FTC Thesis
FTC Thesis
FTC Thesis
JUNE 2005
ii
“I declare that this thesis entitled The Effect of Heat Processing on Triterpene
Glycosides and Antioxidant Activity of Herbal Pegaga (Centella asiatica L.Urban)
Drink is the result of my own research except as cited in the references. The thesis has
not been accepted for any degree and is not concurrently submitted in candidature of any
other degree”
Signature : …………………………………
Name of author : SANIAH BTE KORMIN
Date : …………………………………
iii
ACKNOWLEDGEMENT
First and foremost, thanks to God Almighty for the guidance and help in giving
me the strength to complete this thesis. I would also like to take this opportunity to
express my utmost gratitude to my supervisor, Prof. Dr. Mohd Roji Sarmidi for his
valuable guidance and advice throughout this thesis study.
Finally, I am also forever indebted to my lovely husband, Mohd Azli Sairan for
his continuous encouragement and many sacrifices.
iv
ABSTRACT
The health benefit of herbal pegaga drink, which is associated with triterpene
glycosides content and antioxidant activity attract a lot of interest from the public and
food and herbal industries. The works carried in this research investigated the effect of
heat processing at 65qC/15 minutes, 80qC/5minutes and pasteurization at 80qC/5minutes
followed by canning and boiling at 100qC/10 minutes on these phytochemicals and
compared to untreated herbal pegaga drink or fresh sample. The results revealed that the
untreated pegaga drink exhibited much higher (P<0.05) antioxidant activity than the
heat-treated samples. The Ferric Reducing Ability of Plasm (FRAP) values was 860
µmol/litre for the untreated sample and in the range of 404 - 740 µmol/litre for heat-
treated sample. The untreated drink inhibited about 72% of linoleic acid peroxidation
and the percentage inhibition of heat-treated samples were in the ranged of 26-56%. The
FRAP and Ferric Thiocyanate (FTC) assays were strongly correlated (r=0.93) towards
the assessment of antioxidant activity in pegaga drink samples. The concentration of
ascorbic acid and total polyphenol after heat treatment were 0.7 mg/100ml to 1.76
mg/100ml and 730.27 mg/100ml to 903.23 mg/100ml, respectively. Phenolic compound
was found as the major contributor to the antioxidant activity in pegaga drink. Analysis
of the triterpene glycosides content was performed using an isocratic High Peformance
Liquid Chromatography system (HPLC). Heat processing resulted in a several fold
decreased of total triterpene glycosides. The amount in untreated drink was 10.8 to
17.3% higher than those in heat-treated pegaga drinks. The present study indicated that
the herbal pegaga drinks samples still retain appreciable amount of madecassoside,
madecassic acid, asiaticoside, asiatic acid and polyphenol compounds. These
phytochemicals are good sources of antioxidant.
v
ABSTRAK
Faedah kesihatan bagi minuman herba pegaga yang dikaitkan dengan kehadiran
triterpena glikosida dan aktiviti pengantioksidan telah menarik minat yang tinggi
daripada orang awam dan pengusaha industri herba serta makanan. Kajian ini
dijalankan bagi menyiasat kesan proses pemanasan pada suhu 65qC/15 minit, 80qC/5
minit dan pempasturan pada 80qC/5 minit diikuti dengan pengetinan dan pendidihan
pada 100qC/10 minit ke atas perubahan fitokimia tersebut dan dibandingkan dengan
minuman tanpa rawatan atau sampel segar. Keputusan menunjukkan minuman pegaga
tanpa rawatan menghasilkan aktiviti pengantioksidan yang lebih tinggi (P<0.05)
berbanding sampel yang dipanaskan. Nilai ‘Ferric Reducing Ability of Plasma’ (FRAP)
adalah 860 µmol/liter bagi sampel tanpa rawatan dan dalam julat 404 - 740 µmol/liter
untuk sampel yang dipanaskan. Minuman tanpa rawatan merencat 72% pengoksidaan
asid linoleik dan peratus perencatan bagi sampel yang dipanaskan adalah di antara 26-
56%. Kaedah FRAP dan ‘Ferric Thiocyanate’ (FTC) berkorelasi tinggi (r=0.93) melalui
penilaian aktiviti pengantioksidan di dalam sampel minuman pegaga. Kepekatan asid
askorbik dan jumlah polifenol selepas pemanasan adalah 0.7 mg/100ml hingga 1.76
mg/100ml dan 730.27 mg/100ml hingga 903.23 mg/100ml setiap satunya. Sebatian
fenolik merupakan penyumbang utama kepada aktiviti pengantioksidan. Analisa bagi
kandungan triterpena glikosida dibuat menggunakan sistem isokratik Kromatografi
Cecair Berprestasi Tinggi (HPLC). Proses pemanasan turut menyebabkan penurunan
beberapa kali ganda amaun triterpena glikosida. Amaun di dalam minuman tanpa
rawatan panas adalah 10.8 hingga 17.3% lebih tinggi daripada minuman pegaga yang
dipanaskan. Kajian ini menunjukkan bahawa minuman herba pegaga masih
mengekalkan amaun madekasosida, asid madekasik , asiatikosida, asid asiatik dan
polifenol pada paras yang wajar diterima. Fitokimia ini adalah sumber pengantioksidan
yang baik.
vi
TABLE OF CONTENTS
DECLARATION ii
ACKNOWLEDGEMENT iii
ABSTRACT iv
ABSTRAK v
TABLE OF CONTENTS x
LIST OF PLATE xi
LIST OF TABLES xii
LIST OF FIGURES xiii
LIST OF SYMBOLS xv
LIST OF APPENDICES xviii
1 INTRODUCTION 1
1.1 Objective 9
1.2 Scopes 9
2 LITERATURE RIVIEW 11
3.1 Introduction 54
3.2 Material and Sample Preparation 56
3.2.1 Juice Extraction 56
3.2.2 Preparation of Pegaga Drink 56
3.2.3 Commercial Pegaga Drink Sample 59
3.3 Experimentals and Analytical Methods 59
ix
4.1 Introduction 70
4.2 Physico-chemical Characteristics of Pegaga Drink 71
4.3 Nutrient Composition 74
4.4 Total Polyphenol 77
4.5 Ascorbic Acid Content 81
4.6 Antioxidant Activity 83
x
REFERENCES 126
Appendices 147 – 155
xi
LIST OF PLATE
LIST OF TABLES
LIST OF FIGURES
O2 - Superoxide radical
H2O2 - Hydrogen peroxide
OH. - Hydroxyl radical
LDL - Low debsity lipoprotein
CHO - Carbohydrate
HTST - High temperature short time
RP - Reverse phase
PPO - Polyphenol oxidase
DPPH - Radical scavenging activity
SS - Superoxide free radical scavenging activity
TBHQ - tert-butylhydroquinone
FDA - Food Drug and Administration
TBARS - Thiobarbituric acid reactive species
ORAC - Oxygen radical absorbance capacity
BCBT - E-carotene bleaching test
ABTS - 2.2’, azino-bis(3-ethyl-benz-thiozoline-6-sulfonic acid)
CMC - Carboxy methylcellulose
TSS - Total soluble solid
TA - Total acidity
HCL - Hydrochloric acid
GAE - Gallic acid equivalent
TPTZ - Trypyridyl-s-triazine
UV - Ultraviolet-visible
HCL - Hydrochloric acid
Fe2SO4.7H2O - Ferum sulfate
NaOH - Sodium hydroxide
K2S04 - Pottasium sulfate
EDTA - Ethylenediamine tetra-acetic acid
DMRT - Duncan’s multiple range test
SAS - Statististical Analysis System
CIE - Commision Internationale de L’Eclairage
xv
LIST OF SYMBOLS
Rt - Retention time
L - Linearity
r2 - Correlation coefficient
L* - Colour index for lightness
a* - Colour index for redness
b* - Colour index for yellowness
ppm - part per million
rpm - rotation per minute
HPLC - High Performance Liquid Chromatography
GAE - Gallic acid equivalent (mg/100ml)
TSS - Total soluble solid
TA - Total acidity
qBrix - Unit for total soluble solid
NEB - Non-enzymatic browning
RDA - Recommended Daily Allowance
TLC - Thin Layer Chromatography
FTC - Ferric Thiocyanate
FRAP - Ferric Reducing Ability of Plasma
TBA - Thiobarbituric acid
BHT - Butylated hydroxytoulene
BHA - Butylated hydroxy anisole
MRPs - Maillard Reaction Products
ESR - Electron Spin Resonance Spectroscopy
SO2 - Sodium dioxide
SD - Standard deviation
ROS - Reactive oxygen species
xvii
LIST OF APPENDICES
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).
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
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).
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).
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 most important factor determining the minimum thermal process is the pH
of the product (Noraini, 1984). According to Pederson (1980), for highly acid drink and
juice (the pH is lower than pH 4.2) would normally be processed at 71.1qC to 100qC.
On the other hand, Chuah (1984) reported that the process of pasteurization usually
consists of a process whereby the food is heated to temperature 60-90qC either to
destroy the nonsporing pathogens or to prolong the shelf-life of the food, usually but not
conjunction with some added preservatives which prevent the spores of microorganisms
from germination. High temperature heat processes are unnecessary for acid juices
because the heated spores of spore-forming bacteria are unable to germinate at pH 4.2 or
lower (Pederson, 1980). The heat treatment of beverages held at 60qC for 10-20 minutes
is also recommended for the acidic products (Chuah, 1984). Scalzo (2004) studied the
effect of thermal treatments of blood orange juice at 80qC for 6 minutes on antioxidant
changes compared to non-thermally treated juice. After pasteurization at 80qC for 6
minutes, the inhibition DPPH (%) was reduced from 49.1% (unheated juice) to 43.2%.
The processing of pineapple and “asam jawa” drink at 85 to 90 qC for 1 to 5 minutes still
6
maintained the sensorial quality of products (Che Rahani, 1998). The carrot juice heated
at 82qC for 5 minutes retained 57% of D-carotene (Bao and Chang, 1994). The heating
temperature for canned fruit and vegetables beverage is depended on the microbial level
of the raw materials, the acidity of the products, the size of the can and the thermal
conductivity of the product. Canned mango puree was heated in open steam jacketed
kettle to 80qC for 10minutes. After hot-filling, the sealed cans were immersed in boiling
water for another 20 minutes (Godoy and Rodriguez-Amaya, 1987). In other processing
practice, the guava juice was heated to 87qC for 5 minutes, hot filled and sealed cans
pasteurized in boiling water for 30 minutes. (Padula and Rodriguez-Amaya, 1987). The
authors found that carotene content was maintained after heating at these processing
condition. In other report, Che Rahani (1998) recommended the heat processing of
guava drink at 82qC for 5 minutes, followed by canning and immersed in boiling water
(100qC) for another 10 minutes.
free radical chain breakers (D-tocopherol), reducing agents and oxygen scavengers
(ascorbic acid), chelating agents (citric acid) and ‘secondary’ antioxidant (carotenoids)
may be able to stabilize and prevented oxidation damage in fruits and vegetables.
Pokorny (2000) reported that modification of a recipe during preparation of food and
ready meals improved the stability against oxidation especially with the addition of
spices. Recent studies also indicated that the addition of sulphur dioxide (S02) or
sodium metabisulphite and vitamin C during processing of commercial food products
balanced the depletion of natural antioxidant (Tsai, et al., 2002; Majchrzak, et al., 2004).
The presence of metabisulphite has been demonstrated to control the spoilage and
promote the retention of the natural antioxidant. Sulphites were successfully used to
prevent the non-enzymatic browning in food and vegetables (Sapers, 1993), reduction in
decoloration of pigments, changes in texture and loss of nutritional quality (Lindley,
1998). Other food additives such as citric acid generally enhanced the antioxidant
activity via synergist effect with natural antioxidant like D-tocopherol. Citric acid was
also used as metal chelators to inhibit oxidative reactions (Madhavi, et al., 1996). Citric
acid is widely used as acidulant and preservatives in food system. The high levels of
total soluble solid usually help to stabilize or reduce the deterioration rate of food
products. For example, high sugar concentrations are effectively to protect the
degradation of anthocyanin (Wrolstad, et al., 1990), the strong antioxidant compound in
Roselle (Tsai, et al., 2002) and berry fruits (Skrede, et al., 2000). The effect of sugar
concentration is most likely due to lower in water activity (Skede and Wrolstad, 2002).
The impact of food processing and handling on nutrients such as vitamins and
minerals are well established. However, the stability and the fate of phytochemicals in
processed food have not been investigated to similar extent. It is always believe that
phytochemical from pegaga are depleted by processing, particularly where thermal
treatments are employed. The level of antioxidant activity and the presence of
significant concentration of triterpene glycoside in pegaga are of interest to the herbal
industry. However, the effect of processing parameters on both antioxidant activity and
triterpene glycoside contents of products from pegaga is yet to be investigated
9
1.1 Objective
The main objective of the study was to investigate the effect of heat processing
on the antioxidant activity and triterpene glycosides content of herbal pegaga drink
1.2 Scope
In order to achieve the objective, the scopes of the study are identified as
follows:
1. The herbal pegaga drink was prepared under three different heat
processing conditions; 65qC/15 minutes (A), 80qC/5minutes (B) and
canned process (heat at 80qC/5minutes followed by canning and boiling
at 100qC/10 minutes (C)). The unheated pegaga drink known as fresh
sample (F) and two commercial samples, CM1with no thermal treatment
and CM2, which heat processed at 90qC for 1 minutes were used as
comparison. All pegaga drink samples (F, A, B, C, CM1 and CM2) were
used for further assessment.
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.
CHAPTER 2
LITERATURE REVIEW
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).
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).
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 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).
The chemical constituents of pegaga are classified into main groups including
essential oil, flavone derivatives, triterpenic steroids, triterpenic acids and triterpenic
acid sugar ester or saponin (Brainkhaus, et al., 2000). Pegaga also contains various
important constituents for clinical and pharmaceutical uses (Bonte, et.al., 1994).
Chemicals that were previously investigated from pegaga are brahmic acid,
brahminoside, brahmoside, centellic acid, centelloside, hydrocotyline, 3-
glucosylkaempferol, 3-glucosyl-quercetin, indocentelloside, isobrahmic acid,
isothankunic acid, isothankuniside, madasiatic acid, madecassol, meso-inositol,
oxyasiaticoside, thankunic acid, vallerine; alkaloid, fatty acids, flavonols, polyphenols,
saponins, sterols, sugars, tannins, terpenoids, triterpenes (Goh, et al., 1995). Asiatic
acid, asiaticoside, madecossoside and madecassic acid are the biologically active
constituents in pegaga that have a potential to be promoted as commercial product (Indu
Bala and Ng, 2000).
15
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.
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).
Nutrient composition also plays an important role to promote health. Tee, et al.,
1988 presented a quantitative evaluation of proximate and nutrient composition of fresh
pegaga. Pegaga contained high potassium, calcium and phosphorus levels that
accounted for 391 mg, 171mg and 32 mg per 100g, respectively. Pegaga is not a good
source of protein, carbohydrate and fat. E-carotene and ascorbic acid, known to have
antioxidative activities, are present at appreciable concentration (2649 Pg and 48.5mg,
respectively) in fresh pegaga. E-carotene and carotenoids can act as antioxidant and are
effective quenchers in singlet oxygen. In terms of mechanism, they are preventing the
formation of hydroperoxides (Rajalakshmi and Narasimhan, 1996). Besides the more
popular phytochemical constituents in pegaga, these particular compounds also
17
contributed to the positive health. The nutrition composition in pegaga is shown in table
2.1.
Kcl g g g g g g
Energy Water Protein Fat CHO Fiber Ash
37 87.7 2.0 0.2 6.7 1.6 1.8
Indian
Pennywort 44 Vitamin**
(pegaga);
Hydrocotyle Pg Pg Pg mg mg mg mg
asiatica Retinol Carotene RE B1 B2 Niacin C
0 2649 442 0.09 0.19 0.1 48.5
Mineral**
mg mg mg mg mg
Ca P Fe Na K - -
171 32 5.6 21 391
2 .5 Triterpene Glycosides
(Asiaticoside, Medacosside, Asiatic acid and Madecassic acid)
R4 R5
R3
HO
HO OR2
HOH2C R1
Saponins R1 R2 R3 R4 R5
Asiatic acid -H -H -CH3 -CH3 H
Figure 2.1: Structure of triterpene glycoside: asiatic acid, asiaticoside, madecassic acid,
and madecassoside (Brinkhaus, et al., 2000)
Saponin
Glycone Aglycone
Sugar Sapogenin
Steroids Triterpenoids
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.
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
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
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
Ascorbic acid is a highly soluble compound that has both acidic and strong
reducing properties. At the same time, it is highly sensitive to various modes of
degradation including temperature, salt, sugar concentration, pH, oxygen, enzymes,
metal catalyst and initial concentration of ascorbic acid (Tannenbaum, et al., 1985).
24
Ascorbic acid also can be degraded by active oxygen and by reaction initiated by
transition metals. It removes oxygen in systems where oxygen is present in limited
amounts and gets oxidized to dehydroscobic acid (Jadhav, et al., 1996). Ascorbic acid is
easily destroyed through oxidation, especially at high temperature, and the amount
generally declined during food processing, storage and cooking. Sulfur dioxide
treatment can also affect the ascorbic acid losses during processing, as well as during
storage (Bolin and Stafford, 1974).
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
Total polyphenol was determined in all parts of pegaga including leaves, stem
and root and it shows the highest in the leaves for about 0.23 Pg/mg dried methanol
extract. The high concentration of polyphenol is thought to be responsible for the anti-
inflammatory activity in pegaga (Fezah, et. al., 2000). Zainol et al., (2003) studied the
amount of total polyphenol in four accessions of pegaga extract. The concentration of
total polyphenol varied from 3.23g to 11.7g per 100g of dry sample. They also
suggested that phenolic compounds are the major contributors to the antioxidative
activities of pegaga.
(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 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).
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).
Pegaga is well known to have a high antioxidant activity (Abdul Hamid, et al.,
2001). It has been established that the presence of polyphenol in pegaga extract is
contribute its antioxidative efficiency activity with the correlation of r2=0.9 (Zainol, et
al., 2003). The specific component of phenolic that contributed to antioxidant activity in
this herb is not reported clearly. Vimala, et al., (2003) reported that pegaga leaves were
found to have very high antioxidant activity in three different pathway including
superoxide free radical scavenging activity (86.4%), inhibition of linoleic acid
peroxidation (98.2%) and radical scavenging activity, DPPH (92.7%). The consumption
31
of pegaga was useful to protect the cells from oxidative damage, to destroy excess free
radicals and keep the oxidative stress state in balance. Shukla, et al., 1999 investigated
the role of asiaticoside as antioxidant properties in wound healing activity. Asiaticoside
derived from pegaga has been attributed to increase the antioxidant levels at an initial
stage of healing. Yusuf, et al., (2000) also observed the antioxidative axtivities of
carotenoid and ascorbate peroxidase in herb pegaga. The characteristics of antioxidant
activity in pegaga were previously studied. Pegaga exhibited optimum antioxidant
activity at neutral pH and the activity remained stable up to 50qC. The antioxidative
activities of pegaga extracts increased when concentration was increased from 1000 to
5000ppm(Abdul Hamid, et al., 2001).
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
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).
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
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).
Sugar is widely added in processed food partially to increase the product stability
via lowering the water activity (aw). Addition of sugar also increased the concentration
of products and generally measured by total soluble solid. It has long been recognized
that a relationships exists between water activity and concentration of food products.
Wrolstad, et al., 1990 reported that the concentration of sugar over 20% is preventing
the loss of anthocyanins. Jackman and Smith (1996) also found that the amount of
similar antioxidant compound is considered to be degrading at lower sugar level.
According to Takeoka, et al. (2001), the loss of antioxidant property in tomatoes such
lycopene content is increased at 25-30qBrix of total soluble solid. The longer processing
time required achieving the desired final solid levels also associated with increased
losses of lycopene. The enzymatic and/or chemical oxidation rate of phenolic
compounds are associated with some intrinsic food variables such as water activity (aw)
and it processing condition (Nicoli, et al., 1999). Wrolstad (2000) reported that the
stability of anthocyanin was increases with the decreased water content or with the
decreasing water activity.
35
Herbs and other natural products contain many hundreds compound of natural
antioxidant. Therefore, several methods have been developed to quantify these
compounds individually. The techniques are different in term of mechanism of reaction,
effectiveness and sensitivity (Khal dan Hildrbrant, 1986; Frankel, 1993; Koleva, et al,
2002). Methods that are widely used to measure the antioxidant activity level in herbal
sample, fruits and vegetables, and their products are thiobarbituric acid reactive species
(TBARS) (Roberto, et al, 2000), oxygen radical absorbance capacity (ORAC) (Tsai, et
al. 2002; Wang, et al, 1996; Zheng and Wang, 2001), E-carotene bleaching test (BCBT)
(Markin dan Rossiter, 2001; Gazzani, et al., 1998), ABTS radical-cation (Arena, et al,
37
2000; Miller, et al, 1995), DPPH titration (Imark, et al, 2000), Folin-Ciocalteu
(Donovan, et al, 1998) as well as FTC and FRAP.
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).
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
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.
The main concern of the food industry in thermal processing is to prevent the
growth of bacterial pathogens. The quality and the uniformity of beverages will largely
depend on the degree of control during the heat process, because over-processing may
lead to undesirable changes in flavour, texture and nutritive value. Conversely, under-
processing, which may not destroy all the organisms, leads to spoilage and is a potential
health-hazard. It is therefore important that suitable heat processing schedules be
obtained, taking into consideration the effect of the water activity, pH and thermal
conductivity of the product (Desrosier and Desrosier, 1977). Blanching, dehydration,
sterilization and pasteurization are an example of thermal treatment that commonly
practice in food industry (Pokorny, 2001).
39
100
KILLING
TIME pH 5 to pH 7
(min)
10
pH 4.5
1.0
pH 3.5
0.1
99 110 121
TEMPERATURE (qC)
Figure 2.4: Influence of pH of heating medium on heat resistance of spores
(Desrosier and Desrosier, 1977).
The pasteurized of acid juices and drinks may be filled into plastic bottles, glass
bottles or into cans. Previously pasteurized or sterilized beverages are hot filled between
78-93qC and held in this temperature for 1-3 minutes in containers before cooling
(Noraini, 1984). In other practices, the unheated juice was put in glass bottles, which
were then crowned and pasteurized at 77-82.2qC for 20 to 30 minutes (Pederson, et al.,
1980). According to Mehrlich and Felton (1971), the pasteurization of canned pineapple
42
juice may be handled according to either of two alternatives. The first alternative
procedure for handling the juice is pasteurized the product to approximately 90qC. The
cans are filled with the juice at this temperature and held for 1-3 minutes. In the second
alternative, the juice was first pasteurized at 60qC, filled into cans and the can sealed
was then boiled for certain time according to the size of the can. In canning process of
some juices and drinks, the products are commonly heat-treated at the temperature of 80
to 87qC for 1-10 minutes, filled into cans, sealed and immersed in boiling water in the
range of 10 to 30 minutes (Godoy and Rodriguez-Amaya, 1987; Padula and Rodriguez-
Amaya, 1987; Che Rahani, 1998). According to Luh (1980), the mango juice should be
heat processed at 87.8qC, followed by filling, sealing in processed in water bath.
Processing in the boiling water sterilizes the inner surfaces of the can and lids and
prevents contamination of the product from those surfaces. Although canning processes
result in the losses of sensorial and nutritional quality attributes, the processes are still
widely used, and could be optimized to improve quality retention regarding the specific
of any particular commodity.
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).
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).
melatonin, as well as trace elements such as Cu, Zn, Mn and Se (Zielinski, et al., 2001).
According to Min. et al., (2004), the loss of total selenium content caused by blanching
treatment is greater than the effect of sterilization. The application of moderate
temperatures, up to 100qC, reduces the negative changes of nutritional quality (Pokorny,
2001).
There are many evidences found that industrially processed food and home
prepared significantly change the natural antioxidant. This is based on fact that most of
chemical constituents in food are unstable (Erdman Jr, 1979; Hurt, 1979). Few studies
on the phytochemicals retention including natural antioxidant of processed foods have
been published. The stability of ascorbic acid and some phenolic compound during
processing of foods and beverages are discussed as follows.
Ascorbic acid level in foodstuffs depends not only on the raw material
composition but also on the processing method employed (Marin, et al., 2002). There
are many studies for determining the ascorbic contents under different processing
parameters and storage conditions (Kabasakalis, et al., 2000; Hunter and Fletcher, 2000;
Franworth, et al., 2001; Wong, et al., 2000). The amount of this particular
phytochemical is significantly destroyed in canned peas, pasteurized pineapple and
orange juice as well as processed roselle juice (Lathrop and Leung, 1980; Akinyele,
et.al., 1990; Wong, et al., 2001). Lea (1992) reported that, fresh apple contain up to 100
ppm of vitamin C, but during processing into juice it is rapidly lost. The loss of ascorbic
acid was also found to be highest in medicinal plants dried at 50qC for 9 hour (75.60%)
compared to freeze drying (21.13%) (Mahanom, et al.,1999). Mild (75qC for 30 sec)
and standard pasteurization (95qC for 30 sec) slightly increased the total vitamin C of
orange juice from 143.5 mg to 160.5 and 131.2 to 155.7 mg, respectively, probably due
46
to the contribution from the solid parts (pulp) as a consequence of heat treatment (Gil-
Izquierdo, et al., 2002).
few experiments. The greater loss of total lycopene (35%), major carotenoid pigment
and antioxidant in tomato, was reported when the temperature was increased from 90 to
150qC. The duration of heating below 100qC, however, had little or no effect on the
degradation of lycopene (Shi and Le Maguer, 1999). Thermal processing of tomatoes
into paste partly decreased the concentration of lycopene of 9-28% and it is believed to
be due to longer processing time required to achieve the desired final solid levels
(Takeoka, et al., 2001). Heat is also observed as one of the most destructive factors of
anthocyanins in berry fruit juices (Jackman, et al., 1987). The degradation of
anthocyanin is increased from 30% to 60% after 60 days storage when storage
temperatures were increased from 10qC to 23qC (Cabrita, et al., 2000). Wang, et al.
(2000) also reported that after heat processing and 12 days of storage about 86% of
epigallocatechin gallate, 79% of epigallocatechin and 57% of epicatechin in green tea
extract were lost. Again, carotenoid content in 8 medicinal plants is loss by 27% and
20% after oven drying at 50qC for 9 hours and 70qC for 5 hours (Mahanom, et al.,
1999). The fate of most phytochemicals in processed food products are also notably
influenced by storage conditions. Storage of concentrates of apple juice for 9 months
resulted in 50-60% loss of quercetin and phloretin derivatives (Spanos, et. al., 1990).
Hunter and Flatcher (2002) investigated the antioxidant activity, total polyphenol
and ascorbic acid content in peas and spinach during microwave heating, boiling
treatment for 3 minutes and boiling treatment for 8 minutes (overcooked). The ABTS
49
and FRAP method is used in their assessment. The also studied the antioxidant activity
of peas and spinach at frozen storage and after blanching treatment (97qC for 85 seconds
and 97qC for 90 seconds, respectively). Blanching treatment is found to be useful to
prevent the enzymatic oxidation that usually responsible to the loss of natural
components in raw material or plants (Nicoli, et.al., 1999). However, after blanching of
peas and spinach the level of their antioxidant activity is reduced for about 50% and
20%, respectively, subjected to ABTS assay. The antioxidant activity remained constant
and stable at frozen storage. Boiling peas (100qC for 8 minutes) caused losses in water-
soluble antioxidant activity and ascorbate content of 34% and 61% respectively (Hunter
and Platcher, 2000). The reduction of antioxidant activities in pegaga extract at 70-
90qC is also may associated with the loss of naturally occurring antioxidant (Abdul
Hamid, et al., 2002). Gil-Izquierdo, et al. (2002) studied the effect of pasteurization at
75qC and 95qC on antioxidant activity towards the DPPH method. The antioxidant
activity equivalent to mg L-Ascorbic acid of orange juice increased from 126.8 mg
(before pasteurization) to 135.3 mg after pasteurization at 75qC. However, the activity
was decreased from 150.1 mg to 143.7 mg after standard pasteurization at 95qC for 30
sec.
The loss of antioxidant activity in food products not only associated with the
degradation of natural antioxidant but also due to the formation of compounds with pro
oxidant properties. Pro-oxidant generally appeared in early stages of non-enzymatic
50
browning (Nicoli, et al., 1999). Gazzani, et al. (1998) investigated the effect of thermal
treatment at 2, 25 and 102qC for 10, 20 and 30 minutes on antioxidant activity of
vegetable juice based on E-carotene bleaching test. When prepared at 2qC for 10
minutes, most vegetables juice showed initial pro-oxidant activity. The pro-oxidant
activity was very high in eggplant (-307%), tomato (-621%) and yellow bell pepper (-
432%).
Food processing may also result in the formation of antioxidant compounds such
as Millard reaction products (MRPs) (Madhavi, et al., 1996b). These particular
compounds having antioxidant activity that influenced the antioxidant properties of
food. Formation of advance MRPs during prolonged heating time and storage generally
exhibited strong antioxidant properties (Eichner, 1981; Nicoli, et al., 1999). Millard
reaction products were identified to be active as oxidation inhibitors in tomato puree
(Nicoli, et al., 1997b). The development of non-enzymatic browning reactions, as
occurs in the production of Marsala-type wine, resulted in a great increase in its chain-
breaking activity (Monzocco, et al., 1999b).
(Cornwell and Wostad, 1981) and pigment destruction (Beveridge, et al., 1986). The
rate of NEB is depends on water activiy, pH, temperature and chemical composition of
the food system (Whistler and Daniel, 1985; Potter, 1986). The brown colour is
developed in c. asiatica drink during heat processing but it is not clear which reactions
are involved to enhance NEB. The influenced of NEB to antioxidant capacity is already
discussed in a few papers (Manzocco, et al., 2000; Nicoli, et al., 1999; Morales and
Jimenez-Perez, 2001; Manzocco, et al., 1999). Although the concentration of natural
antioxidant is significantly reduces as a result of thermal treatments, the overall
antioxidant properties of process products are maintained by the development of NEB
such Millard reactions (Nicoli, et al., 1997b). They also described the correlation
between the developments of Millard reaction products with relative antioxidant activity.
The correlation of relative antioxidant activity with heating time and heating temperature
is shown as figure 2.5.
Gazzani, et al. (1998) reported that heat treatment of carrot juice, cauliflower
juice and zucchini juice at 102qC for 10 minutes exhibited higher antioxidant activity.
The antioxidant activity (based on E-carotene bleaching test) also increased with the
increasing of heating time and heating temperature. For example, the antioxidant
activity of carrot juice at 25qC for 10 minutes was 24% and it was increased to 75% after
30 minutes of heating. They suggested that pro-oxidant activity, which is due to
peroxidases, are inactivated at high temperature. Wang, et al. (1996), have observed
that commercial tomato and grape juice had much higher antioxidant activity than fresh
materials but the reason for the increase in antioxidant as a consequence of food
processing was not evaluated.
52
12
T3
11
Relative antioxidant activity
10
8
T2
7
5
T1
4
Heating time
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
This chapter presents the material and methods used for the overall experiments.
This work was aimed at investigating the antioxidant activity and the fate of triterpene
glycosides content of herbal pegaga drink as affected by heat treatment. The physico-
chemical characteristics of pegaga drinks were also observed. The information obtained
from the study could be used as a guideline for designing thermal processes to reduce
the phytochemical degradation of the products. Besides, the factors that may contribute
to the antioxidant activity in unheated pegaga drink were also studied.
3.1 Introduction
Thermal treatment is generally applied to extend shelf life of fruit and vegetable
products. However, heating processes can affect the nutrient and phytochemical loss,
which leads to consumer dissatisfaction. In this study, the three different heat
processing treatment applied on pegaga drink were 65qC/15 minutes, 80qC/5 minutes
and in canning process (heat at 80qC/5minutes, canned and followed by boiling at
100qC/10 minutes before cooling process). The heat processing parameters were based
on pasteurization methods of acidified foods (Chuah, 1984; Scalzo, et al., 2004; Che
Rahani, 1998). The canning process of herbal pegaga drink was followed the procedures
55
of high acid canned beverages as previously done on fruit and vegetable juice. (Godoy
and Rodriguez-Amaya, 1987; Luh, 1980; Che Rahani, 1998).
Traditionally, pegaga juice was prepared by blending the whole parts of pegaga
with certain amount of water, before it is consumed fresh as cooling drink. In this study,
the untreated drink, known as fresh sample, was used in order to compare the status of
antioxidant activity and triterpene glycosides content before heating and without
addition of any food additives and food ingredients.
Recently the demand of herbal pegaga drink by the consumers is on the increase
mostly due to the health benefit and the phytochemical presence in the drinks.
Therefore, the current status of nutrient content, antioxidant activity and active
constituents in commercial pegaga drink available in the market are important to be
studied. The results obtained are useful as a reference for consumers and researchers.
The factors influence to the antioxidant activity was also investigated. The used
of citric acid (Dziezak, 1986; Sherwin, 1990) and sodium metabisulphite (Tsai, et al.,
2000) is reported to increase the antioxidant activity in several food products. However,
the effect of these food additives and total soluble solid via sugar addition on antioxidant
activity of pegaga drink is still unclear. The range of citric and total soluble solid used
in this study was based on consumer acceptances as previously reported in many
research works (Pederson, 1980; Lea, 1991; Henrix, 1995), while the range of sodium
metabisulphite was followed the level permitted in Malaysian Food Act 1983 and Food
Regulation1985. The study on effect of addition of citric acid (0-0.3%w/v), sodium
metabisulphite (0-350ppm) and sugar (in the range of 1 to 15q Brix) on antioxidant
activity of fresh pegaga drink was carried out. Citric acid addition varied in accordance
with acidities of raw materials and consumer acceptance. Citric acid in the range of
0.1%-0.3%w/v are usually added into fruit and vegetable juices to increase the acidity
for the flavour and preservative purposes (Pederson, 1980). Vegetable juice acidified
56
with 0.4%w/v citric acid was too sour. According to Malaysian Food Act 1983 and
Food Regulation1985, the maximum level of sodium metabisulphite permitted in fruit
and vegetable drinks is about 350 part per million (ppm). Therefore, the effect of
sodium metabisulphite at concentration of 0-350ppm on antioxidant activity was used in
this study. The amount of sugar added to fruit and vegetable drink mainly based on
sensory test or consumer acceptance. However, the total soluble solid in ready-to-drink
of fruit beverages is widely varied from 5-15qBrix (Lea, 1991; Henrix, 1995)
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.
for the preparation of pegaga drink is shown in Figure 3.1. The product was pasteurized
at three different heat-processing temperatures; 65qC/15 minutes, 80qC/5 minutes and in
canning process (heat at 80qC/5minutes, canned and followed by boiling at 100qC/10
minutes before cooling process). Fresh sample (F) or non-thermally treated drink
without added sugar and food additives was also prepared. Each product was then kept
at 4qC.
Pegaga sample (Centella asiatica)
Analysis
Figure 3.1: Flowchart of the preparation of pegaga drink
Samples Fresh sample Heat-treated samples Commercial sample
(Sample F) (Sample A, B and C) (Sample CM1 and CM2)
pH, Total acidity, Colour index L*, a* Ferric thiocyanate assay Asiaticoside content,
and b* values, Total soluble solid, (FTC) and Ferric Reducing Madecassoside content, Asiatic
Proximate composition, Microelement, Ability of Plasma (FRAP) acid content and Madecassoside
Total polyphenol and Ascorbic acid assay content
content
The two commercial samples were obtained from Loo Pegaga Enterprises,
Taman Anggerik Johor Baharu (CM1) and HPA Sdn Bhd, Kuala Perlis, Perlis (CM2). .
The pegaga drink of CM1 was prepared without any thermal treatment. The second
commercial sample (CM2) was prepared in squash form and pasteurized at
90żC/1minutes. This sample was first diluted into drink prior to analysis. The squash
sample was diluted three times according to direction on the label. All samples were
kept at 4qC.
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.
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.
Total Soluble Solid (TSS) and pH was measured with a hand held refractometer
(Atago) and pH-meter (EcoMet), respectively.
3.3.2.1 Moisture
10-15 g of homogenized sample was placed into glass dish before dried in a
105qC oven for five hours. The dish was then removed from oven (Memmert,
61
Germany), cooled in dessicator and weighed soon after attaining room temperature. The
steps were repeated until constant weight was obtained (AOAC, 1984).
% Moisture by weight = loss of weight in gramme of the sample x 100 (3.2)
Weight in gramme of sample
3.3.2.2 Ash
2.5-3 g sample was weighed into crucible. The sample was charred on heating
mantle until no smoke evolves. Ashing was carried out in muffle furnace (Memmert,
Germany) at 550qC for about 8 hours or until grey ash was obtained. Sample was then
cooled in dessicator. The ash was calculated after constant weight was obtained
(AOAC, 1984).
% of Ash = Weight of ash / Weight of sample x 100 (3.3)
3.3.2.3 Protein
The Kjeldahl method for determining total nitrogen was based on Tecator Kjeltec
System 1026 and David Pearson (1976) was used.
Reagent: Concentrated sulphuric acid (A.R Grade), Sodium hydroxide (A.R Grade
40%), 0.05M Hydrochloric acid, 4% Boric acid with bromocresol green indicator and
catalyst, Kjeltabs (1.5 g K2S04 and 0.0075 g Se) were used.
Assay: 0.2-1 g sample was weighed and mixed with 2 pieces of Kjeltabs and 10 ml of
sulphuric acid in digestion tube. The mixture was digested for 1 hour or until a clear
solution was obtained at 420qC. The sample was cooled and distilled using Kjeltec 1026
Distilling Unit with 25 ml of 4% boric acid solution. Bromocresol indicator was placed
62
on receiver flask. The sample was then titrated with 0.05M Hydrochloric acid (HCL) to
neutral grey.
Calculation:
% N = 14.01 x (ml of titrant of sample – ml of titrant of blank) x conc. of standard acid
g of sample x 10
% Protein = % N x factor specific for different product (6.25) (3.4)
3.3.2.4 Fat
Assay: Sample (3-4g) was placed into an extraction thimble. Thimble was then placed
in a beaker and dried in an electric oven for 5 hours at 70-80qC. Dried sample was
extracted with petroleum ether using Soxhlet extraction apparatus for 6-8 hours. The
solvent was evaporated and the residue was dried in an electric oven for 30 minutes at
105qC. The sample weight was then measured (AOAC, 1980).
% Fat = (W2-W1) x 100 (3.5)
Sample weight in g
W1 = weight of evaporating flask
W2 = weight of evaporating flask + content after drying
3.3.2.5 Fibre
Reagent: 0.255N Sulphuric acid (A.R Grade), 0.313N Sodium hydrochloride (A.R
Grade), Hydrochloric acid (1% in water v/v) were used.
63
Assay: Defatted sample (1-3g) was weighed (W0) and placed in beaker. 200ml of
sulphuric acid was added and boiled for 30 minutes. The sample was filtered with
Whatman paper no. 1 and the residue was washed with hot water until free from acid.
The residue was then washed with 200 ml of warmed sodium hydroxide (0.313N),
boiled for 30 minutes and filtered through crucible. The residue was washed with hot
water, 1% HCL and hot water again until neutral, then followed by ethanol. The sample
was dried in oven at 105qC for 1 hour. The crucible with residue was weighed (W1) and
ignited in muffle furnace at 450qC for 4 hours. The cooled crucible was weighed again
(W2) (AOAC, 1984) .
% Crude fiber = W1 - W2 / W0 x 100 (3.6)
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
Assay: 1 ml of sample was digest with 5 ml of Aqua Regia solution for 30 minutes at
70-80qC. Aqua Regia solution was prepared from mixing of 1N Nitric acid and 1N
Hydrochloic acid (3:1). The sample was then filtered using Wathman no. 540 Hardened
Ashless. The filtrate was added with deionized water and make up to 100ml. The
sample was again filtered through a nylon filter Wathman 0.2 Pm before injected on
Mass Spectrometer. The calculation of microelement was based on multi-element
calibration standard.
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
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.
Preparation of sample: Each sample of pegaga drink was filtered using Bunchner
funnel with Whatman no.4 filter paper. The extract was kept at 4qC before assay.
Reagent: 300 mmol/litre buffer acetate, p.H 3.6 ; 10 mmol/litre TPTZ (2,4,6-
trypyridyl-s-triazine, Fluka Chemicals) in 40 mmol/litre HCL (BDH); 20 mmol/litre
Fe3.6H2O (BDH). FRAP reagent was prepared by mixing 25 ml buffer acetate, 2.5 ml
TPTZ solution and 2.5 ml Fe3.6H2O solution (Fluka, Chemicals).
Assay: Antioxidant activity was analyzed according to procedure of Benzie and Strain
(1996) with slight modification as described by Gardner, et al. (2000). Freshly prepared
FRAP reagent was warmed to 37qC. The Reagent blank reading was taken at 593 nm. 1
ml of diluted 10-fold sample was added into 3 ml of FRAP reagent. Absorbance reading
was taken after 4 minutes. Results were calculated from calibration curve prepared from
Fe2SO4.7H2O (Fluka, Chemicals) solution in the range of 0.1mM to 10mM.
The FTC model method will be used according to modified method of Kikuzaki dan
Nakatani (1993).
67
Reagent: 2.51% linoleic acid in 99.8% ethanol. 0.05M phosphate buffer (pH 7). 30%
ammonium thiocynate. 0.02 M ferrous chloride in 3.5% HCl.
Assay: 1 ml sample (0.02% in 99.5% ethanol) was mixed with 2 ml of linoleic acid, 4.0
ml phosphate buffer (pH 7.0) and distilled water (3.0 ml). The sample was kept in cap
screwed container in dark condition at temperature of 40qC.
0.1 ml of sample was added with 75% ethanol (9.7ml) and 0.1 ml of 30% ammonium
thiocyanate. 3 minutes after addition of 0.1 ml of ferrous chloride to the reaction
mixture, the absorbance of red colour was measured at 500 nm until absorbance of
control (blank reagent) reach maximum. D-tocopherol and ascorbic acid were used as
standard sample.
Sampel preparation:
Water extract: Pegaga drink (20ml) was centrifuged at 4000 rpm for 15 min. The
supernatant was filtered through a Milipore filter (0.45-µm) before injection into the
High Performance Liquid Chromatography (HPLC) (Shui & Leong, 2002).
Methanol extract: Pegaga drink (20ml) was centrifuged at 4000 rpm for 15 min. The
sample was then concentrated using vacuum evaporator and dissolved in 20 ml of
69
methanol-water (90:10) (Inamdar, et al., 1996). The sample was vortexed for 5
minutes, centrifuged and filtered using a Milipore filter (0.45-µm) and a known amount
of extract was subjected to HPLC under the above conditions. The contents of triterpene
glycosides were calculated based on water extract and methanol extract with the aid of
calibration graph obtained using a stock solution of each component.
CHAPTER 4
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
The results for pH, total soluble solids (TSS), % of total acidity (TA) expressed
as citric acid, L*, a* and b* values of different samples are shown in Table 4.1. The low
pH (3.72-3.79) of the heat-treated drink (sample A, B and C) was accompanied by a
high acidity (14.37-14.72%) calculated as citric acid. The addition of citric acid (0.12%)
in heat-treated pegaga drink is responsible for the low of pH and by a high acidity as
compared to untreated sample (F). The pH and total acidity (TA) of fresh or untreated
sample are 5.93 and 3.85%, respectively. The pH was higher than those obtained in two
commercial samples (CM1 and CM2). The organic acid may added to both commercial
samples as preservative to extend the shelf life of products. For comparison, the titrable
acidity of apple juice is 0.2-0.7% (Lea, 1991). The herbal pegaga drink had a rather low
pH. However, it is higher than apple juice (3.5-3.8) (Lea, 1991), orange juice (3.3-3.8)
and grape juice (2.8-3.0) (Henrix, 1995). The content of soluble solids in pegaga drink
was between 1.0qBrix (fresh) and 11.2-11.8qBrix (heat-treated drink), which is almost
similar to orange juice (9-15 qBrix) and apple juice (11-14qBrix). The total soluble solid
in CM2 was significantly lower (7.6qBrix) than CM1 (12.6qBrix) and heat-treated drink.
For heat-treated samples (65qC/15 minutes, 80qC/5 minutes and canned, the parameters
of pH, TSS and %TA shows small changes. According to Kaanane, et al. (1988), the
minimal change in pH can be explained by relationship existing between pH and free
acid content.
as b* values. The increase in b* value was used to indicate the development of a brown
colour. The data on L*, a* and b* values are shown in table 4.1. Results shows that all
heat-treated samples (65qC/15 minutes, 80qC/5 minutes and canned) gradually turned
brownish during processing and their b* values steadily increased from 4.88 + 0.06
before heating (F) to in the range of 6.03 + 0.18 - 6.88 + 0.18 after heat processing.
Heating at 65qC/15 minutes shows higher development of browning followed by canned
and pasteurization at 80qC/5 minutes. The results showed that heat treatments
significantly increased (P < 0.05) the brown colour development. CM1 shows greenish
in colour as good as fresh sample (F). Both sample were not involved heating process.
The whiteness value (L*) of the products was significantly different between F and
sample A, B and C. This shows that heat treatment affects the colour of the products.
Samples pH TSS TA L* a* b*
(Brix) (%) +sd +sd +sd
F (Fresh) 5.93 1.0 3.85 24.43+0.21c 2.83+0.04a 4.88+0.06b
AA (65qC/15min) 3.72 11.2 14.37 27.84+0.31ab 2.17+0.08b 6.88+0.18a
B (80qC/5min) 3.79 11.8 14.72 28.86+0.48a 2.09+0.08bc 6.03+0.18ab
C (Canned) 3.72 11.2 14.38 27.04+2.57ab 2.67+0.24a 6.56+2.27a
CM1 3.89 12.6 7.71 26.90+0.77ab 2.22+0.16b 4.80+0.21b
CM2 4.86 7.6 4.21 26.35+0.21bc 1.96+0.03c 5.40+0.03ab
Mean values in each column with the same letter (a, b, c) are not significantly different
(p>0.05) according to LSD test; sd = standard deviation
According to Labuza and Baisier (1992), the rate of formation of brown pigment
is increased with the increase of the heating temperatures. The longer heating time and
other complex reaction between components during initial stages of browning, may be
associated with the increase of colour.
73
Table 4.2 shows the proximate values of pegaga drink in all sample tested.
Generally, proximate values and elements of heat-treated samples were almost higher
than those obtained in fresh drink, except for moisture. Fresh drink contained 99.62% of
moisture that is significantly higher than heat-treated samples (approximately 88%).
This indicate evaporations of water occurred during the heating process as well as sugar
addition. A higher amount of carbohydrate was detected in all heat-treated samples (in
the range of 10.99% to 11.40%), which mostly due to addition of sugar. The fresh drink
provide only about 0.22% of carbohydrate. Most of metabolizable carbohydrate used by
humans comes from sucrose or starch. However, sucrose is present in relatively minor
quantities in most plant foods and sucrose isolated from sugarcane generally added to
commercial foods (Whistler and Daniel, 1985). Similar results were found in crude fiber
content that only 0.01% detected in fresh and approximately 0.015% in heat-treated
samples, respectively. The nondigestible polysaccharides (fiber) are beneficial for a
healthy intestinal activity. There were no significant effect of ash and protein content
after heating processed that the amount in heat-treated was approximately 0.07% and
0.1%, respectively. As can be observed, the amount of nutrient components in pegaga
drink was very low and/or below human requirements. For example, staple foods with
75
protein content below 3% do not meet the protein requirements in human, but a diet of
cereals with an 8-10% protein content, provided enough to supply caloric requirements
of adults (Cheftel, et al., 1985). Fats serve as concentrated source of energy compared
to protein and carbohydrate. Unfortunately, no fats were detected both in fresh and heat-
treated samples. Similarly, Prasad, et al., (2000) reported that the fruit based products
such as pineapple beverage powder contained negligible amounts of both protein and fat.
Fresh pegaga drink contained only 0.06% amount of total ash, which was 0.01% less
than other samples.
As shown in the data, herbal pegaga drink provides a good source of mineral and
trace elements. Potassium was found as major components (347.99-469.91mg) in herbal
pegaga drink, followed by sodium (12.06-82.01 mg) and phosphorus (28.91-40.70mg).
Generally, the amount of minerals and trace elements in fresh and heat-treated samples
were greater than commercial samples. According to food U.S RDA (1980), the
optimum daily dietary intakes of adults for phosphorus, magnesium, iron, zinc, sodium
and potassium are about 800mg, 300-350mg, 10-18mg, 15mg, 1100-3300mg and 1875-
5625 mg, respectively. Consumption of one liter of herbal pegaga drink daily could
contribute appreciable amounts of minerals to the body. The calculation indicates about
9.3%-12.5% of RDA for potassium being contributed from 500 ml of sample, followed
by phosphorus (1.8%-2.5%) and sodium (0.5%-3.7%). Potassium (intracellular cation)
and sodium (extracellular ion) are regulated osmotic equilibrium and pressure, and also
maintained body-fluid volume. Phosphorus is involved in the enzymes-controlled
energy-yielding reactions of metabolism and helps control the acid-alkaline reaction of
the blood (Potter, 1986). 500 ml of pegaga drink also provided about 6.7%-11.2% of
iron for daily requirement.
Table 4.2 also demonstrated that the amount of zinc traced in pegaga drink was
in a range of 1.08-1.83 mg. The amount of zinc in pegaga drink was accounted about
7.2-12.2% of Recommended Daily Allowance (RDA).
76
Table 4.2: The nutritional value and trace element of pegaga drink
Sample of
pegaga drink
Proximate value
Fresh A B C CM1 CM2
Calorie (Kcal) 1.20 45.50 46.00 44.30 1.21 49.56
Moisture (%) 99.62 88.53 88.41 88.83 99.64 87.50
Ash (%) 0.063 0.070 0.073 0.070 0.055 0.089
Protein (%) 0.093 0.100 0.101 0.091 0.083 0.090
Crude fiber (%) 0.009 0.015 0.014 0.015 0.008 0.020
Fat (%) ND ND ND ND ND ND
Carbohydrate (%) 0.215 11.285 11.402 10.994 0.220 12.301
Minerals (mg/L)
Zinc 1.09 1.83 1.16 1.41 1.28 1.63
Phosphorus 33.74 30.71 28.91 40.70 18.53 16.60
Iron 4.04 3.17 2.55 2.41 2.81 2.58
Sodium 12.06 82.01 71.47 68.24 8.43 52.75
Potassium 469.91 446.10 372.42 347.99 273.28 131.94
Element (mg/L)
Selenium ND 0.01 ND 0.01 ND ND
Aluminium 149.38 3.45 3.24 1.13 0.97 6.55
Plumbum 0.44 2.18 0.87 0.56 0.45 0.33
Magnesium 10.27 10.19 9.38 8.28 6.86 4.69
* ND – Not detected
No selenium was detected in most samples except for sample A and C. The
concentration of selenium in sample A and C was only 0.01mg each. At low levels of
occurrence, zinc, selenium and manganese are essential to life, which usually function as
miscellaneous antioxidant. Zinc, one of the essential nutrients, strongly inhibits lipid
peroxidation, which is possibly to be due to altering or preventing iron binding. On the
other hand, selenium plays a major role in the synthesis and activity of glutathione
peroxidase, a primary cellular antioxidant enzyme (Madhavi and Salunkhe, 1996).
Since the intakes of trace elements may caused toxicity, the maximum levels of
selenium for adults should not exceeded 0.05-0.2 mg (Potter, 1986). Potentially harmful
77
metals such as lead, mercury, cadnium, zinc and selenium naturally present in soil, water
and plant foods. However, according to Potter (1986), some undesirable minerals and
certain natural toxicants are largely removed or inactivated when foods are processed.
Heat treatment applied during preparation of herbal pegaga drink still retains
appreciable amount of total polyphenol. After canning processed with the temperature
up to 100qC, about 50% of total polyphenol in pegaga drink remain. The loss of total
polyphenol content under heat processing treatment at 65qC for 15 minutes and 80qC for
5 minutes was 45% and 49%, respectively. In agreement with previous investigation,
phenolic compounds contained in food were significantly loss during heat processing.
This finding supported by Fezah, et al. (2000), who noted that the air-dried treatment at
room temperature of pegaga leaf contained about 0.111 mg pyrogallol per mg dried
MeOH extract, which is significantly lower than fresh sample. The total polyphenol in
leaf and underground part of pegaga was reduced by 52% and 50%, respectively. Gil-
Izquierdo, et al. (2002) reported that pasteurization led to degradation of several
phenolics such as caffeic acid, vicenin 2 and narirutin in orange pulp. Boiling of onion
bulbs considerably affected the content of quercetin, yielding losses of 43.2%. The 60-
min boiling had more severe effects in terms of flovonol loss in onion and asparagus.
This treatment resulted in 20.5% and 43.9% decrease in total flavonol content,
respectively (Markis & Rossiter, 2001). Similarly, Crozier, et al. (1997) reported that
cooking lowered the quercetin content of both tomatoes and onion. In contrast, 80% of
total phenolics in Roselle remained, even after drying at 75ºC and storage for 15 weeks
at 40ºC (Tsai, et al., 2002). Since the amount of total polyphenol was significantly
reduced after thermal treatment, the unstable phenolic compound may present in pegaga
drink as major component.
For comparison, Zainol, et al. (2003) reported that 100g of pegaga leaf extract
contained 8130-11700mg of total polyphenol in all accession tested. Fezah, et al. (2000)
79
observed slightly high total polyphenol content (23000mg per 100 g) in similar herb. It
can also be monitored that the phenolic content of pegaga drink was significantly lower
than raw material. This is mainly due to dilution process and the reduction of naturally
occuring phenolic compounds during preparation of raw material into drink.
Nevertheless, polyphenols composition in foods and processed products, is influenced
by the source of raw materials, variety and procedure used of sample preparation as well
as by the analytical methods employed to quantify polyphenols (Peleg, et al., 1991). As
previously observed, processing treatment of pegaga drinks at the high temperatures,
potentially causing thermal decomposition of some phenolic antioxidant. On the other
hand, processing steps such as cutting, blending and storage are expected to contribute
the degradation and/ or transformation of it biologically active component. Extraction of
pegaga juice is performed using industrial food processor and filtered by muslin-cloth.
The residue may contained some phenolic compounds and markedly decrease the
amount of this component in juice extract. Skrede and Wrolstad (2002) found that the
extensive loss of polyphenolic compounds occurred during processing single strength
juice. The industrial processing of pasteurized highbush blueberry recovered only 32%
of anthocyanin, whereas 18% remained in press-cake residue after pressing the pulp.
Similarly, Koo and Suhaila (2001) noticed that at high temperatures certain phenolics
decompose or combine with other plant components. Moreover, it was probably due to
the degradation of these compounds, as the best substrate for polyphenol oxidase (PPO),
for browning process.
catechin were contributed to the antioxidative activities in plant materials (Bors and
Saran, 1987). Component of phenolic antioxidants in herbs include catechins in tea
extract (Yen & Chen, 1995; Wang, et al., 2000), curcumin in C. longa (Ruby, et al.,
1995) and quercetin in Polygonum hydropiper (Haraguchi, et al., 1992).
d
CM2
b
Pegaga drink sample
CM1
e
C
B d
A c
a
F
Figure 4.1: Total phenolic compounds (as ferulic acid and gallic acid
equivalents) of different sample of pegaga drink (n=3). Key: F (fresh sample); A
(65qC/15 min); B (80qC/5 min); C (canned); CM1 (commercial sample -Loo Ent.); CM2
(commercial sample-HPA). Values with same letter (a,b,c) are not significantly different
(P>0.05) between samples.
Figure 4.2 shows the ascorbic acid content in different samples of pegaga drink.
The amount of ascorbic acid was reduced significantly after heat treatment. Unheated
samples contained the highest amount of ascorbic acid tasted (4.23mg/100ml), followed
by heat sample at 65ºC/15 minutes and 80ºC/5 minutes (1.76mg/100ml each) and the
lowest concentration was observed in canned drink (0.7mg/100ml). The commercial
pegaga drink (CM1) contains much higher ascorbic acid (2.11 mg/100ml) than CM2
(1.41mg/100ml), however its amount was found to be lower than unheated or fresh
sample. The amount of ascorbic acid in fresh drink, however is significantly lower than
those determined from guava juice (80.1mg/100g), passion juice (39.1 mg/100g) and
lemon juice (10.5 mg/100g) but almost similar to G. schomburgkiana juice (4.6
mg/100g) (Suntornsuk, et al., 2002). The residual ascorbic acid content in heat-treated
82
drinks, was lower than the unheated product. This observation is in agreement with the
reported by Mahanom, et al. (1999), that the loss of ascorbic acid in dried herbal tea,
dried at 50ºC for 9 hours and 70ºC for 5 hours, is about 75.60% and 34.19%,
respectively. In addition, the concentration of ascorbic acid in tomato puree and tomato-
oil samples was reduced to 46% and 55%, subjected to heat treatment at 95ºC for 30 min
(Nicoli, et al., 1997b). Freeze-dried of guava juice and emblic myrobolan juice also
cause the decrease amount of ascorbic acid up to 41.4% and 20.4%, respectively
(Suntornsuk, et al., 2002)
4.5 a
Concentration of ascorbic acid
4
3.5
3
(mg/100ml)
2.5 b
2 c c
d
1.5
e
1
0.5
0
F A B C CM1 CM2
Sample
Figure 4.2: Ascorbic acid content of different sample of pegaga drink (n=3).
Key: F (fresh sample); A (65qC/15 min); B (80qC/5 min); C (canned); CM1
(commercial sample -Loo Ent.); CM2 (commercial sample-HPA). Values with same
letter (a,b,c) are not significantly different (P>0.05) between samples.
The positive and negative effect of heat treatment of foods on their antioxidative
activities was previously reported. Since pegaga was found to have antioxidant activiy,
the present of antioxidant compounds in fresh and heat-treated pegaga drink may delay
oxidation of linoleic acid and exhibited the antioxidative activity. The Ferric
thiocyanate assay was used to evaluate the antioxidant activity of pegaga drink, only at
primary state of oxidation.
Generally, the oxidative activity of linoleic acid is markedly inhibited by any samples of
pegaga drink compared to control assay. The results showed that the level of antioxidant
activity is reduced when the temperature is increased. Fresh sample of pegaga exhibited
much higher (P<0.05) antioxidant activity than heat-treated samples. The % of
inhibition of peroxidation is 72.98% for fresh and 53.73, 64.80% and 69.88% for sample
C, B and A respectively (Figure 4.3). The lipid peroxidation inhibitory activity of all
pegaga drink samples significantly lower than raw pegaga leaves (98.2%) as previously
reported by Vimala, et al. (2003). However, the antioxidant activity of fresh pegaga
drink was comparable to those reported for oolong tea and higher than green tea. Yen &
Chen (1995) in their investigation indicated that oolong tea and green tea exhibited
73.6% and 40% inhibition of linoleic acid peroxidation, respectively. Our finding is also
similar to the work of Duh and Yen (1997), who reported that the addition of herbal
extracts significantly increased the inhibition the linoleic acid peroxidation. In terms of
mechanism, it is prolongs the induction period by the lowering rate of accumulation of
oxidative products.
All pegaga drink samples exhibited higher activity than natural antioxidant such
as D-tocopherol and ascorbic acid but lower than synthetic antioxidant, butylated
hydroxytoulene (BHT) at concentration of 200ppm. In agreement with our result, earlier
studies by Abdul Hamid, et al. (2002) revealed that the activity of pegaga evaluated
from similar method, is significantly lower than BHT. However, the antioxidant activity
of D-tocopherol at concentration of 300ppm and above is not significantly different from
that exhibited by leaves and roots extract of pegaga. Observation of antioxidant activity
under Ferric Thiocynate (FTC) assay, done by Mohd Zin et al. (2002) showed that ethyl
acetate extract of mengkudu exhibited significant activity, which are comparable to that
of both D-tocopherol and BHT.
in that particular important component. The reduction of the natural antioxidants could
be due to evaporation and transformation of the food component during processing that
could have pro-oxidant activity (Pokorny et al., 2001b; Nicoli et al., 1999). A brownish
colour was disserved in heat-treated drink, as a result of Millard reactions or degradation
of chlorophyll pigment. The reduction of antioxidant activity in heat-treated samples
can be attributed to the formation of compounds with pro-oxidants properties during
processing. Namiki and Hayashi (1983) reported that highly reactive radicals having
pro-oxidant properties might be formed in early stages of the Millard reactions, which
the formation of both pro-oxidant and antioxidant properties are always depend upon the
intensity and the duration of heat treatment (Nicoli, et al., 1999).
BHT a
Vit.C h
Pegaga drink and standard
Vit. E g
CM2 f
e
sample
CM1
f
C
d
B
c
A
b
Fresh
40.00% 50.00% 60.00% 70.00% 80.00% 90.00%
% inhibition of linoleic acid peroxidation
Figure 4.3: % Inhibition of peroxidation as mean (n=3) in pegaga drinks and standard
sample. Key: F (fresh sample); A (65qC/15 min); B (80qC/5 min); C (canned); CM1
(commercial sample -Loo Ent.); CM2 (commercial sample-HPA); Vit.E (D-tocopherol),
Vit.C (ascorbic acid) and BHT (Butylated hydroxy toluene). Values with same letter are
not significantly different at P=0.05
86
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.
The results of antioxidant activity from linoleic acid peroxidation was compared
with FRAP value. Figure 4.4 demonstrates the FRAP values of pegaga drink as a
consequence of processing procedures. The FRAP value was interpolated from a
standard calibration curve with the linear regression was y = 7.387e-5x + 0.002. Heat
processing studied showed negative effects thus resulted in a decrease in the antioxidant
potential of the pegaga drink. The greatest FRAP value was observed in fresh sample
(860 µmol/liter) followed by A (65qC/15 minutes), B (80qC/5minutes) and C (canned).
FRAP values of heat-treated samples are in the range of 404 - 740 µmol/litre. Two
commercial samples (CM1 and CM2) showed the appreciable amount of antioxidant
activity, which were able to reduce about 620 µmol/litre and 370 µmol/litre of Fe (III),
respectively. The antioxidant activity, however, is significantly lower than value
reported by Gardner, et al., (2000), who observed that the ability of vegetable and
orange juice to reduce Fe(III) are approximately 1.2mM and 6mM, respectively. These
results indicated a similar trend as to the FTC assay. Tsai, et al. (2002) found that the
87
FRAP activity of roselle extract and green tea was 2 mmol/litre and 8 mmol/litre,
respectively. The FRAP value was obtained from 1 g of roselle and green tea, extracted
in 300ml water at 100qC for 3 min. Results showed that, the level of antioxidant activity
in pegaga drink is slightly lower than green tea and roselle extract. Our results differ
from previous report on green tea beverages. The optimum activity was obtained when
it was prepared at high infusion temperature with long infusion time. The tea beverages
prepared at 20-70qC of infusion temperature was significantly lower than at the infusion
temperature of 90qC Lingley-Evans (2000). However, the antioxidant compounds in
black tea is ideally extracted at 70-90qC in 1-2 minutes infusion time.
Commercial antioxidant at 200 ppm (vitamin E) showed the highest FRAP value
(569.37 µmol/litre) followed by synthetic antioxidant, specifically BHT (543.75
µmol/litre), at the same concentration. The FRAP value of vitamin C was 398.36
µmol/litre. In contrast with FTC assay, BHT exhibited lower antioxidant activity than
fresh and heat-treated pegaga drink. Our result is similar to the previous studies, which
reported that the water extract of Chrysanthemum and Roselle exhibited a greater
reducing power than 200 ppm of D-tocopherol and BHA (Duh and Yen, 1997).
activity of the tomato samples increased with the increase in heating time Nicoli, et al.
(1997b). Nicoli et al. (1997a) also reported that the antioxidant activity did not
increased linearly with the increasing of roasting time of coffee brews.
e
Pegaga drink and standard
BHT
Vit.C f
d
Vit. E
g
CM2
sample
CM1 c
f
C
d
B
A b
Fresh a
Figure 4.4: FRAP activity as mean (n=3) in different thermal processing of pegaga
drinks. Key: F (fresh sample); A (65qC/15 min); B (80qC/5 min); C (canned); CM1
(commercial sample-Loo Ent.); CM2 (commercial sample-HPA). Values with same
letter are not significantly different at P=0.05
heat-processed tomato juice and grape juice had a much higher antioxidant activity than
fresh products, however the mechanism for the increase in activity is not clear. Thermal
treatment is responsible to induce the increase in the amount of phenolic antioxidant,
particularly anthocyanin and total cinnamates (Scalzo, et al., 2004). Results obtained
from previous studies also noted that the increased in antioxidant activity of plant foods
during prolonged heat treatments is because of the formation of Millard reactions
product (Nicoli et al., 1999). Monzocco, et al., 1999 found that the increase of chain-
breaking activity in Marsala-type wine is related to the development of non-enzymatic
browning (MRPs). Nicoli et al. (1997a) was reported a similar result in their
investigation on the antioxidant properties of coffee brew in relation to the roasting
degree. Millard products, especially melanoidins, can also bind iron and copper ions
into inactive macromolecular complexes (Pokorny, 2001b). In our research, the relation
of antioxidant activity with the formation of browning during processing of pegaga drink
was not investigated specifically.
Other factors such the synergism with other food components or chelating agents
particularly citric acid could be attributed to the appreciable level of antioxidant activity
in heat-treated samples. It has been reported that most natural antioxidative compounds
often work synergically with each other to provide a broad spectrum of antioxidant
activtiy that creates an effective defense system against free radical attack (Lu and Foo,
1995).
concentration. Under these conditions, reaction between components can occur and
affect the antioxidant property.
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)
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)
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.
Preliminary data on FRAP assay showed that the decrease in antioxidant activity
is mainly due to the decrease of phenolic compounds. Similar antioxidant activity has
been described for phenolic-rich beverages such as grape wines and teas (Frankel, et al.,
1995; Rice-Evans, et al., 1996). The decrease in phenolic total polyphenol is always
associated with significant decrease of antioxidant activity (Tsai, et al., 2002). The
finding also supported by Duh & Yen (1997), who noticed that the water extracts of
Chrysanthemum and Roselle possessed high contents of phenolic compounds and had
effective activities as radical scavengers. They also concluded that herbal water extracts
have effective activities as hydrogen donors and as primary antioxidants by reacting
with the lipid radical.
1600
(gallic acid eqv. mg/100ml)
Total polyphenol content
1400
1200
1000
800
600
300 400 500 600 700 800 900
FRAP value (umol/L)
Figure 4.7: Correlation coefficient of antioxidant activity (FRAP assay) and total
polyphenol content
The correlation between antioxidant activity and their phenolic compounds was
successfully established in a few studies. For example, Lunder (1992) reported that
there was a good correlation between the antioxidant activity and the epigallocatechin
gallate (EGCg) content. In other study, Gardner, et al. (1997), noted that the antioxidant
95
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)
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.
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
Figure 4.8 illustrate the effect of addition of citric acid at various concentrations
on their ability to inhibit the linoleic acid peroxidation in pegaga drink. Increased
absorbance of sample and the reaction mixture indicated decreased % inhibition of
linoleic acid (Yen and Chen, 1995). The oxidative activity of linoleic acid was inhibited
by pegaga drink sample with addition of any concentration of citric acid. The increasing
amount of citric acid up to 0.2% (200ppm) added to pegaga drink result in the increase
of the antioxidant activity. The antioxidant activity was slightly reduced at concentration
of 0.3%.
99
0.45
0.4 d
Absorbance at 500nm
0.35
0.3
c
0.25
0.2 a b
0.15
0.1
0.05
0
Control 0% 0.10% 0.20% 0.30%
Concentration of citric acid
Figure 4.8: The effect of citric acid on the antioxidant activity (FTC assay) of
pegaga drink (n=3). Values with same letter are not significantly different (p>0.05)
0.04 6.5
FRAP value (Absorbance at 593 nm) a
6.0
0.02
5.5
0.00 5.0
4.5
pH
-0.02 4.0
b 3.5
-0.04 c
b 3.0
FRAP
-0.06 2.5
-0.05 0.05 0.15 0.25 0.35 pH
Concentration of citric acid (%)
Figure 4.9: The effect of citric acid on the antioxidant activity (FRAP assay) of pegaga
drink (n=3). Values with same letter are not significantly different (p>0.05)
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
The total soluble solid (TSS) in pegaga drink was increased by the addition of
sugar at different concentration. To evaluate the effect of TSS on antioxidant activity in
pegaga drink, the level of TSS was increased from 1qBrix to 15qBrix. Figure 4.10
shows the antioxidant activity of pegaga drink at different level of total soluble solid
(TSS). The antioxidant activity of pegaga drink strictly depended on total soluble solid
(TSS), which high absorbance value of FRAP assay was observed at 15qBrix (0.057
nm) followed by 10q Brix (0.050 nm), 5qBrix (0.044 nm) and control sample (0.032
nm). A slightly contrast, the antioxidant activity was gradually increased at 5qBrix and
10qBrix by FTC assay, but declined thereafter with the addition of sugar up 15qBrix
(figure 4.11). Pegaga drink at concentration of 15qBrix, however shows the lowest
antioxidant activity compared to control sample (1qBrix) and other samples tasted.
According to Takeoka, et al. (2001), the increase in total soluble solid level up to 25-30
Brix appeared to influence the loss of antioxidant property in tomatoes such lycopene
content. They reported that the longer processing time, required to achieve the desired
final solid levels, might be associated with increased losses of lycopene. In our study,
the role of total soluble solid on overall antioxidant activity was still unclear. However,
it was because of different rates in chemical oxidation of phenolic compounds, which
are depending on some intrinsic food variables and it processing condition such as water
activity (aw) (Nicoli, et al., 1999).
102
a
0.06
b
0.05 c
0.04 d
FRAP Values
(Absorbance at 0.03
593 nm)
0.02
0.01
0
1 Brix 5 Brix 10 Brix 15 Brix
o
Total soluble solid ( Brix)
Figure 4.10: The effect of total soluble solid on the antioxidant activity (FRAP assay)
of pegaga drink (n=3). Values with same letter are not significantly different (p>0.05).
The increase in total soluble solid through the addition of sugar also reduced the
aw value in pegaga drink. The relationship between food products stability and water
activity was previously investigated (Tannenbaum, et al., 1985; Karel and Yong, 1981;
Labuza, 1985). The rate of chemical reactions such as lipid oxidation and degradation
of vitamin C, generally increased as water is added up to a higher aw value with
maximum rates typically occur in the range of intermediate moisture foods (0.7-0.9 aw)
(Fennema, 1985). Karel and Young (1981) have suggested that the present of free water
may accelerate oxidation by increasing the solubility of oxygen and by macromolecules
to swell, thereby exposing more reactions.
103
0.45 c
0.4 b
0.35 a a
0.3
Absorbance at 0.25
500nm 0.2
0.15
0.1
0.05
0
Control 1 Brix 5 Brix 10 Brix 15 Brix
Total soluble solid (Brix)
Figure 4.11: The effect of total soluble solid on the antioxidant activity (FTC assay) of
pegaga drink (n=3). Values with same letter are not significantly different at p>0.05.
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).
0.032 nm to 0.062 nm, 0.097 nm, 0.109 nm and 0.122nm at 200ppm, 250ppm, 300ppm
and 350ppm, respectively.
Similar results were observed in FTC assay that the % inhibition of linoleic
peroxidation was also increased accordingly. The sample of pegaga drink markedly
inhibited the oxidation of linoleic acid with the addition of sodium metabisulphite. The
% inhibition of linoleic acid oxidation was increased about 22.68% at a concentration of
350ppm sodium metabisulphite compared to control. Manzocco, et al. (2001) reported
that the addition of SO2 contributed to the retention most of the original chain breaking
activity of the dried apple cubes. In similar finding, Wang, et al. (1996) observed that
commercial tomato and grape juice had much higher antioxidant activity than fresh
materials. The high antioxidant activity was also found in the commercial wine and
juice sample, partially due to the presence of food preservatives such as sodium
metabisulphite and vitamin C, which is commonly, added to commercial food products
(Tsai, et al., 2002).
0.14 a
b
0.12 c
0.1
FRAP Values 0.08 d
(Absorbance at
593 nm) 0.06
e
0.04
0.02
0
0 200 250 300 350
Concentration of sodium metabisulphite (ppm)
0.14
0.12
(Absorbance at 593 nm)
0.10
FRAP values
0.08
0.06
0.04
0.02
-50 0 50 100 150 200 250 300 350 400
Concentration of
Sodium metabisulphite (ppm)
0.45
0.4 e
0.35 d
c
Absorbance at 500 nm
0.3 b a
0.25
0.2
0.15
0.1
0.05
0
Control 0 200 250 300 350
Concentration of sodium metabisulphite (ppm)
Figure 4.14: The effect of sodium metabisulphite on the antioxidant activity (FTC
assay) of pegaga drink (n=3). Values with same letter are not significantly different
(p>0.05).
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
Preliminary study was carried out to choose the best combination of methanol-
water that commonly used as mobile phase for analysis of saponins including triterpene
glycosides using High Peformance Liquid Chromatography (HPLC) (Court, et al., 1996;
Inamdar, et al., 1996; Verma, et al., 1999). Beside, a few different variables including
eluent strength, column and flow-rate were studied in order to accomplish optimum
separation of four active components of pegaga. The separation of active constituents in
pegaga was performed at the room temperature (Morganti, et al., 1999; Burnouf-
Radosevich and Delfel, 1986) with using methanol-water as a mobile phase in isocratic
HPLC system. Due to difference in polarity of the triperpene acids and its glycosides,
different concentrations of methanol (in the range of 10-90%) were used to get the better
eluent. From a few series of experiment it was observed that no peak of both triterpene
acids and its glycosides were detected at very low concentration of methanol including
20:80 and 10:90 of methanol:water. The optimum separation for madecassoside and
asiaticoside were obtained at ratio of 80:20 methanol:water after 7.87 and 8.53 minutes
(tR), respectively. The concentration of 90% methanol was observed to be excellent in
triterpene acids separation. Similarly, in isocratic HPLC assessment, Inamdar, et al.
(1999) reported that the two triterpene acids were separated by using high concentration
of methanol or acetonitrile but their glycosides needs low concentration of methanol or
acetonitrile. The chromatographic separation was peformed with a Genesis C18, flow
rate at 0.4ml/min and attenuation of 1 AUSF. The chromatograms corresponding to the
standard of asiaticoside and madecassoside are shown in figure 4.16 and 4.17.
108
4000
3500
3000
Area(1E3 mV.s)
2500
y = 8675.9x + 94.036
2000
1500
1000
500
0
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45
Concentration of standard solution (mg/ml)
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)
The peak for madecassic acid and asiatic acid were identified by using methanol-
water at concentration of 90:10 as mobile phase. The retention times (tR) of madecassic
acid and asiatic acid were 9.11 and 11.51, respectively. Figure 4.20 and 4.21 represent
the standard peak for madecassic acid and asiatic acid.
111
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
7000
6000
5000
Area (1E3 mV.s)
3000
2000
1000
0
0.0 0.1 0.2 0.3 0.4
Concentration of standard solution (mg/ml)
Figure 4.21: Calibration curve for madecassic acid (area vs concentration of standard
madecassic acid)
113
14000
12000
10000
Area (1E3 mV.s)
y = 31621x + 1.2049
8000
6000
4000
2000
0
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45
Concentration of standard solution (mg/ml)
Figure 4.22: Calibration curve for asiatic acid (area vs concentration of asiatic acid)
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.
Asiaticoside content shows a different trend that its concentration in fresh drink
was significantly lower (3.92 mg/100 ml) than in sample A (4.32 mg/100ml). However,
was significantly higher than other heat-treated samples. The concentration of
asiaticoside was remarkably reduced to 8-22.5% when exposed to high temperature up
to 80qC as in sample B (3.61 mg/100ml) and sample C (3.03 mg/100ml). Therefore, it
may be concluded that the heat treatment at moderate temperature (65qC) is likely to
increase the ability of water (as a medium) to dissolve the asiaticoside. In accordance
with the report of Vongsangnak, et al., (2003), which obtained the maximal saponin
yield when the extraction temperature was controlled around 50qC. On the other
research, Pan, et al., (2002) noticed that the application of high temperature (20-50qC)
enhanced the extraction efficiency. This is a result of an increased in diffusivity of the
solvent into cells and at the same time it increased the ability of components to adsorb
from the cells.
116
Means with the same letter (a, b, c) in each column are not significantly different at p=0.05
117
Asiatic acid was not stable at the higher temperature and this resulted in a
decreasing amount in all heat-treated sample. The concentration of asiatic acid varies in
the range of 2.45-0.97mg/100 ml. Asiatic acid content dropped to 10.7%, 16.3 % and
17.3% in A, B and canned sample, respectively. Pasteurization processed at
65qC/15min (A) to 100qC (C) resulted in a significant change of asiatic acid content in
pegaga drink. Again, the asiatic acid content of commercial sample, CM1 (10.18
mg/ml) was significantly higher than CM2 (8.06 mg/ml).
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.
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.
60
50
40
Triterpene glycosides s
Madecassoside
30
content (%)
Asiaticoside
20 Madecassic acid
Asiatic acid
10
0
F A B C CM1 CM2
Pegaga drink sample
Figure 4.23: Triterpenoid fraction (%) of pegaga extract from drink samples
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.
CHAPTER 5
5.1 Conclusion
x Fresh pegaga drink (F) contained about 1470.14 mg/100ml of total polyphenol
(GAE equivalent), which was significantly higher than pasteurized sample at
65qC/15 minutes (903.23 mg/100ml) and 80qC/5 minutes (805.54 mg/100ml);
and canned pegaga drink (730.27 mg/100ml).
x 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
x The two assays (FRAP and FTC) were strongly correlated (r=0.93) at p=0.05.
However, very low correlation was obtained (r=0.54) when antioxidative activity
of pegaga drink, synthetic antioxidant (BHT) and natural antioxidant (ascorbic
acid and vitamin E) were taken into account.
x The average content of madecassic acid was higher in canned drink (3.22
mg/100ml) followed by B (3.02mg/100ml), A (2.70 mg/100ml) and fresh sample
(2.56 mg/100ml).
x The heat processing of pegaga drink resulted lower amount of asiatic acid, where
the asiatic acid content varies in the range of 2.45-0.97 mg/100ml. The non-
thermally treated drink or fresh sample (F) contained higher amount of total
124
x Previous works by Vimala, et al. (2003) reported that pegaga leaves extract
contain a high antioxidant activity towards superoxide free radical scavenging
activity (SS) and radical scavenging activity (DPPH). The similar assessment
should be carried out for pegaga drink in order to evaluate its ability to reduce
the excess free radical and to determine the level of prevention of tissue and cells
damage. Scavenging of DPPH radical determines the antioxidant potential of the
test sample, which shows its effectiveness, prevention, interception and repair
mechanism against injury in biological system.
x 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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APPENDIX A1
APPENDIX A2
APPENDIX A3
APPENDIX B1
APPENDIX B2
APPENDIX B3
APPENDIX C
APPENDIX D
0.12
0.11
0.10
0.09
0.08
0.07 y=7.387E-5x + 0.002
0.06
0.05
0.04
0.03
Absorbance at 593nm
0.02
0.01
0.00
-0.01
-200 0 200 400 600 800 1000 1200 1400
APPENDIX E
4.5
4.0
3.5
3.0
2.5
2.0
1.5
Absorbance at 750nm
1.0
0.5
0.0
0.0 0.2 0.4 0.6 0.8 1.0 1.2