities of carotenoid and ascorbate peroxidase in herb pegaga. The characteristics of antioxidant activity in pegaga were previously studied. Pegaga exhibited optimum antioxidant activity at neutral pH and the activity remained stable up to 50 C. The antioxidative activities of pegaga extracts increased when concentration was increased from 1000 to 5000ppm(Abdul Hamid, et al., 2001).

2.8.3

The Role of Synergistic or Secondary Antioxidants

Synergistic antioxidant generally classified as chelators and oxygen scavenger. Chelator such as citric acid is basically used as acidulant and stabilizer in some food products. It is first suggested for the stabilization of edible oil where it acts as a synergist of tocopherols. In food industries, it is commonly added in foods and In terms of antioxidant activity, citric acid beverages (e.g. fruit and vegetable juice or drink) in order to produce sour taste as well as lowering the pH in such products. provides an acidic medium that improves the stability of primary antioxidants. It is also act as metal-chelating agent in some food systems (Lindsay, 1985).

Heavy metals such as iron (Fe) and copper (Cu) are strong important promoter of lipid oxidation as they catalyse the decomposition of lipid hydroperoxides into free radicals. Chelating heavy metal, by chelating agents such as citric acid and EDTA, into inactive complexes improved the stability of fats, oils and food lipids (Pokorny, 2001b).

32 2.8.3.1 Effect of citric acid

Chelators like citric acid and phosphate are not antioxidant, but highly effective as synergists with both primary antioxidants and oxygen scavengers. For example, the addition of citric acid generally enhances the activity of primary antioxidant such as BHT and TBHQ, and the combination is used in vegetables oils, shortenings and animal fats. The application of 0.02% citric acid with TBHQ is effective in the improvement of oxidative stability of olive oil from 7 to 12 hours. In further investigation, addition of citric acid was found to increase the stability to 58 hours (Sherwin, 1990). Mixtures of citric acid and erythrobic acid are used to retard the browning of bananas. Santerre, et al. (1988) reported that application of citric acid can prevent browning of sliced apple and, thus, extend shelf life. Besides, the combination of citric acid with oxygen scavenger such as ascorbic acid exhibited more beneficial effects (Pizzocaro, et al., 1993). Citric acid prevents discoloration of some fruits and vegetables such as canned pear, sliced beets, onions and potatoes. In meat products, the combination of citric acid with BHA and phenolic antioxidants generally applied to increased stability, retarding oxidative rancidity and preserved the flavour (Madhavi, et al., 1996b).

2.8.3.2 Effect of sulphites

Sulphites are weak antioxidant and are known as oxygen scavenger. Its also have been used for food preservatives in commercial food production. Currently, the forms employed include SO2 gas, and the sodium or potassium salt sulphite, bisulphite or metabisuphite. It is most effective as an antimicrobial agent in acid media, which is optimum from below pH 3.0. Generally, the production of brown pigments by enzyme and catalyzed oxidation of phenolic compounds can lead to a various quality problem during the handling of some fresh fruits and vegetables. However, the use of sulphite or metabisulphite sprays or dips with or without added citric acid provides effective control

33 of enzymatic browning in prepeeled or presliced fruits and vegetables (Lindsay, 1985). Sulphites are effective in preventing enzymatic browning and preserve freshness in raw packaged and unpackaged fruits, vegetables, fruit juices and beer. According to Taylor, et al. (1986), the mechanism protection of sulphites may involve several reactions including directly inhibit the enzymes, interact with intermediates of reaction or act as reducing agents promoting the formation of phenols from quinones. In the prevention of non-enzymatic browning, sulphites react with carbonyl intermediates and preventing their participation in reactions leading to the formation of brown pigments (Madhavi, et al., 1996b). Sulphites functions as an antioxidant in variety of food products. For example, the addition of sulphur dioxide (SO2) in dried apple cubes was contributed to inhibit the development of enzymatic oxidation of phenols during the drying process. SO2 has an ability to retain most of the original antioxidant activity (Manzocco, et al., 1998). In other cases, the high antioxidant activity was also observed in the commercial wine and juice sample, partially due to the presence of vitamin C or preservative such as metabisulphite (Tsai, et al., 2002). The used of sulphites, however, associated with asthma in some individuals. Since 1986, Food Drug Administration (FDA) banned the use of synthetic preservative from sulphites in raw packaged or unpackaged fruits and vegetables because of some adverse reactions reported in sulphites-sensitive individuals.

2.8.4

Effect of enzymatic oxidation on antioxidant activity

Chemical and enzymatic oxidations are the main caused of the reducing of polyphenol antioxidant properties. Green tea was found to have higher phenol and chain-breaking activity than those observed in black tea (Manzocco, et al., 1998; Yen and Chen, 1995). The enzymatic oxidation of polyphenols during processing of black tea was reduced the antioxidant properties. However, polyphenols with an intermediate oxidation state have a higher radical scavenging efficiency than the non-oxidized polyphenol. For example, antioxidant properties are increased and higher in semi fermented tea as compared to fermented tea and non-fermented tea (Yen and Chen,

34 1995). Pokorny (1987) reported that oxidation of polyphenols leads to the formation of stable intermediates or macromolecular compounds, which can still maintain strong antioxidant activity. The chain-breaking efficiency during processing of beverages is also attributed to the increased stability of partially oxidized polyphenols (Manzocco, et al., 1998).

2.8.5

Effect of concentration and sugar content

Sugar is widely added in processed food partially to increase the product stability via lowering the water activity (aw). Addition of sugar also increased the concentration of products and generally measured by total soluble solid. It has long been recognized that a relationships exists between water activity and concentration of food products. Wrolstad, et al., 1990 reported that the concentration of sugar over 20% is preventing the loss of anthocyanins. Jackman and Smith (1996) also found that the amount of similar antioxidant compound is considered to be degrading at lower sugar level. According to Takeoka, et al. (2001), the loss of antioxidant property in tomatoes such lycopene content is increased at 25-30 Brix of total soluble solid. The longer processing time required achieving the desired final solid levels also associated with increased losses of lycopene. The enzymatic and/or chemical oxidation rate of phenolic compounds are associated with some intrinsic food variables such as water activity (aw) and it processing condition (Nicoli, et al., 1999). Wrolstad (2000) reported that the stability of anthocyanin was increases with the decreased water content or with the decreasing water activity.

35 2.8.6 Mechanism of antioxidant activity

Antioxidants help to prevent the occurrence of oxidative damage to biological macromolecules caused by reactive oxygen species (Lindley, 1998). Reactive oxygen species (ROS) are constantly generated in vivo, both by “accidents of chemistry” and for specific purposes (Wang, et al., 1996). Active oxygen forms superoxide, hydrogen peroxide (H2O2) and hydroxy radicals (OH) is a by- product of normal metabolism and attacks biological molecules, leading to cell or tissue injury. Active oxygen and free radicals are produced by certain chemical carcinogens and play a role in carcinogenic process (Cerutti, 1985). The higher antioxidant properties of certain compounds are related to their increased ability to donate a hydrogen atom to free radicals. Antioxidants reduce the primary radicals to non-radical chemical species and are thus converted to oxidize antioxidant (Gordon, 2001).

The mechanism protection by phenolic antioxidants as peroxy radical scavenger is more effectives during the propagation stage of oxidation. It is prevent the formation of hydroperoxides, so that it can stop the chain reactions and provide a longer shelf life of the foods.

According to the chemical structure, antioxidant activities could be categorized into four types including free radical chain breakers such as tocopherol, reducing agents and oxygen scavengers such as ascorbic acid, chelating agents likes citric acid and other secondary antioxidant such as carotenoids (Lindley, 1999).

Primary antioxidants, for example phenolic compounds react with peroxyl radicals and unsaturated lipid molecules and converted them to more stable products. Whereas, secondary antioxidant or preventive are compounds that retard the rate of chain initiation by various mechanism. This antioxidant reduce the rate of autoxidation of lipids by such processes as binding metal ions, scavenging oxygen and decomposing

36 hydroperoxides to non radical products (Gordon, 1990). Secondary or synergistic may function as electron or hydrogen donors to primary antioxidant radicals, thereby regenerating the primary antioxidant. Chelating agents remove prooxidant metals and preventing metal catalyzed oxidations. The oxygen scavenger such as ascorbic acid is able to scavenge oxygen and prevent oxidation of foods, regenerate phenolic or fatsoluble antioxidant, maintain sulfhydryl groups in –SH form and act synergistically with chelating agents (Madhavi, et al., 1996b).

The mechanism of Millard reaction products (MRPs) as antioxidant properties is not clearly observed. In such cases, MRPs can act as metal ion chelators, which is bind heavy metals into inactive compounds (Pokorny, et. al., 2001). Metal chelating is an example of a secondary antioxidant mechanism by which many natural antioxidants can influence the oxidation process. Metal chelators can stabilize the oxide forms of metals that are reduced redox potential, thus preventing metals from promoting oxidation (Hall, 2001).

2.8.7

Assessment of antioxidant activity

Herbs and other natural products contain many hundreds compound of natural antioxidant. Therefore, several methods have been developed to quantify these compounds individually. The techniques are different in term of mechanism of reaction, effectiveness and sensitivity (Khal dan Hildrbrant, 1986; Frankel, 1993; Koleva, et al, 2002). Methods that are widely used to measure the antioxidant activity level in herbal sample, fruits and vegetables, and their products are thiobarbituric acid reactive species (TBARS) (Roberto, et al, 2000), oxygen radical absorbance capacity (ORAC) (Tsai, et al. 2002; Wang, et al, 1996; Zheng and Wang, 2001), -carotene bleaching test (BCBT) (Markin dan Rossiter, 2001; Gazzani, et al., 1998), ABTS radical-cation (Arena, et al,

37 2000; Miller, et al, 1995), DPPH titration (Imark, et al, 2000), Folin-Ciocalteu (Donovan, et al, 1998) as well as FTC and FRAP.

2.8.7.1 Ferric Reducing Ability of Plasma (FRAP)

Ferric Reducing Ability of Plasma (FRAP) is a novel method for assessing antioxidant power through their reduction of ferric to ferrous ion at low pH. colour and it is detected at wavelength of 593 nm (Benzie and Strain, 1996). The combination or complex of ferrous-tripyridyltriazine is caused to formation of blue

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, underprocessing, which may not destroy all the organisms, leads to spoilage and is a potential health-hazard. It is therefore important that suitable heat processing schedules be obtained, taking into consideration the effect of the water activity, pH and thermal conductivity of the product (Desrosier and Desrosier, 1977). Blanching, dehydration, sterilization and pasteurization are an example of thermal treatment that commonly practice in food industry (Pokorny, 2001).

39 The pasteurization process is firstly established for the preservation of milk. They are two types of pasteurization procedures namely batch or vat pasteurization and flash pasteurization. Flash pasteurization is used to designate High Temperature Short Time treatment or HTST (Potter, 1986). Recently, the flash pasteurization at 88 C for 1 minutes, 100 C for 12 seconds and 121 C for 2 seconds are common practice in fruit juice industry, where the bacterial destruction effect is very nearly equivalents (Veldhuis, 1971). HTST practice is effectively retained the flavour and nutritional value of juices. However, the short holding time required special equipment, which is more expensive than the batch process. The flash pasteurization of milk at 71.1 C for 15 sec is equivalent in bacterial destruction to batch method at 62.8 C for 30 minutes (Potter, 1986).

The time and temperature relationship of pasteurization process required the knowledge D and Z value for the destruction of the target organisms. The D value is a measure of heat resistance of a microorganism. It is the time in minutes at a given temperature required to destroy 1 log cycle (90%) of the target microorganism. The Z value reflects the temperature dependence of the reaction. It is defined as the temperature change required to change the D value by a factor of 10 (Potter, 1986; Desrosier and Desrosier, 1977). The times and temperatures however vary according to the heat sensitivities of the foods and the effects of the different foods on microorganism survival (Noraini, 1984; Potter, 1986). For example, a study done by Mazzota (2001) has resulted in a recommended general thermal process of 3 seconds at 71.1 C for achieving a 5-log reduction for E. coli, Salmonella and Listeria monocytogenes in apple juices with the pH adjusted to pH of 3.9. However, a study done by Mak, et al. (2001) has shown that treatments of 68.1 C for 14 seconds and 71.1 C for 6 seconds are capable of achieving a 5-log reduction of acid adapted E. coli in apple cider (pH 3.3).

The heat resistance of the microorganisms and their spores is affected by a number of factors which include; 1) Age and previous history of the organisms or spores, 2) composition of the medium in which the organisms or spores are grown,

40 heated and recovered, 3) pH and water activity of heating medium 4) heating temperatures and 5) initial concentration of organisms and spores (Chuah, 1984). As shown in Figure 2.2, Desrosier and Desrosier (1977) were reported the effect of pH on heating temperature and the time required to kill the heat resistance of spores. Thermal processes for low acid foods are designed to in activate the spore of Clostridium botulinum. Low acid foods usually processed in steam under pressure at temperatures of 116 C or 121 C and sometime in steam at temperatures of about 140 C (Noraini, 1984). Several low acid beverages are acidified with organic acid, such as citric acid and malic acid, to reduce their pH to less than 4.6 so that pasteurization processes may be used. However the addition of acid promotes the formation of coagulation of suspended solids. Thus, thickening agents such as methyl cellulose have been used to increase stability via holding the solids in suspension (Pederson, 1986).

In pasteurization certain acid juices, the industry formerly used treatments at 63 C for 30 minutes (Potter, 1986). The high acid (below pH 4.2) beverages could also be process at the temperature as low as 60 C for about 10-20 minutes (Chuah, 1984). According to Pederson (1980), the heating treatment at the temperature as low as 71.1 C is high enough to kill the vegetative bacteria, since the spore-forming bacteria are unable to germinate at pH 4.2 or lower. In this type of beverages, the preservatives such as benzoic acid, sodium metabisulphite and sobic acid, sometime is added (Moyer and Aitken, 1971).

41 100 KILLING TIME (min) 10 pH 4.5 1.0 pH 3.5 0.1 99 110 TEMPERATURE ( C) Figure 2.4: Influence of pH of heating medium on heat resistance of spores (Desrosier and Desrosier, 1977). 121 pH 5 to pH 7

Depending on the types of beverages, the pasteurization process is applied at different combination of time and temperatures. For example, the pineapple and “asam jawa” drink was prepared at 85 to 90 C for 1 to 5 minutes (Che Rahani, 1998), but the orange juice was thermally treated at slightly low temperature, 80 C for 6 minutes (Scalzo, 2004). For the heat processing of guava drink and carrot juice, the pasteurization at 82 C for 5 minutes was recommended (Bao and Chang, 1994). In other study, the mango juice was processed by 4 different methods by Muhammed, et al. (1965). Of their processing methods, the one employing the least heat (pasteurize at 87.8 C for 1 minutes) gave the best quality.

The pasteurized of acid juices and drinks may be filled into plastic bottles, glass bottles or into cans. Previously pasteurized or sterilized beverages are hot filled between 78-93 C and held in this temperature for 1-3 minutes in containers before cooling (Noraini, 1984). In other practices, the unheated juice was put in glass bottles, which were then crowned and pasteurized at 77-82.2 C for 20 to 30 minutes (Pederson, et al., 1980). According to Mehrlich and Felton (1971), the pasteurization of canned pineapple

42 juice may be handled according to either of two alternatives. The first alternative procedure for handling the juice is pasteurized the product to approximately 90 C. The cans are filled with the juice at this temperature and held for 1-3 minutes. In the second alternative, the juice was first pasteurized at 60 C, filled into cans and the can sealed was then boiled for certain time according to the size of the can. In canning process of some juices and drinks, the products are commonly heat-treated at the temperature of 80 to 87 C for 1-10 minutes, filled into cans, sealed and immersed in boiling water in the range of 10 to 30 minutes (Godoy and Rodriguez-Amaya, 1987; Padula and RodriguezAmaya, 1987; Che Rahani, 1998). According to Luh (1980), the mango juice should be heat processed at 87.8 C, followed by filling, sealing in processed in water bath. Processing in the boiling water sterilizes the inner surfaces of the can and lids and prevents contamination of the product from those surfaces. Although canning processes result in the losses of sensorial and nutritional quality attributes, the processes are still widely used, and could be optimized to improve quality retention regarding the specific of any particular commodity.

2.9.1

The retention of nutrient and phytochemical during processing of foods

Exposure of food components to temperature above ambient condition during heat processing) is a major cause of detectable changes, no only on nutritional quality, but also of phytochemical contents (Pokorny, 2001b). Phytochemical is a food The components that are derived from natural occurring ingredients and are actively being investigated for their health-promoting potential (Bloch and Thomson, 1995). 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, -Dglycosides hydrolyzed less rapidly than -D-glycosides. During processing, the starch molecule undergoes several physical modifications depending on the type of contained starch and severity of the conditions employed (Goni, 1996) leading to the formation of resistant starch (RS) that escapes digestion and absorption in the intestine (Annison and Topping, 1994). Oxidation or degradation of lipid and protein leads to the development of off-flavour, rancidity, softening, loss of solubility and loss of nutritive value (Cheftel, et al., 1985).

The effect of storage and food processing on nutrients particularly in vitamins and minerals are well known. The Arrhenius activation energy (Ea) for ascorbic acid degradation in canned peas at 110-132 C was calculated to be 41 kcal/mol (Lathrop and Leung, 1980). The amount of potassium, sodium and phosphorus in spinach were reduced by 56%, 43% and 36%, respectively after blanching treatment (Bengtsson, 1969). Similarly, Meiners, et al. (1976) reported that cooking process of navy bean caused to the decrease the amount of iron, zinc, magnesium and phosphorus in the range of 50-65%. Extrusion of cereal at high temperatures caused a significant decreased the amount of biologically active compounds including tocopherols, reduced glutathione,

45 melatonin, as well as trace elements such as Cu, Zn, Mn and Se (Zielinski, et al., 2001). According to Min. et al., (2004), the loss of total selenium content caused by blanching treatment is greater than the effect of sterilization. 2001). The application of moderate temperatures, up to 100 C, reduces the negative changes of nutritional quality (Pokorny,

2.9.3

Effect of heat processing on natural antioxidant

There are many evidences found that industrially processed food and home prepared significantly change the natural antioxidant. This is based on fact that most of chemical constituents in food are unstable (Erdman Jr, 1979; Hurt, 1979). Few studies on the phytochemicals retention including natural antioxidant of processed foods have been published. The stability of ascorbic acid and some phenolic compound during processing of foods and beverages are discussed as follows.

Ascorbic acid level in foodstuffs depends not only on the raw material composition but also on the processing method employed (Marin, et al., 2002). There are many studies for determining the ascorbic contents under different processing parameters and storage conditions (Kabasakalis, et al., 2000; Hunter and Fletcher, 2000; Franworth, et al., 2001; Wong, et al., 2000). The amount of this particular phytochemical is significantly destroyed in canned peas, pasteurized pineapple and orange juice as well as processed roselle juice (Lathrop and Leung, 1980; Akinyele, et.al., 1990; Wong, et al., 2001). Lea (1992) reported that, fresh apple contain up to 100 ppm of vitamin C, but during processing into juice it is rapidly lost. The loss of ascorbic acid was also found to be highest in medicinal plants dried at 50 C for 9 hour (75.60%) compared to freeze drying (21.13%) (Mahanom, et al.,1999). Mild (75 C for 30 sec) and standard pasteurization (95 C for 30 sec) slightly increased the total vitamin C of orange juice from 143.5 mg to 160.5 and 131.2 to 155.7 mg, respectively, probably due

46 to the contribution from the solid parts (pulp) as a consequence of heat treatment (GilIzquierdo, et al., 2002).

Several studies on the effect of home and industrial processing on polyphenol stability have been carried out (Gil-Izquierdo, et al., 2001; Mannzocco, et al., 1998; Takeoka, et al., 2000; Wang, et al., 2000). Their influence on total polyphenol is dependent on the types of processing employed and the stability of individual phenolic compounds. In fact, the concentration of phenolic compounds is not necessarily reduced as a consequence of heat processing. For example, Spanos, et al. (1990) reported that a high temperature during initial processing of apple juice produced up to a 5-fold increased in amount of phloretin glucosides as compared to that obtained in pressed juice without temperature elevation. The concentration of anthocyanins of pasteurized (80 C/1minutes) blood orange juice was higher than non-thermally treated juice (Scalzo, et al., 2004). They also reported that a higher antioxidant capacity of thermally treated juice can be ascribed to the extraction, during processing, of antioxidant compounds, such as free and bound hydrocinnamic acids and anthocyanins. After drying at 75 C and storage for 15 weeks at 40 C, 85% of the total phenolic in Roselle extract remained (Tsai, et al., 2002).

Many studies, however, indicated that most food processing procedures significantly reduced the concentration of phenolic compounds. Boilling for 60 min caused overall flavonol losses of 20.6% and 43.9% in onions and asparagus, respectively (Makris and Rossiter, 2001). The level of polyphenol content in air-dried sample of pegaga is also lower than the fresh sample. The difference is expected due to oxidoreduction of polyphenol compound during processing and storage (Fezah, et. al., 2000). During extraction process, phenolic compounds are usually sensitive to acidic solution and high temperature. According to Julkunen-Garcia (1997), drying at below 50 C yields the highest amount of total phenolics in the sample. Increasing the temperature above 60 C, however, lowered the phenolic compounds considerably. The negative effect of thermal treatments on some phenolic antioxidant is also widely reported in a

47 few experiments. The greater loss of total lycopene (35%), major carotenoid pigment and antioxidant in tomato, was reported when the temperature was increased from 90 to 150 C. The duration of heating below 100 C, however, had little or no effect on the degradation of lycopene (Shi and Le Maguer, 1999). Thermal processing of tomatoes into paste partly decreased the concentration of lycopene of 9-28% and it is believed to be due to longer processing time required to achieve the desired final solid levels (Takeoka, et al., 2001). Heat is also observed as one of the most destructive factors of anthocyanins in berry fruit juices (Jackman, et al., 1987). The degradation of anthocyanin is increased from 30% to 60% after 60 days storage when storage temperatures were increased from 10 C to 23 C (Cabrita, et al., 2000). Wang, et al. (2000) also reported that after heat processing and 12 days of storage about 86% of epigallocatechin gallate, 79% of epigallocatechin and 57% of epicatechin in green tea extract were lost. Again, carotenoid content in 8 medicinal plants is loss by 27% and 20% after oven drying at 50 C for 9 hours and 70 C for 5 hours (Mahanom, et al., 1999). The fate of most phytochemicals in processed food products are also notably influenced by storage conditions. Storage of concentrates of apple juice for 9 months resulted in 50-60% loss of quercetin and phloretin derivatives (Spanos, et. al., 1990).

In other investigation, blanching and boiling treatment significantly affected the amount of quercetin and kaempferol in onion. The amount of quercetin in onion was reduced from 41mg/100g in fresh to 25mg/100g after steam blanching and 22mg/100g after boiling for 3 minutes. Since quercetin previously has been reported to be heat stable, the great loss of these compounds may occur during pre-processing like peeling, chopping and trimming (Ewald, et al., 1999). Similarly, chopping significantly reduced the amount of rutin in asparagus up to 18.5% (Makris and Rossiter, 2001).

48 2.9.4 Effect of heat processing on antioxidant activity

The change in antioxidant activity, particularly during thermal processing, is mainly due to the loss of naturally occurring antioxidant properties, the presence of very heat stable natural antioxidant, the presence of polyphenols and formation of novel compounds having pro-oxidant and antioxidant activity (Nicoli, et al., 1999). Thus, the antioxidative activities processed foods can be loss, remain stable or unchanged and even enhanced.

During processing of fruits and vegetables, several oxidation-reduction may occurred in which electron is removed from atom or molecule and presence as oxidized form. It is known that, this particular reaction is susceptible to colour such as browning, flavour, odor, texture and nutritional change if processed and stored in high temperature. For example, during pasteurization, the colour deterioration in fruit juice is mainly due to enzymatic browning of polyphenolic, catalysed by polyphenoloxidases in the presence of dissolved oxygen (Pokorny, 2001b). (Dziezak, 1986). Generally, the physico-chemical change was indicated through the degradation of vitamins and essential fatty acid 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 physicochemical as well as the presence of catalyst (Frankel and Meyer, 2000). Heating and a high oxygen pressure cause an acceleration of the chain initiation and propagation of the oxidation process, and hence a decrease in the oxidation stability, or in the activity of the present antioxidant (Yanishlieva-Maslarove, 2001).

Hunter and Flatcher (2002) investigated the antioxidant activity, total polyphenol and ascorbic acid content in peas and spinach during microwave heating, boiling treatment for 3 minutes and boiling treatment for 8 minutes (overcooked). The ABTS

49 and FRAP method is used in their assessment. The also studied the antioxidant activity of peas and spinach at frozen storage and after blanching treatment (97 C for 85 seconds and 97 C for 90 seconds, respectively). Blanching treatment is found to be useful to prevent the enzymatic oxidation that usually responsible to the loss of natural components in raw material or plants (Nicoli, et.al., 1999). However, after blanching of peas and spinach the level of their antioxidant activity is reduced for about 50% and 20%, respectively, subjected to ABTS assay. The antioxidant activity remained constant and stable at frozen storage. Boiling peas (100 C for 8 minutes) caused losses in watersoluble antioxidant activity and ascorbate content of 34% and 61% respectively (Hunter and Platcher, 2000). The reduction of antioxidant activities in pegaga extract at 7090 C is also may associated with the loss of naturally occurring antioxidant (Abdul Hamid, et al., 2002). Gil-Izquierdo, et al. (2002) studied the effect of pasteurization at 75 C and 95 C on antioxidant activity towards the DPPH method. The antioxidant activity equivalent to mg L-Ascorbic acid of orange juice increased from 126.8 mg (before pasteurization) to 135.3 mg after pasteurization at 75 C. However, the activity was decreased from 150.1 mg to 143.7 mg after standard pasteurization at 95 C for 30 sec.

Processing treatment sometimes did not affect or caused insignificant change to the content and activity of naturally occurring antioxidant. As previously observed, carotenoids content such as lycopene and –carotene, are very heat stable even after prolonged heat treatments (Elkin, 1979; Miki & Akatsu, 1971).

2.9.4.1 Development of pro-oxidant during heat processing

The loss of antioxidant activity in food products not only associated with the degradation of natural antioxidant but also due to the formation of compounds with pro oxidant properties. Pro-oxidant generally appeared in early stages of non-enzymatic

50 browning (Nicoli, et al., 1999). Gazzani, et al. (1998) investigated the effect of thermal treatment at 2, 25 and 102 C for 10, 20 and 30 minutes on antioxidant activity of vegetable juice based on -carotene bleaching test. When prepared at 2 C for 10 minutes, most vegetables juice showed initial pro-oxidant activity. The pro-oxidant activity was very high in eggplant (-307%), tomato (-621%) and yellow bell pepper (432%).

2.9.4.2 Development of heat-induced antioxidant

Food processing may also result in the formation of antioxidant compounds such as Millard reaction products (MRPs) (Madhavi, et al., 1996b). These particular compounds having antioxidant activity that influenced the antioxidant properties of food. Formation of advance MRPs during prolonged heating time and storage generally exhibited strong antioxidant properties (Eichner, 1981; Nicoli, et al., 1999). Millard reaction products were identified to be active as oxidation inhibitors in tomato puree (Nicoli, et al., 1997b). The development of non-enzymatic browning reactions, as occurs in the production of Marsala-type wine, resulted in a great increase in its chainbreaking activity (Monzocco, et al., 1999b).

The development of non-enzymatic browning (NEB) in foodstuffs is caused positive and negative impact to the food industry and consumers. For example, it is important for some types of food processing (baking, cocoa and coffee roasting) but often has negative impact due to changes in sensorial aspects (colour and aroma) in other food products such as fruit juice (Carabasa-Giribet and Ibarz-Ribas, 2000). Browning due to thermal treatments are the results of several reactions. Non-enzymatic browning reactions between amino acids and reducing sugars are the basis of the Millard reaction, which usually appears during thermal process (Whistler and Daniel, 1985). Beside, the reactions are included caramellisation, ascorbic acid browning process

51 (Cornwell and Wostad, 1981) and pigment destruction (Beveridge, et al., 1986). The rate of NEB is depends on water activiy, pH, temperature and chemical composition of the food system (Whistler and Daniel, 1985; Potter, 1986). The brown colour is developed in c. asiatica drink during heat processing but it is not clear which reactions are involved to enhance NEB. The influenced of NEB to antioxidant capacity is already discussed in a few papers (Manzocco, et al., 2000; Nicoli, et al., 1999; Morales and Jimenez-Perez, 2001; Manzocco, et al., 1999). Although the concentration of natural antioxidant is significantly reduces as a result of thermal treatments, the overall antioxidant properties of process products are maintained by the development of NEB such Millard reactions (Nicoli, et al., 1997b). They also described the correlation between the developments of Millard reaction products with relative antioxidant activity. The correlation of relative antioxidant activity with heating time and heating temperature is shown as figure 2.5.

Gazzani, et al. (1998) reported that heat treatment of carrot juice, cauliflower juice and zucchini juice at 102 C for 10 minutes exhibited higher antioxidant activity. The antioxidant activity (based on -carotene bleaching test) also increased with the increasing of heating time and heating temperature. 30 minutes of heating. For example, the antioxidant activity of carrot juice at 25 C for 10 minutes was 24% and it was increased to 75% after 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

Relative antioxidant activity

11 10 9 8 7 6 5 4 3

T3

T2

T1

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 physicochemical characteristics of pegaga drinks were also observed. The information obtained from the study could be used as a guideline for designing thermal processes to reduce the phytochemical degradation of the products. Besides, the factors that may contribute to the antioxidant activity in unheated pegaga drink were also studied.

3.1

Introduction

Thermal treatment is generally applied to extend shelf life of fruit and vegetable products. However, heating processes can affect the nutrient and phytochemical loss, which leads to consumer dissatisfaction. In this study, the three different heat processing treatment applied on pegaga drink were 65 C/15 minutes, 80 C/5 minutes and in canning process (heat at 80 C/5minutes, canned and followed by boiling at 100 C/10 minutes before cooling process). The heat processing parameters were based on pasteurization methods of acidified foods (Chuah, 1984; Scalzo, et al., 2004; Che Rahani, 1998). The canning process of herbal pegaga drink was followed the procedures

55 of high acid canned beverages as previously done on fruit and vegetable juice. (Godoy and Rodriguez-Amaya, 1987; Luh, 1980; Che Rahani, 1998).

Traditionally, pegaga juice was prepared by blending the whole parts of pegaga with certain amount of water, before it is consumed fresh as cooling drink. In this study, the untreated drink, known as fresh sample, was used in order to compare the status of antioxidant activity and triterpene glycosides content before heating and without addition of any food additives and food ingredients.

Recently the demand of herbal pegaga drink by the consumers is on the increase mostly due to the health benefit and the phytochemical presence in the drinks. Therefore, the current status of nutrient content, antioxidant activity and active constituents in commercial pegaga drink available in the market are important to be studied. The results obtained are useful as a reference for consumers and researchers.

The factors influence to the antioxidant activity was also investigated. The used of citric acid (Dziezak, 1986; Sherwin, 1990) and sodium metabisulphite (Tsai, et al., 2000) is reported to increase the antioxidant activity in several food products. However, the effect of these food additives and total soluble solid via sugar addition on antioxidant activity of pegaga drink is still unclear. The range of citric and total soluble solid used in this study was based on consumer acceptances as previously reported in many research works (Pederson, 1980; Lea, 1991; Henrix, 1995), while the range of sodium metabisulphite was followed the level permitted in Malaysian Food Act 1983 and Food Regulation1985. The study on effect of addition of citric acid (0-0.3%w/v), sodium metabisulphite (0-350ppm) and sugar (in the range of 1 to 15 Brix) on antioxidant activity of fresh pegaga drink was carried out. Citric acid addition varied in accordance with acidities of raw materials and consumer acceptance. Citric acid in the range of 0.1%-0.3%w/v are usually added into fruit and vegetable juices to increase the acidity for the flavour and preservative purposes (Pederson, 1980). Vegetable juice acidified

56 with 0.4%w/v citric acid was too sour. According to Malaysian Food Act 1983 and Food Regulation1985, the maximum level of sodium metabisulphite permitted in fruit and vegetable drinks is about 350 part per million (ppm). Therefore, the effect of sodium metabisulphite at concentration of 0-350ppm on antioxidant activity was used in this study. The amount of sugar added to fruit and vegetable drink mainly based on sensory test or consumer acceptance. However, the total soluble solid in ready-to-drink of fruit beverages is widely varied from 5-15 Brix (Lea, 1991; Henrix, 1995)

3.2

Material and sample preparation

3.2.1

Juice extraction

C. asiatica from species ‘pegaga ubi’ or also known as ‘pegaga biasa’ that was recommended for commercial production (Indu Bala & Ng, 2000) was used in preparation of pegaga drink. Local supplier from Johor Bahru supplied the plant material for this study. 400 g of pegaga including leaves, stolon and root was cleaned under running tap water. The clean sample was blend with 2 litre deionised water by using food processor. The juice extract then was filtered using muslin-cloth.

3.2.2

Preparation of pegaga drink

Pegaga drink was prepared based on formulation that was developed by Malaysian Agricultural Research & Development Institute (MARDI). The juice extract was mixed with 11% (w/v) sugar, 0.12% (w/v) citric acid, 0.11% (w/v) carboxyl methylcellulose (CMC) and made up to a volume of 2.3 L with deionised water. The flow chart

57 for the preparation of pegaga drink is shown in Figure 3.1. The product was pasteurized at three different heat-processing temperatures; 65 C/15 minutes, 80 C/5 minutes and in canning process (heat at 80 C/5minutes, canned and followed by boiling at 100 C/10 minutes before cooling process). at 4 C. Pegaga sample (Centella asiatica) Cleaned and washed under running tap water Homogenized in deionized water Filtered through muslin-cloth Pegaga juice extract Add water Add sugar, CMC, citric acid and water Fresh sample (F) Heat treatment Commercial sample CM1 (unheated) CM2 (90 C/1min) Fresh sample (F) or non-thermally treated drink without added sugar and food additives was also prepared. Each product was then kept

65 C/15 min (A)

80 C/5 min (B)

Canning process (C) (heat at 80 C/5 min, canned, boiled at 100 C/10 min)

Packed in LDPE bottle

Analysis Figure 3.1: Flowchart of the preparation of pegaga drink

Samples Fresh sample (Sample F) Heat-treated samples (Sample A, B and C) Commercial sample (Sample CM1 and CM2)

Assessments

Physico-chemical characteristics Antioxidant activity Triper

Triterpene glycosides content

pH, Total acidity, Colour index L*, a* and b* values, Total soluble solid, Proximate composition, Microelement, Total polyphenol and Ascorbic acid content

Ferric thiocyanate assay (FTC) and Ferric Reducing Ability of Plasma (FRAP) assay

Asiaticoside content, Madecassoside content, Asiatic acid content and Madecassoside content

Output

Contribution of total polyphenol and ascorbic acid on antioxidant activity using correlation coefficient, r

Correlation of FRAP and FTC measurement of antioxidant activity (correlation coefficient, r)

Contribution of asiaticoside on antioxidant activity

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 90 C/1minutes. This sample was first diluted into drink prior to analysis. The squash sample was diluted three times according to direction on the label. All samples were kept at 4 C.

3.3

Experiments and Analytical Methods

The heat-treated sample (A, B and C), fresh sample or non-thermally treated (F) sample and two commercial samples (CM1 and CM2) were subjected to analysis of physico-chemical characteristic, antioxidant activity and triterpene glycosides content. Three replicates sample of pegaga drink for each treatment were used for each analysis. The data was presented as means and were analyzed by ANOVA. Figure 3.2 presented the layout of experiments.

3.3.1

Physico-chemical characteristics

3.3.1.1 Colour Index

Colour analyses were carried out pegaga drink samples using Minolta Chromameter CR-300 (Minolta Camera Co. Ltd., Osaka, Japan). The instrument was

60 standardized against a white tile before each measurement. Colour was expressed in L*, a* and b* Hunter scale parameters (Nicoli, et al., 1996). Hunter L* denotes lightness with 0 being black and 100 being white, while a* denotes a red hue when positive or a green hue when negative, and b* denotes a yellow hue when positive and blue hue when negative.

3.3.1.2 Total Soluble Solid (TSS) and pH

Total Soluble Solid (TSS) and pH was measured with a hand held refractometer (Atago) and pH-meter (EcoMet), respectively.

3.3.1.3 Total Acidity (TA)

Total acidity, expressed as citric acid monohydrate was calculated in percent after titration of 10 mL sample against 0.1N NaOH to end point, pH 8.2 (AOAC, 1980). % TA = Titrate (ml) x 0.007009 g/ml x 100 Sample (ml) (3 .1)

3.3.2

Proximate Composition and Microelements Analysis

3.3.2.1 Moisture

10-15 g of homogenized sample was placed into glass dish before dried in a 105 C oven for five hours. The dish was then removed from oven (Memmert,

61 Germany), cooled in dessicator and weighed soon after attaining room temperature. The steps were repeated until constant weight was obtained (AOAC, 1984). % Moisture by weight = loss of weight in gramme of the sample x 100 Weight in gramme of sample (3.2)

3.3.2.2 Ash

2.5-3 g sample was weighed into crucible. The sample was charred on heating mantle until no smoke evolves. Ashing was carried out in muffle furnace (Memmert, Germany) at 550 C for about 8 hours or until grey ash was obtained. Sample was then cooled in dessicator. (AOAC, 1984). % of Ash = Weight of ash / Weight of sample x 100 (3.3) The ash was calculated after constant weight was obtained

3.3.2.3 Protein

The Kjeldahl method for determining total nitrogen was based on Tecator Kjeltec System 1026 and David Pearson (1976) was used. Reagent: Concentrated sulphuric acid (A.R Grade), Sodium hydroxide (A.R Grade 40%), 0.05M Hydrochloric acid, 4% Boric acid with bromocresol green indicator and catalyst, Kjeltabs (1.5 g K2S04 and 0.0075 g Se) were used. Assay: 0.2-1 g sample was weighed and mixed with 2 pieces of Kjeltabs and 10 ml of sulphuric acid in digestion tube. The mixture was digested for 1 hour or until a clear solution was obtained at 420 C. The sample was cooled and distilled using Kjeltec 1026 Distilling Unit with 25 ml of 4% boric acid solution. Bromocresol indicator was placed

62 on receiver flask. The sample was then titrated with 0.05M Hydrochloric acid (HCL) to neutral grey. Calculation: % N = 14.01 x (ml of titrant of sample – ml of titrant of blank) x conc. of standard acid g of sample x 10 % Protein = % N x factor specific for different product (6.25) (3.4)

3.3.2.4 Fat

Reagent: Petroleum ether BP 40 – 60 C Assay: Sample (3-4g) was placed into an extraction thimble. Thimble was then placed in a beaker and dried in an electric oven for 5 hours at 70-80 C. Dried sample was extracted with petroleum ether using Soxhlet extraction apparatus for 6-8 hours. The solvent was evaporated and the residue was dried in an electric oven for 30 minutes at 105 C. The sample weight was then measured (AOAC, 1980). % Fat = (W2-W1) x 100 Sample weight in g W1 = weight of evaporating flask W2 = weight of evaporating flask + content after drying (3.5)

3.3.2.5 Fibre

Reagent: 0.255N Sulphuric acid (A.R Grade), 0.313N Sodium hydrochloride (A.R Grade), Hydrochloric acid (1% in water v/v) were used.

63

Assay: Defatted sample (1-3g) was weighed (W0) and placed in beaker. 200ml of sulphuric acid was added and boiled for 30 minutes. The sample was filtered with Whatman paper no. 1 and the residue was washed with hot water until free from acid. The residue was then washed with 200 ml of warmed sodium hydroxide (0.313N), boiled for 30 minutes and filtered through crucible. The residue was washed with hot water, 1% HCL and hot water again until neutral, then followed by ethanol. The sample was dried in oven at 105 C for 1 hour. The crucible with residue was weighed (W1) and ignited in muffle furnace at 450 C for 4 hours. The cooled crucible was weighed again (W2) (AOAC, 1984) . % Crude fiber = W1 - W2 / W0 x 100 (3.6)

3.3.2.6 Carbohydrate and Energy

Total carbohydrate was estimated according to Nergiz and Otles (1993). Energy was calculated using the factors 4.0, 4.0 and 9.0 kcal/g for protein, carbohydrate and fat, respectively (Abdurahman, et al., 1998). Calculation: Total carbohydrate (%) = 100% - (moisture content (%) + ash (%) + fat(%) + protein(%) + crude fiber(%) ). Energy (Kcal)= (4 kcal/g x amount of protein, g) + (4 kcal/g x amount of carbohydrate, g) + (9 kcal/g x amount of fat, g)

3.3.2.7 Microelement

Instrument: The analysis of micronutrient element (selenium, aluminium, plumbum and magnesium) was conducted using Elan 6100 Inductively Coupled Plasma Mass

64 Spectrometer Method (Perkin Elmer, Canada) and multi-element calibration standard diluted into 10ppm. Assay: 1 ml of sample was digest with 5 ml of Aqua Regia solution for 30 minutes at 70-80 C. Aqua Regia solution was prepared from mixing of 1N Nitric acid and 1N Hydrochloic acid (3:1). The sample was then filtered using Wathman no. 540 Hardened Ashless. The filtrate was added with deionized water and make up to 100ml. The sample was again filtered through a nylon filter Wathman 0.2 m before injected on Mass Spectrometer. calibration standard. The calculation of microelement was based on multi-element

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 FolinCiocalteu phenol reagent (Merck-Schuchardt, Hohnenburn, Germany). The mixture was than allowed to stand for 5 min and 2 ml of sodium carbonate were added to the mixture. The absorbance was measured using UV-Vis spectrophotometer (Interscience). The absorbance of blue complex was analysed at 750 nm in a cuvette of 1 cm. The total phenolic content of pegaga drink was calculated from the calibration curve prepared from the absorbance of gallic acid standard (Fluka, Chemicals) solutions and ferullic acid standard (Fluka, Chemicals) solutions. Results are expressed as equivalents mg of gallic acid (GAE) per 100ml of sample.

66 3.3.5 Antioxidant Assay

Preparation of sample: Each sample of pegaga drink was filtered using Bunchner funnel with Whatman no.4 filter paper. The extract was kept at 4 C before assay.

3.3.5.1 Ferric reducing ability of plasm (FRAP) Assay

Reagent:

300 mmol/litre buffer acetate, p.H 3.6 ; 10 mmol/litre TPTZ (2,4,6-

trypyridyl-s-triazine, Fluka Chemicals) in 40 mmol/litre HCL (BDH); 20 mmol/litre Fe3.6H2O (BDH). FRAP reagent was prepared by mixing 25 ml buffer acetate, 2.5 ml TPTZ solution and 2.5 ml Fe3.6H2O solution (Fluka, Chemicals). Assay: Antioxidant activity was analyzed according to procedure of Benzie and Strain (1996) with slight modification as described by Gardner, et al. (2000). Freshly prepared FRAP reagent was warmed to 37 C. The Reagent blank reading was taken at 593 nm. 1 ml of diluted 10-fold sample was added into 3 ml of FRAP reagent. Absorbance reading was taken after 4 minutes. Results were calculated from calibration curve prepared from Fe2SO4.7H2O (Fluka, Chemicals) solution in the range of 0.1mM to 10mM.

3.2.5.2 Ferric thiocyanate method (FTC)

The FTC model method will be used according to modified method of Kikuzaki dan Nakatani (1993).

67 Reagent: 2.51% linoleic acid in 99.8% ethanol. 0.05M phosphate buffer (pH 7). 30% ammonium thiocynate. 0.02 M ferrous chloride in 3.5% HCl. Assay: 1 ml sample (0.02% in 99.5% ethanol) was mixed with 2 ml of linoleic acid, 4.0 ml phosphate buffer (pH 7.0) and distilled water (3.0 ml). The sample was kept in cap screwed container in dark condition at temperature of 40 C.

0.1 ml of sample was added with 75% ethanol (9.7ml) and 0.1 ml of 30% ammonium thiocyanate. 3 minutes after addition of 0.1 ml of ferrous chloride to the reaction mixture, the absorbance of red colour was measured at 500 nm until absorbance of control (blank reagent) reach maximum. standard sample. % Inhibition = 100 – Absorbance increase of sample (max) x 100 Absorbance increase of control (max) (3.7) -tocopherol and ascorbic acid were used as

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. 2. 3. Citric acid at concentrations of 0% (control), 0.1%w/v, 0.2%w/v, 0.3%w/v) Sodium metabisulphite at concentrations of 0 ppm (control), 200ppm, 250ppm, 300ppm and 350ppm Sucrose was also added onto fresh pegaga drink up to total soluble solid 5 Brix, 10 Brix and 15 Brix. Fresh drink (1 Brix) without sucrose added was used as control.

68 3.3.7 Determination of Triterpene glycoside

Determination of bioactive constituents such as triterpene acid (asiatic acid, madecassic acid) and its glycosides (madecassoside and asiaticoside) were according to modified method as reported by Inamdar, et.al. (1996). HPLC Conditions: Isocratic HPLC system by Waters 2487 Dual Absorbance

Detection was used. Chromatographic separation was performed with a Genesis, C18, 4 cm 120 with a methanol-water mobile phase (90:10) for triterpene acid and (80:20) for its glycosides, UV detection at 220nm, flow rate at 0.4ml/min. A 10 sample was injected onto the column. Preparation of Standard Triterpene Glycosides: Standard triterpene acids; l volume of

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. 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 The solutions were filtered through 0.45- m membrane filter and 10 l of each standard was

69 methanol-water (90:10) (Inamdar, et al., 1996). The sample was vortexed for 5

minutes, centrifuged and filtered using a Milipore filter (0.45-µm) and a known amount of extract was subjected to HPLC under the above conditions. The contents of triterpene glycosides were calculated based on water extract and methanol extract with the aid of calibration graph obtained using a stock solution of each component.

3.4

Statistical analysis

Experimental data was analyzed by analysis of Variance (ANOVA) and the significant differences among means was determined by Duncan’s multiple range test (DMRT) using the Statistical Analysis System (SAS V.8) computing program (SAS, Cary,NC)

70

CHAPTER 4

RESULTS AND DISCUSSION

4.1

Introduction

This chapter presents the results on the effect of thermal processing on the physico-chemical characteristics of pelage drink. The thermal processing parameters considered in this study were the preservation temperature and time of the treatment and canning process. The physical and chemical analysis was carried out to determine the product characteristics including acidity, soluble solid content and the color index in herbal pelage drinks and, hence, compare them with some other commercial samples, which is highly consumed locally. The level of nutrient compositions and trace elements were also examined. The antioxidant activity was assessed using two different methods and their correlation was discussed. The factors influenced to the antioxidant activity in pelage drink were also investigated. In addition, the concentration of total polyphenol and ascorbic acid was demonstrated and their contribution to antioxidant activity was predicted through the coefficient of correlation (r). Herbal pegaga drink prepared by different heat treatments was analyzed for their triterpene glycosides content using High Performance Liquid Chromatography (HPLC).

71 4.2 Physico-chemical characteristics of pegaga drink

The results for pH, total soluble solids (TSS), % of total acidity (TA) expressed as citric acid, L*, a* and b* values of different samples are shown in Table 4.1. The low pH (3.72-3.79) of the heat-treated drink (sample A, B and C) was accompanied by a high acidity (14.37-14.72%) calculated as citric acid. The addition of citric acid (0.12%) in heat-treated pegaga drink is responsible for the low of pH and by a high acidity as compared to untreated sample (F). The pH and total acidity (TA) of fresh or untreated sample are 5.93 and 3.85%, respectively. The pH was higher than those obtained in two commercial samples (CM1 and CM2). The organic acid may added to both commercial samples as preservative to extend the shelf life of products. For comparison, the titrable acidity of apple juice is 0.2-0.7% (Lea, 1991). The herbal pegaga drink had a rather low pH. However, it is higher than apple juice (3.5-3.8) (Lea, 1991), orange juice (3.3-3.8) and grape juice (2.8-3.0) (Henrix, 1995). The content of soluble solids in pegaga drink was between 1.0 Brix (fresh) and 11.2-11.8 Brix (heat-treated drink), which is almost similar to orange juice (9-15 Brix) and apple juice (11-14 Brix). The total soluble solid in CM2 was significantly lower (7.6 Brix) than CM1 (12.6 Brix) and heat-treated drink. For heat-treated samples (65 C/15 minutes, 80 C/5 minutes and canned, the parameters of pH, TSS and %TA shows small changes. According to Kaanane, et al. (1988), the minimal change in pH can be explained by relationship existing between pH and free acid content.

To elucidate the formation of browning, absorbance (A) at 280 and/or 420 nm on a UV-Vis spectrophotometer has been extensively used by the other researchers for measurement of brown colour in fruit juices (Buedo, et al., 2000). The formation of advanced Millard products was also monitored by CIE values such as L* (ligntness), a* (redness) and b* (yellowness) values of the sample (Carabasa-Giribet and Ibraz-Ribas, 1999). Since original colour of pegaga was dark green, the absorbance at 280 and/or 420 nm, as well as the L* value were not suitable in indicating the colour changes. In this cases, the changes in green colour of pegaga drink during processing was expressed

72 as b* values. The increase in b* value was used to indicate the development of a brown colour. The data on L*, a* and b* values are shown in table 4.1. Results shows that all heat-treated samples (65 C/15 minutes, 80 C/5 minutes and canned) gradually turned brownish during processing and their b* values steadily increased from 4.88 + 0.06 before heating (F) to in the range of 6.03 + 0.18 - 6.88 + 0.18 after heat processing. Heating at 65 C/15 minutes shows higher development of browning followed by canned and pasteurization at 80 C/5 minutes. The results showed that heat treatments significantly increased (P < 0.05) the brown colour development. CM1 shows greenish in colour as good as fresh sample (F). Both sample were not involved heating process. The whiteness value (L*) of the products was significantly different between F and sample A, B and C. This shows that heat treatment affects the colour of the products.

Table 4.1: Physico-chemical characteristic of pegaga drink Samples F (Fresh) AA (65 C/15min) B (80 C/5min) C (Canned) CM1 CM2 pH 5.93 3.72 3.79 3.72 3.89 4.86 TSS (Brix) 1.0 11.2 11.8 11.2 12.6 7.6 TA (%) 3.85 14.37 14.72 14.38 7.71 4.21 L* +sd 24.43+0.21c 27.84+0.31ab 28.86+0.48a 27.04+2.57ab 26.90+0.77ab 26.35+0.21bc a* +sd 2.83+0.04a 2.17+0.08b 2.09+0.08bc 2.67+0.24a 2.22+0.16b 1.96+0.03c b* +sd 4.88+0.06b 6.88+0.18a 6.03+0.18ab 6.56+2.27a 4.80+0.21b 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 heattreated 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 18755625 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 Fresh Calorie (Kcal) 1.20 Moisture (%) 99.62 Ash (%) 0.063 Protein (%) 0.093 Crude fiber (%) 0.009 Fat (%) ND Carbohydrate (%) 0.215 Zinc Phosphorus Iron Sodium Potassium Selenium Aluminium Plumbum Magnesium 1.09 33.74 4.04 12.06 469.91 ND 149.38 0.44 10.27 A 45.50 88.53 0.070 0.100 0.015 ND 11.285 1.83 30.71 3.17 82.01 446.10 0.01 3.45 2.18 10.19 Proximate B 46.00 88.41 0.073 0.101 0.014 ND 11.402 Minerals 1.16 28.91 2.55 71.47 372.42 Element ND 3.24 0.87 9.38 value C 44.30 88.83 0.070 0.091 0.015 ND 10.994 (mg/L) 1.41 40.70 2.41 68.24 347.99 (mg/L) 0.01 1.13 0.56 8.28 CM1 1.21 99.64 0.055 0.083 0.008 ND 0.220 1.28 18.53 2.81 8.43 273.28 ND 0.97 0.45 6.86 CM2 49.56 87.50 0.089 0.090 0.020 ND 12.301 1.63 16.60 2.58 52.75 131.94 ND 6.55 0.33 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. polyphenols are in the range of 147.92-1413.49mg/100ml. The total The amount of total

polyphenol in commercial sample (CM1) was higher (1140.24 mg/100ml), than sample A, B, and C but the concentration in CM2 (797.53 mg/100ml) was insignificant with sample B. Generally, it can be observed that all pegaga drink sample contained high

78 amount of phenolic compounds as compared to orange juice (75.5 mg/100ml), pineapple juice (35.8 mg/100ml) and vegetable juice (29.3 mg/100ml) (Gardner, et al., 2000). However, based on other finding reported by Manzocco, et al. (1998), the amount of phenolic compounds in heat-treated drink was still lower than observed in green tea and black tea beverages (953.84 mg/L and 801.16 mg/L GAE equivalents, respectively).

Heat treatment applied during preparation of herbal pegaga drink still retains appreciable amount of total polyphenol. After canning processed with the temperature up to 100 C, about 50% of total polyphenol in pegaga drink remain. The loss of total polyphenol content under heat processing treatment at 65 C for 15 minutes and 80 C for 5 minutes was 45% and 49%, respectively. In agreement with previous investigation, phenolic compounds contained in food were significantly loss during heat processing. This finding supported by Fezah, et al. (2000), who noted that the air-dried treatment at room temperature of pegaga leaf contained about 0.111 mg pyrogallol per mg dried MeOH extract, which is significantly lower than fresh sample. The total polyphenol in leaf and underground part of pegaga was reduced by 52% and 50%, respectively. GilIzquierdo, 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 60min 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).

CM2 Pegaga drink sample CM1 C B A F 0 200 400 600 800

d b e d c
a

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 (65 C/15 min); B (80 C/5 min); C (canned); CM1 (commercial sample -Loo Ent.); CM2 (commercial sample-HPA). Values with same letter (a,b,c) are not significantly different (P>0.05) between samples.

As previously investigated, Velioglu et al. (1998) reported that phenolic compounds are responsible for the antioxidative activity in selected vegetables, fruits and grains. In a similar finding, Yen and Chen (1995) noticed that tea leaf exhibited marked antioxidative activity as it contain significant amount of polyphenols. Nicoli et al. (1999), also found that the antioxidative effectiveness in most plant materials is

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 tomatooil 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 Concentration of ascorbic acid (mg/100ml) 4 3.5 3 2.5 2 1.5 1 0.5 0 F

a

b c c e d

A

B Sample

C

CM1

CM2

Figure 4.2: Ascorbic acid content of different sample of pegaga drink (n=3). Key: F (fresh sample); A (65 C/15 min); B (80 C/5 min); C (canned); CM1 (commercial sample -Loo Ent.); CM2 (commercial sample-HPA). Values with same letter (a,b,c) are not significantly different (P>0.05) between samples.

As expected, heat treatment dramatically reduced (41.6-83.45%) the ascorbic acid content in all heat-treated pegaga drink samples. The losses of ascorbic acid maybe attributed to the thermal treatment applied (Yang and Atallah, 1985). Ascorbic

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. primary state of oxidation. The Ferric thiocyanate assay was used to evaluate the antioxidant activity of pegaga drink, only at

The individual antioxidant activity of samples or the effect of heat treatments on the peroxidation of linoleic acid is shown in figure 4.3. Each sample of herbal pegaga drink showed a low absorbance values at 500 nm, which indicated high level of antioxidant activity. The exposure of food components to high temperatures can cause negative change not only to nutritional quality, but also their antioxidant activity.

84 Generally, the oxidative activity of linoleic acid is markedly inhibited by any samples of pegaga drink compared to control assay. The results showed that the level of antioxidant activity is reduced when the temperature is increased. Fresh sample of pegaga exhibited much higher (P<0.05) antioxidant activity than heat-treated samples. The % of inhibition of peroxidation is 72.98% for fresh and 53.73, 64.80% and 69.88% for sample C, B and A respectively (Figure 4.3). The lipid peroxidation inhibitory activity of all pegaga drink samples significantly lower than raw pegaga leaves (98.2%) as previously reported by Vimala, et al. (2003). However, the antioxidant activity of fresh pegaga drink was comparable to those reported for oolong tea and higher than green tea. Yen & Chen (1995) in their investigation indicated that oolong tea and green tea exhibited 73.6% and 40% inhibition of linoleic acid peroxidation, respectively. Our finding is also similar to the work of Duh and Yen (1997), who reported that the addition of herbal extracts significantly increased the inhibition the linoleic acid peroxidation. In terms of mechanism, it is prolongs the induction period by the lowering rate of accumulation of oxidative products.

All pegaga drink samples exhibited higher activity than natural antioxidant such as -tocopherol and ascorbic acid but lower than synthetic antioxidant, butylated hydroxytoulene (BHT) at concentration of 200ppm. In agreement with our result, earlier studies by Abdul Hamid, et al. (2002) revealed that the activity of pegaga evaluated from similar method, is significantly lower than BHT. However, the antioxidant activity of -tocopherol at concentration of 300ppm and above is not significantly different from that exhibited by leaves and roots extract of pegaga. Observation of antioxidant activity under Ferric Thiocynate (FTC) assay, done by Mohd Zin et al. (2002) showed that ethyl acetate extract of mengkudu exhibited significant activity, which are comparable to that of both -tocopherol and BHT.

In the previous studies the changes of antioxidant activity in relation to processing and storage was carried out in different food system. In most cases, the exposure of natural antioxidant to high processing temperatures caused to the decreased

85 in that particular important component. The reduction of the natural antioxidants could be due to evaporation and transformation of the food component during processing that could have pro-oxidant activity (Pokorny et al., 2001b; Nicoli et al., 1999). A brownish colour was disserved in heat-treated drink, as a result of Millard reactions or degradation of chlorophyll pigment. The reduction of antioxidant activity in heat-treated samples can be attributed to the formation of compounds with pro-oxidants properties during processing. Namiki and Hayashi (1983) reported that highly reactive radicals having pro-oxidant properties might be formed in early stages of the Millard reactions, which the formation of both pro-oxidant and antioxidant properties are always depend upon the intensity and the duration of heat treatment (Nicoli, et al., 1999).

BHT Pegaga drink and standard sample Vit.C Vit. E CM2 CM1 C B A Fresh 40.00% 50.00% 60.00% 70.00% h g f e f d c b 80.00%

a

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 (65 C/15 min); B (80 C/5 min); C (canned); CM1 (commercial sample -Loo Ent.); CM2 (commercial sample-HPA); Vit.E ( -tocopherol), Vit.C (ascorbic acid) and BHT (Butylated hydroxy toluene). Values with same letter are not significantly different at P=0.05

86 The antioxidant capacity of herb extract is composed of a mixture of antioxidants, which generally include phenolics, carotenoids and tocopherol. The antioxidant content and profile varied greatly when it was exposed to different environment and processes. As a result, the antioxidant activity of herbs in different processing treatment may differ considerably from one to another.

It clearly that drink samples under study showed the decreased of % inhibition with increasing of processing temperature, in agreement with the results reported by Abdul Hamid et al. (2002), who studied the characterization of antioxidative activities of various extracts of pegaga. They noted that the antioxidant activity of pegaga extract was stable up to 50ºC of incubation temperature and reduced significantly at 70 to 90ºC.

4.6.2

Antioxidant activity by Ferric reducing ability of plasma (FRAP assay)

The results of antioxidant activity from linoleic acid peroxidation was compared with FRAP value. Figure 4.4 demonstrates the FRAP values of pegaga drink as a consequence of processing procedures. The FRAP value was interpolated from a standard calibration curve with the linear regression was y = 7.387e-5x + 0.002. Heat processing studied showed negative effects thus resulted in a decrease in the antioxidant potential of the pegaga drink. The greatest FRAP value was observed in fresh sample (860 µmol/liter) followed by A (65 C/15 minutes), B (80 C/5minutes) and C (canned). FRAP values of heat-treated samples are in the range of 404 - 740 µmol/litre. Two commercial samples (CM1 and CM2) showed the appreciable amount of antioxidant activity, which were able to reduce about 620 µmol/litre and 370 µmol/litre of Fe (III), respectively. The antioxidant activity, however, is significantly lower than value reported by Gardner, et al., (2000), who observed that the ability of vegetable and orange juice to reduce Fe(III) are approximately 1.2mM and 6mM, respectively. These results indicated a similar trend as to the FTC assay. Tsai, et al. (2002) found that the

87 FRAP activity of roselle extract and green tea was 2 mmol/litre and 8 mmol/litre, respectively. The FRAP value was obtained from 1 g of roselle and green tea, extracted in 300ml water at 100 C for 3 min. Results showed that, the level of antioxidant activity in pegaga drink is slightly lower than green tea and roselle extract. Our results differ from previous report on green tea beverages. The optimum activity was obtained when it was prepared at high infusion temperature with long infusion time. The tea beverages prepared at 20-70 C of infusion temperature was significantly lower than at the infusion temperature of 90 C Lingley-Evans (2000). However, the antioxidant compounds in black tea is ideally extracted at 70-90 C in 1-2 minutes infusion time.

Commercial antioxidant at 200 ppm (vitamin E) showed the highest FRAP value (569.37 µmol/litre) followed by synthetic antioxidant, specifically BHT (543.75 µmol/litre), at the same concentration. The FRAP value of vitamin C was 398.36 µmol/litre. In contrast with FTC assay, BHT exhibited lower antioxidant activity than fresh and heat-treated pegaga drink. Our result is similar to the previous studies, which reported that the water extract of Chrysanthemum and Roselle exhibited a greater reducing power than 200 ppm of -tocopherol and BHA (Duh and Yen, 1997).

After heat treatments, the antioxidant capacity were reduced by 14 – 53.59% evaluated by FRAP assay. The polyphenols, which are present in the pegaga drink, can be destroyed or transformed into other phytochemicals during heat treatment and processing. Transformation of existing structure (Kikuzaki and Nakatani, 1993), oxidation of phenolic compounds during processing steps and interaction of phenolic antioxidant with other food components (Nicoli, et al., 1999) may also explain the reduction in the value of antioxidant activity in pegaga drink. Results also indicated that the heating period did not significantly affect the antioxidant properties in pegaga drink. Sample A with prolong heating period (15 min) was still shows higher antioxidant activity compared to sample B and Sample C. Skorikova and Lyashenko (1972) previously mentioned a negative correlation between the heating period and the phenol content of apple and pear juices. However, the browning as well as the antioxidant

88 activity of the tomato samples increased with the increase in heating time Nicoli, et al. (1997b). Nicoli et al. (1997a) also reported that the antioxidant activity did not increased linearly with the increasing of roasting time of coffee brews.

Pegaga drink and standard sample

BHT Vit.C Vit. E CM2 CM1 C B A Fresh 0 200

e f g f d b a 400 600 800 1000 c d

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 (65 C/15 min); B (80 C/5 min); C (canned); CM1 (commercial sample-Loo Ent.); CM2 (commercial sample-HPA). Values with same letter are not significantly different at P=0.05

Although the antioxidant activity of pegaga drink was reduced as a consequence to heat processing, the antioxidant activity of the samples were still considerably high as they exhibited more than 50% inhibition of linoleic acid peroxidation. The trend is almost similar to the antioxidant activity determined by FRAP assay. The high capacity of antioxidant in heat-treated drink is probably due to development of new component having antioxidant properties. Processing steps such ageing, oxygenation and heat treatments can promote progressive polymerisation of phenols to form brown coloured macromolecular products, which are expected to possess the same antioxidant activity of the original phenols (Manzocco, et al., 1999). Wang, et al. (1996), also observed that

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 chainbreaking 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

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

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, Flavonoids function as primary antioxidants, chelators and inflammation, free radicals, hepatotoxins, microbes, ulcers, viruses and tumor (Marcia Zimmerman, 2001). 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. 1996; Namiki, 1990; Larrauri, et al., 1996). Similarly, it is clear that quercetin and kaempferol exhibit a strong antioxidant capacity (Hertog and Hollman, 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 FRAP FTC 0.8078
a

Ascorbic acid 0.8364b 0.7461b

0.6525b

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 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 Control 0%

Absorbance at 500nm

d

c a b

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 FRAP value (Absorbance at 593 nm) 0.02 0.00 -0.02 -0.04 -0.06 -0.05 6.5 6.0 5.5 5.0 pH 4.5 4.0

a

b b
0.05 0.15 0.25

c

3.5 3.0 2.5 0.35 FRAP 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 1 Brix to 15 Brix. Figure 4.10 shows the antioxidant activity of pegaga drink at different level of total soluble solid (TSS). The antioxidant activity of pegaga drink strictly depended on total soluble solid (TSS), which high absorbance value of FRAP assay was observed at 15 Brix (0.057 nm) followed by 10 Brix (0.050 nm), 5 Brix (0.044 nm) and control sample (0.032 nm). A slightly contrast, the antioxidant activity was gradually increased at 5 Brix and 10 Brix by FTC assay, but declined thereafter with the addition of sugar up 15 Brix (figure 4.11). Pegaga drink at concentration of 15 Brix, however shows the lowest antioxidant activity compared to control sample (1 Brix) and other samples tasted. According to Takeoka, et al. (2001), the increase in total soluble solid level up to 25-30 Brix appeared to influence the loss of antioxidant property in tomatoes such lycopene content. They reported that the longer processing time, required to achieve the desired final solid levels, might be associated with increased losses of lycopene. In our study, the role of total soluble solid on overall antioxidant activity was still unclear. However, it was because of different rates in chemical oxidation of phenolic compounds, which are depending on some intrinsic food variables and it processing condition such as water activity (aw) (Nicoli, et al., 1999).

102

0.06 0.05 0.04 FRAP Values (Absorbance at 0.03 593 nm) 0.02 0.01 0 1 Brix 5 Brix

a b c d

10 Brix
o

15 Brix

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 0.4 0.35 0.3 Absorbance at 0.25 500nm 0.2 0.15 0.1 0.05 0 Control 1 Brix 5 Brix 10 Brix

c b a a

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). a c b

0.14 0.12 0.1 FRAP Values 0.08 (Absorbance at 0.06 593 nm) 0.04 0.02 0 0 200 250 300 d e

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 FRAP values (Absorbance at 593 nm) 0.12 0.10 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 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

e

Absorbance at 500 nm

d

c

b

a

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 various types of mobile phase at different concentrations. Absorbance Detection using

Preliminary study was carried out to choose the best combination of methanolwater 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; BurnoufRadosevich 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 2000 1500 1000 500 0 0.00 0.05

y = 8675.9x + 94.036

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 methanolwater 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

Area (1E3 mV.s)

5000 4000 3000 2000 1000 0 0.0 0.1 0.2 0.3 0.4

y = 14125x + 75.092

Concentration of standard solution (mg/ml)
Figure 4.21: Calibration curve for madecassic acid (area vs concentration of standard madecassic acid)

113

14000 12000

Area (1E3 mV.s)

10000 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

y = 31621x + 1.2049

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).
al., 1999)

Methanol and aqueous methanol are effectively use as an

extraction solvent of triterpene glycosides in pegaga (Inamdar, et al., 1996; Verma, et

114 Table 4.4: The results of HPLC analysis Compound Calibration curve Madecassoside Asiaticoside Asiatic acid y=8675.9x+94.036 0.05 – 0.4 y=6767.1x+110.94 0.05 – 0.4 0.025 – 0.4 0.05 – 0.4 y=31621x+1.2049 L (mg/ml) Retention time (tR) (minute) 7.87 8.53 9.11 11.51 Correlation Coefficient (r2) 0.9989 0.9973 0.9996 0.9999

Madecassic acid y=1415x+75.092

4.9.2 Quantitative determination of triterpene glycosides in pegaga drink

Herbal drink such as tea is widely consumed due to its desirable taste as well as their antixidative, antimicrobial and anticarcinogenic properties (Osawa and Namiki, 1981). At the same time, there is now an increased interest in herbal drink or tea from local plant. Herbal drink from pegaga was developed for similar purposes. Traditionally, pegaga is commonly used as herbal tea or herbal drink for it cooling effect especially among the Chinese. The commercial production of pegaga into processed food or value-added products increased the market potential and usage. This assessment was conducted to evaluate the triterpene glycosides content before and after heat processing treatment of pegaga drink.

Table 4.5 illustrates the results of individual component of triterpene glycosides (mg/100ml) content in pegaga drinks and commercial products. The total amount of triterpene glycoside in every sample was tabulated. The average contents of madecassoside in fresh drink were 12.2-22.1% higher than those in the corresponding heat-treated samples. The level of this component dropped from 3.12 mg/100ml (F) to 2.70mg/100ml (A) and 2.43 mg/100ml (B). The amount of madecassoside was first declined at 65 C/15 minutes and 80 C/5 minutes, however, it slightly increased when

115 the temperature was further increased to 100 C as observed in sample C (2.74 mg/100ml). It could be assumed that the target constituent was extracted and dissolved easily at high temperature during heating process of pegaga drink. Madecassoside content of commercial sample (CM1) was almost similar with fresh or control sample (2.93 mg/ml) and significantly higher than other commercial sample, CM2 (2.54 mg/100ml).

Asiaticoside content shows a different trend that its concentration in fresh drink was significantly lower (3.92 mg/100 ml) than in sample A (4.32 mg/100ml). However, was significantly higher than other heat-treated samples. The concentration of asiaticoside was remarkably reduced to 8-22.5% when exposed to high temperature up to 80 C as in sample B (3.61 mg/100ml) and sample C (3.03 mg/100ml). Therefore, it may be concluded that the heat treatment at moderate temperature (65 C) is likely to increase the ability of water (as a medium) to dissolve the asiaticoside. In accordance with the report of Vongsangnak, et al., (2003), which obtained the maximal saponin yield when the extraction temperature was controlled around 50 C. On the other

research, Pan, et al., (2002) noticed that the application of high temperature (20-50 C) enhanced the extraction efficiency. This is a result of an increased in diffusivity of the solvent into cells and at the same time it increased the ability of components to adsorb from the cells.

116

Table 4.5: Results for triterpene glycosides assay

___________________________________________________________________________________________________ Asiaticoside +SD +SD 3.91 + 0.10b 4.32 + 0.05a 3.61 + 0.07c 3.03 + 0.05d 2.60 + 0.30e 3.10 + 0.14d 1.37 + 0.10d 2.79 + 0.10bc 3.22 + 0.33a 0.97 + 0.05c 1.86 + 0.28b + 1.05 + 0.09c 3.02 + 0.20ab 1.02 + 0.07c 2.70 + 0.03bc 1.03 + 0.09c 2.56 + 0.20c 2.45 + 06a 12.04 + 0.14a 10.75 + 0.14b 10.08 + 0.17cb 9.96 + 0.68c 10.18+ 0.46b 8.06+ 0.32d acid +SD acid + SD Madecassic Asiatic Triterpene glycosides

Triterpene glycosides content (mg/100ml) of methanol extracts of pegaga drink

Sample

Madecassoside

+S D

F

3.12 +0.10a

A

2.70 + 0.05cd

B

2.43 + 0.07e

C

2.74 + 0.08c

CM1

2.93 + 0.13b

CM2

2.54 + 0.09de

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 30-50 C (36mg/100g dry weight), previously reported by Verma (1999). After heat treatment, the quantity of madecassic acid is not significantly different over a range of temperature of 0 C (control) to 100 C (C). However, data showed that the lowest amount of this particular compound was observed in fresh drink followed by A, B and C, in which the concentrations were 2.56, 2.70, 3.02 and 3.22 mg/100ml, respectively. The madecassic acid content in commercial samples, CM1 and CM2 were 2.79 and 1.37 mg/100ml, respectively.

Asiatic acid was not stable at the higher temperature and this resulted in a decreasing amount in all heat-treated sample. The concentration of asiatic acid varies in the range of 2.45-0.97mg/100 ml. Asiatic acid content dropped to 10.7%, 16.3 % and 17.3% in A, B and canned sample, respectively. pegaga drink. Pasteurization processed at 65 C/15min (A) to 100 C (C) resulted in a significant change of asiatic acid content in Again, the asiatic acid content of commercial sample, CM1 (10.18 mg/ml) was significantly higher than CM2 (8.06 mg/ml).

The total amount of triterpene glycosides in individual sample was recorded. The overall result shows a different trend from individual assessment. Still heat treatment has a great influence on the concentration of total triterpene glycosides. Heat treatment at 65 C/15 minutes, 80 C/5 minutes and up to 100 C in canned drink caused significant changes in the amount of total triterpene glycosides. The degree of reduction were in the order of: F (12.04 mg/100ml) > A (10.75 mg/100ml) >B (10.08 mg/100ml) >C (9.96 mg/100ml). The changes in phytochemical contents occurred due to the chemical degradation and conversions of some thermolabile asiaticoside and madecassoside to another components during heat treatments. Similar results were reported in ginsenosides during steaming process of P. ginseng (Ren and Chen, 1999). The levels of four active constituents of both commercial samples are always lower than observed in fresh and heat-treated sample. CM1 and CM2 contained about 10.18 and 8.06 mg/100ml of total triterpene glycosides, respectively. The variations of triterpene

118 glycosides content in different samples occur due to various factors such as species, geographical source, cultivation, harvest, storage, as well as preparation method of herb.

It can be seen that the increase in heating temperature decreases the concentration of madecassoside (65 C and up to 80 C), asiaticoside (80 C and up to 100 C) and asiatic acid under all heating conditions. It could be explained that the used of heat may also slightly reduced the concentration of saponins (Court, et al., 1996). According to Choi, et al. (1982), a little thermal degradation of saponin occurred during microwave extraction at 80 C. The effect of heating treatment on active constituents such as triterpene glycosides in pegaga has not been investigated in previous reports. However, the effect of steaming process on Panax notoginseng containing saponins investigated as reported by Lau, et al., (2003). This information might be useful to relate the chemical stability and characteristics of other saponin components such asiaticoside, madecassoside and its triterpene acids. For example, the notoginsenoside R1, ginsenoside Rg1, Re, Rb1, Rc and Rd was degraded after exposure at high temprature during steaming process. prolong steaming duration. The amount was significantly declined upon

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). pharmaceutical preparation. The amount is depended on it formulation and the

120

60 50 Triterpene glycosides s content (%) 40 30 20 10 0 F A B C CM1 CM2 Pegaga drink sample

Madecassoside Asiaticoside Madecassic acid Asiatic acid

Figure 4.23: Triterpenoid fraction (%) of pegaga extract from drink samples

Daily consumption of 60mg-120mg of standardized extracts of pegaga containing up to 100% total triterpenoids is suggested in modern herbal medicine (Murry, 1995; WHO, 1999). The used about 60 mg/day or 120 mg/day of pegaga extract is effective in the treatment of venous insufficiency (Pointel, et al., 1997). It has further been proposed that oral application of total triterpenoid fraction of Centella
asiatica (TTFCA) with dosage of 60 mg/day for about 10 weeks is suggested for venous

hypertension treatment (Belcero, et al., 1990).

No significant side effects are

experienced with internal or topical used of pegaga except for person who allergic to this herb (Murray, 1995; Danese, et al., 1994). It may be possible to suggest that the consumption of 500 ml, accounted for 60.2 mg of total triterpene glycosides, in once or twice a day of fresh pegaga drink is good enough for our health benefits. On the other hand, consumption of 600 ml or more heat-treated herbal drink containing about 59.76 – 64.56 mg triterpene glycosides daily could also contribute appreciable amount of active constituents to the body.

121

Rush, et al. (1993) reported that asiaticoside is converted in vivo to asiatic acid by hydrolytic cleavage of the sugar moiety. Similarly, Grimaldi, et al. (1990) explained that asiaticoside is transformed into asiatic acid in vivo through metabolic interaction. They also suggested that the therapeutic effects of asiaticoside might be mediated through conversion to asiatic acid. Since the actual absorption of these phytochemicals on our body is still unclear, a further investigation is needed to prove their significant role on pharmacological activity and toxicological effect through the consumption of pegaga as herbal drink.

4.10

Antioxidant activity of asiaticoside

Shukla, et al., (1999a) reported the antioxidant effect of asiaticoside. They reported that topical application of 0.2% asiaticoside solution twice daily for 7 days to skin wounds shows an increased in both enzymatic and non-enzymatic antioxidant activity namely superoxide dismutase (35%), catalase (67%), glutathione peroxidase (49%), vitamin E (77%) and ascorbic acid (36%) in newly formed tissue. It also results in several fold decrease in lipid peroxide levels (60%) as measured in terms of their thiobarbituric acid reactive substance (TBARS). Jayasharee, et al. (2003) also reported that oral treatment of extract of Centella asiatica for 14 days significantly increased antioxidant enzymes like superoxide dismutase, catalase and glutathione peroxidase. Results showed that asiaticoside was associated (r=0.63471 and r=0.879 towards FRAP and FTC, respectively) with antioxidant activity of pegaga drink. However, the concentration of asiaticoside in pegaga drink was relatively low (3.03-4.32 mg/100ml) as compared to the amount required for antioxidant effect (0.2g/100ml). Shukla, et al., (1999) also reported that lower concentrations of asiaticoside (0.05% and 0.1%) were found to have no significant effect on wound healing activity. Results obtained from the study assumed that the presence of very small amount of asiaticoside in pegaga drink indicates a relatively low or negligible contribution on total antioxidant activity.

122

CHAPTER 5

CONCLUSION AND RECOMMENDATION

5.1

Conclusion

Fresh pegaga drink (F) contained about 1470.14 mg/100ml of total polyphenol (GAE equivalent), which was significantly higher than pasteurized sample at 65 C/15 minutes (903.23 mg/100ml) and 80 C/5 minutes (805.54 mg/100ml); and canned pegaga drink (730.27 mg/100ml). The degradation of ascorbic acid occurred at a higher rate in canned pegaga drink, followed by pasteurization at 80 C/5 minutes and 65 C/15 minutes. The concentration in fresh pegaga drink was significantly higher, which is about 4.23mg/100ml. Antioxidant assay results revealed that the control sample (F) of pegaga drink exhibited much higher (P<0.05) antioxidant activity than heat-treated samples. The FRAP values of 860 µmol/litre was obtained from untreated or fresh sample (F) and the activity from 404 to 740 µmol/litre were observed in heat-treated drinks. The % inhibition of peroxidation was 72% for fresh sample (F) and in the ranged of 26-56% for heat-treated samples. The reduction of natural occurring antioxidants in pegaga drink could be due to the transformation of the

123 food component during processing into compound that possessed pro-oxidant property. The two assays (FRAP and FTC) were strongly correlated (r=0.93) at p=0.05. However, very low correlation was obtained (r=0.54) when antioxidative activity of pegaga drink, synthetic antioxidant (BHT) and natural antioxidant (ascorbic acid and vitamin E) were taken into account. Initial results suggested that total polyphenol is a major contributor to the antioxidant activity of herbal pegaga drink, which significantly correlated (r=0.8071) at p<0.05 towards FRAP assay. The analysis of individual component of triterpene glycosides shows a different trend. The concentration of madecassoside in fresh sample (F) was 12.2-22.1% higher than heat-treated drink. minutes (B). The maximum amount of asiaticoside (4.32 mg/100ml) was observed during processing at 65 C/15 minutes (A). 3.92mg/100ml. The amount in fresh sample was This result suggested that the heat processing at moderate Canning process retains higher level of madecassoside as compared to pasteurization at 65 C/15 minutes (A) and 80 C/5

temperature (65 C) and longer heating period (15minutes) enhanced the extraction efficiency of asiaticoside. The average content of madecassic acid was higher in canned drink (3.22 mg/100ml) followed by B (3.02mg/100ml), A (2.70 mg/100ml) and fresh sample (2.56 mg/100ml). The heat processing of pegaga drink resulted lower amount of asiatic acid, where the asiatic acid content varies in the range of 2.45-0.97 mg/100ml. The nonthermally treated drink or fresh sample (F) contained higher amount of total

124 triterpene glycosides.followed by sample A, B and C. The change in these components is due to chemical degradation and conversions of some thermolabile asiaticoside and madecassoside to another components.

5.2

Recommendations and further works

Changes in antioxidant activities and other constituents in pegaga drink are already demonstrated. However, the specific components, structures and mechanism that involved in antioxidant activity should also be studied in detail. The future research works should be focuses on flavonoid content such as quercetin and kaempferol and their contribution to antioxidant activity in pegaga drink. Previous works by Vimala, et al. (2003) reported that pegaga leaves extract contain a high antioxidant activity towards superoxide free radical scavenging activity (SS) and radical scavenging activity (DPPH). The similar assessment should be carried out for pegaga drink in order to evaluate its ability to reduce the excess free radical and to determine the level of prevention of tissue and cells damage. Scavenging of DPPH radical determines the antioxidant potential of the test sample, which shows its effectiveness, prevention, interception and repair mechanism against injury in biological system. Further research should be oriented to the optimisation of antioxidant activity and triterpene glycosides content in herbal pegaga drink in order to understand the factors controlling the retention of these phytochemicals. Beside, the data on the potential interactions of natural bioactive constituents with other food components during industrial processing and home preparation of food and beverages is very limited. Therefore, the optimum retention of these phytochemicals under various processing parameters and their interactions with

125 other food components should also be studied in future. Furthermore, their stability under different parameters such as storage conditions, packaging, light, water activity, degree of oxidation and High Temperature Short Time (HTST) processing technology need also be evaluated in future. On the other hand, consumers believe that herbal pegaga products that were assumed rich in antioxidants and triterpene glycosides may afford a degree of protection against free radical damage and higher in pharmacological activity. The data on their adsorption in blood stream, pharmacological benefit and toxicity over the range of studies of still remain unknown and further information should be provided.

126

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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

Absorbance at 593nm

0.03

0.02

0.01

0.00 0 200 400 600 800 1000 1200 1400

-0.01 -200

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

Absorbance at 750nm

1.5

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)

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