ANTIOXIDANT ACTIVITIES OF Metroxylon sagu EXTRACT

ITS
AND
EXTRACT
OF Metroxylon
ACTIVITIES
ANTIOXIDANT
THERAPEUTIC
SMOKE
CIGARETTE
;:
EXPOSED
PETER
LAURA
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EFFECTS
MICE
DABBI
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uhtINUS;
SABAH
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TESIS
INI
SYARAT
DIKEMUKAKAN
UNTUK
MEMENUHI
MEMPEROLEHI
IJAZAH
SARJANA
SAINS
SCHOOL
UNIVERSITY
(BIOKIMIA)
OF MEDICINE
MALAYSIA
2007
SABAH
sagu
ON
UNIVERSITI
BORANG
JUDUL:
ANTIOXIDANT
ITS
SESI:
Saya,
2003
LAURA
disimpan
PETER
3.
pertukaran
TIDAK
4.
ON
(BIOKIMIA)
2007
DABBI,
Sarjana
ini
tesis
mengaku
membenarican
Universiti
Malaysia
Sabah
dengan
syarat-syarat
hakmilik
Universiti
Universiti
Malaysia
Malaysia
Sabah
Sabah
dibenarkan
pengajian
saya
dibenarkan
membuat
salinan
tinggi
antara institusi pengajian
membuat
ini
tesis
salinan
sebagai
bahan
TERHAD
Disahkan
(Penulis:
EXTRACT
AND
sagu
CIGARETTE
SMOKE
berikut:
seperti
untuk tujuan
Perpustakaan
EFFECTS
SAINS
- JAN
1. Tesis adalah
2. Perpustakaan
TESIS41
OF Metroxylon
ACTIVITIES
di Perpustakaan
kegunaan
STATUS
MICE
SARJANA
NOV
SABAH
PENGESAHAN
THERAPEUTIC
EXPOSED
IJAZAH:
MALAYSIA
LAURA
PETER
DABBI)
oleh
(TANDATANGAN
PUSTAKAWAN)
Alamat
Peti
Surat
89507
704,
Penampang,
Sabah
OF.
(Pen
DR.
PERUMAL
RAMASAMY)
Tarikh:
0ý,,
to
1co
"1
1b'
Tarikh:
CATATAN:
Sarjana
dan
OTesis
secara
penyelidikan,
dimaksudkan
sebagai
disertasi
penyelidikan
atau
Projek
atau laporan
tesis
bagi
Sarjana
Qb
Ijazah
U
-ý
Doktor
pengajian
secara
Muda (LPSM)
Falsafah
kerja
dan
kursus
DECLARATION
The
in
material
summaries
and
this
thesis
references,
is
which
original
have
except
been
duly
for
quotations,
excerpts,
equations,
acknowledged.
0
ýýý
LAURA
'r
PETER DABBI
PSO3-016-002
ACKNOWLEDGEMENT
to my supervisor,
my deepest
gratitude
and appreciation
express
for all his advice,
Dr. Perumal
Ramasamy,
guidance
and never-ending
support
help throughout
He has been a great
in the completion
the years and
of this thesis.
during the thesis-writing
to embark
period. Thank you for giving me the opportunity
on
this research
on the given subject.
and enhance
my knowledge
like
I would
Professor
to
Dr. Hj. Kamaruzaman
Prof. Datuk
my internal
examiner,
for their comments,
Shanmugam
Ampon
Prof. Dr. Balabaskaran
examiner,
and external
Further
ideas and advice during the Viva voce session.
my
more, I would like to extend
Special
thanks
go
to
data, not
to Dr. Khin Saw Naing who guided me in the analysis of statistical
appreciation
forgetting
Dr. How Siew Eng, Dr. Md. Lutfor Rahman
and Mr. Mustafa for their valuable
Many
knowledge
in assisting
me with the interpretation
of FT-IR and NMR spectrum.
thanks
help
go to Prof.
in my thesis-writing.
also
Madya
Dr.
Nur
Ashikin
Miss Jayanthy
and
who
attributed
their
Dr.
of Science and Technology),
Prof. Datin Dr.
Mohd.
Technology),
of Engineering
and Information
(Institute
for Tropical
Maryati
Mohamed
Biology and Conservation)
and to all their staffs
for their kind assistance
facilities.
in providing
me with the laboratory
I wish to thank
Dr.
Yunus Hamid (School
Zaleha
In addition,
(B-006-18-ER
also
I would
/U082)
Abd.
like
Aziz
to thank
(School
the
UMS
for
the
Fundamental
Research
Furthermore,
I
this research.
me to conduct
which enabled
Beaufort
the staff of Pejabat
Pertanian
and the villagers
also wish to thank Mr. Tarmudzi,
in Kg. Gerama
for providing
Beaufort
sagu) pith
and Gadong,
me the sago (Metroxylon
samples.
Grant
My
contributions
throughout
heartfelt
for their
countless
go to all my friends
and seniors,
their
such as supporting
advice
and experiences
me, sharing
opinions,
the completion
of this task and most of all their friendship
which I cherished
thanks
the most.
Finally,
strongest
and
that
to
support,
understanding
I dedicate
this
my
beloved
parent,
family
emotionally
and spiritually.
during
those tough times
research
for they
have been
and Ranique
my
love
Thank you for the encouragement,
it the most. It is to them
when I needed
work.
11
ABSTRAK
Antioksidan
Aktiviti
Metroxylon
ekstrak
pendedahan
Secara
amnya
bersifat
yang
asap
terdapat
kepelbagaian
antioksidan
melalui
dan
sagu
rokok
komponen
pada
tindakannya
Metroxylon
penyakit.
sagu
Pepejal-Cecair.
Sebanyak
diperolehi.
SAE
aktiviti
antioksidan
2,2'-Azinobis-(3-etilbenzotiazolin-6-sulfonat)
(FTC),
Tiosianat
esei
logam.
SAE berpotensi
pengikatan
tindakannya
menjadi
dalam
menghalang
proses
2,2'-Azinobis-(3-etilbenzotiazolin-6-sulfonat)
Tiga
terapeutiknya
sumber
tumbuh-tumbuhan
diekstrak
semasa
pertama,
bebas
radikal
menggunakan
dan
air
melalui
(SAE)
2.87%
akueus
sagu
ekstrak
dilakukan
kaedah
Ferric
menerusi
(ABTS)
dan
aktiviti
berdasarkan
antioksidan
semulajadi
asid linoleik,
memerangkap
pengoksidaan
dan memiliki
keupayaan
dengan
masa retensi
pada 2.61 min, 4.30
min dan
SAE oleh
HPLC menggunakan
kolum
C18 fasa
pemisahan
SAE (Puncak
1) dipilih
kajian terperinci
untuk
menggunakan
puncak
pada
tikus
semulajadi
pada
dalam
menentang
menghalang
pelbagai
kaedah
pengestrakan
Penentuan
kesan
radikal
logam.
mengikat
4.70 min
dikesan
berbalik.
Puncak
FT-IR
dan
NMR
FT-IR,
memandangkan
puncak tersebut
adalah paling stabil. Berdasarkan
pada spekrum
kumpulan
1) adalah kumpulan
alkana, alkena,
asas yang dikenalpasti
pada SAE (Puncak
dan alkohol.
1) menggunakan
Pengenalpastian
SAE (Puncak
alifatik tak tepu, karbohidrat
'H
berbeza,
keseimbangan
NMR
terdapat
yang
mendapati
sepuluh
proton
(kawasan
bagi
berkemungkinan
berasal
dari
kumpulan
dan
karbohidrat
alifatik
13C NMR bagi
komponen
berkenaan
lebih kurang b 0.8 sehingga
6 4.2). Spektra
terletak
SAE
(Puncak
terletak
pada
1)
memaparkan
julat 6 60.3 -6
kehadiran
dua
belas
81.5,
dan selebihnya
berkemungkinan
karbon
pada
karbon
sepuluh
dan 6 103.8. Komponen
dengan
6 92.3
karbohidrat,
dengan
tersebut
yang memiliki
struktur
sepuluh
adalah
karbon
karbon
bukan
C-1 pada
dan selebihnya
berkemungkinan
adalah
anomerik
terminal
dan C-1 yang terlibat
Artemia
penurunan
dalam ikatan glikosidik.
Ujian toksisiti
SAE adalah
dibandingkan
dengan
salina
menunjukkan
tidak
toksik
apabila
potasium
dikromat
dengan toksisiti
iaitu 0.004. Bagi penentuan
relatifnya
enzim
aktiviti antioksidan
dismutase,
(superoxid
kawalan
dan katalase),
kumpulan
glutation
peroxidase
negatif
menunjukkan
berbanding
menunjukkan
manakala
berbanding
aktiviti
kumpulan
glutation
kajian.
lebih
tinggi
tisu
peroxidase
pada
paru-paru
yang
kajian
Bagi tisu hepar
dan buah
kumpulan
pinggang,
lebih
berbanding
kumpulan
kawalan
rendah
negatif
katalase
aktiviti
kumpulan
kajian
pada tisu otak,
memiliki
kumpulan
kawalan
Penambahan
negatif.
dalam
mengurangkan
tisu
membahayakan
bebas
radikal
yang
berkenaan.
Oleh
itu,
katalase
aktiviti
SAE mempunyai
dibebaskan
jumlah
aktiviti
yang
asap
glutation
kerosakan
yang diperlukan
adalah minimum
untuk menghalang
daripada
Berdasarkan
tisu berkenaan
asap rokok.
yang berpunca
katalase
pada
kajian,
melalui
yang lebih tinggi
kesan pelindung
pengambilan
terdedah
pada
SAE dalam
asap
diet
mampu
melindungi
perokok
rokok
sebelum
dan
peroxidase
yang
lebih
teruk
pada keputusan
dan bukan
perokok
rokok.
I11
ABSTRACT
Activities
Antioxidant
on
It
is known
Metroxylon
of
Cigarette
sagu
Smoke
extract
Exposed
and its therapeutic
Mice
effects
that
in the plant
kingdom
a vast number
of natural
compounds
possess
that can combat
the deleterious
properties
antioxidant
effects
of free radicals
and thus
Metroxylon
prevent
a number
of diseases.
sagu was extracted
with water
using the
Liquid-Solid
2.87%
(SAE)
extraction
method.
yield
of sago
aqueous
extract
was
Determination
obtained.
of the antioxidant
(FTC)
Ferric
Thiocyanate
2,2'
method,
(ABTS) assay and Metal Chelating
Activity.
activities
of the SAE was carried
out
Azinobis-(3-ethylbenzothiazoline-6-sulphonate)
by the
natural
shown to be a potential
based
in inhibiting
the peroxidation
source,
on its action
of linoleic
acid,
the 2,2'-Azinobis-(3-ethylbenzothiazoline-6-sulphonate)
radicals and its metal
antioxidant
scavenging
SAE has been
Three
times of 2.61 min, 4.30 min and 4.70
peaks with retention
min, respectively,
were detected
on separation
of SAE by HPLC using C18 reverse phase
The first peak, SAE (Peak 1) was further
investigated
column.
using FT-IR and NMR as it
bands identified
in
the principal
was the most stable peak. Based on the FT-IR spectrum,
SAE (Peak
1) were
belonging
those
to alkanes,
aliphatics,
alkenes,
unsaturated
chelating
properties.
ten
of SAE (Peak 1) using 1H NMR found
to aliphatic
protons,
regions
which could belong
and carbohydrate
13C NMR
(as the region indicative
5
4.2).
0.8
to
is approximately
of these compounds
(5
spectrum
of SAE (Peak 1) shows the presence
of twelve
carbons
of which ten carbons
two at b 92.3 and b
were located
within the range of 5 60.3 -b 81.5, and the remaining
103.8,
The
be
these
respectively.
possible
could
compound
structures
with
carbohydrates
and
different
equivalent
carbohydrates,
signals from
alcohols.
Identification
ten
brine
two probably
non-anomeric
are
carbons,
and the remaining
C-1 at a reducing
lingkage.
The
terminus
in a glycosidic
and C-1 involved
lethality
test shows
that SAE is non-toxic
to potassium
when compared
toxicity
For the determination
with
relative
of 0.004.
of free
radical
scavenging
enzyme
shrimp
dichromate
with
(superoxide
activities
dismutase,
and
glutathione
peroxidase
higher glutathione
peroxidase
the negative
control
group showed
significantly
in the lung tissue compared
In liver and kidney
to the experimental
activities
group.
the experimental
tissues,
lower catalase
group showed
a significantly
activity
compared
to the negative
higher
tissue,
control
group
and in brain
catalase
activities
were
in the experimental
Thus, SAE
to the negative
observed
group
compared
controls.
has protective
in reducing
those free radicals
that are released
supplementation
effects
catalase),
from
cigarette
oxidative
of glutathione
induced
from
sidestream
a lesser amount
results,
stress
SAE supplementation
non-smokers
exposed
before
smoke
causing
damage
extensive
peroxidase
and catalase
activity
the cigarette
in those
smoke
might
to sidestream
have
a beneficial
cigarette
smoke.
role
to the tissues.
Thus,
is needed
to combat
the
tissues.
Based on these
in protecting
smokers
and
iv
TABLE
OF
CONTENTS
Page
TITLE
i
DECLARATION
ACKNOWLEDGEMENT
ii
ABSTRAK
III
ABSTRACT
IV
TABLE
OF CONTENTS
V
LIST
OF FIGURES
ix
LIST
OF TABLES
xi i
LIST
OF ABBREVIATIONS
XIV
LIST
OF SYMBOLS
xvi
CHAPTER
ONE
1.1
Introduction
1.2
Objectives
CHAPTER
2.1
1
3
of the Research
TWO
LITERATURE
REVIEW
Free Radical
2.1.1
2.2
1
INTRODUCTION
Types
4
of Free Radicals
2.1.1.1
Superoxide
2.1.1.2
Hydrogen
2.1.1.3
Hydroxyl
2.1.1.4
Peroxyl
2.1.1.5
Nitric
2.1.1.6
Hypochlorous
5
Radical
(O2' -)
5
Peroxide
(H202)
6
Radical
Radical
Oxide
(OH')
6
(ROO')
7
(NO')
2.1.2
Formation
2.1.3
Free Radical
Chain
2.1.4
Free Radical
in Cigarette
8
acid (HOO)
of Free Radicals
Intracellular
8
9
Reaction
10
Smoke
12
Antioxidants
2.2.1
4
16
antioxidant
defence
systems
17
V
2.2.2
2.2.1.1
Superoxide
2.2.1.2
Catalase
2.2.1.3
Glutathione
Extra-cellular
Ascorbic
2.2.2.2
Vitamin
Antioxidant
2.2.4
Natural
2.2.5
Metroxylon
2.2.6
Free radicals,
2.2.7
Determination
Sago
2.2.9
CHAPTER
defence
acid (Vitamin
21
systems
C)
22
23
25
as a source
of Antioxidants
29
Antioxidant
enzyme
34
and Diseases
and free
of antioxidant
radical
35
Detection)
36
activities
(UV/Vis
and identification
(SAE)
Extract
Aqueous
26
Rottb.
sago
2.2.8.1
High
Performance
2.2.8.2
Fourier
transform
2.2.8.3
Nuclear
Magnetic
Liquid
in
of Components
separation
Scope
20
E
Spectrophotometer
Isolation,
(GSH-Px)
Interrelationships
scavenging
2.2.8
17
19
peroxidase
compounds
2.2.7.1
(SOD)
(CAT)
antioxidant
2.2.2.1
2.2.3
dismutase
Chromatography
(FT-IR)
infrared
spectroscopy
(NMR)
Resonance
(HPLC)
spectroscopy
37
39
41
42
of this Research
THREE
37
METHODOLOGY
44
3.1
Materials
44
3.2
Methods
48
3.2.1
Liquid-Solid
(Nagai
3.2.2
Determination
(i) Ferric
1993;
Metal
of antioxidant
Thiocyanate
Rahmat
(ii) ABTS assay
(iii)
of Metroxylon
extraction
et al. 2003)
components
49
activities:
(FTC)
48
(Kikuzaki
& Nakatani,
49
et al. 2003)
(Cano
Chelating
Yen & Wu,
Method
sagu
et al. 1998;
Activity
(Decker
Yu et a1.2004)
& Welch,
1990;
50
51
1999)
vi
3.2.3
(SAE)
3.2.4
in Sago Aqueous
Extract
and Separation
of Components
by High Performance
Liquid Chromatography
(HPLC)
Isolation
in Sago Aqueous
of Components
Transform
Infrared
(FT-IR)
spectroscopy
Identification
Fourier
3.2.5
of Components
Magnetic
Resonance
in Sago
Identification
Nuclear
3.2.6
Brine
Shrimp
3.2.7
Sago
Aqueous
radical
Lethality
Extract
scavenging
side-stream
Extract
(SAE)
by
55
Extract
(SAE)
by
56
spectroscopy
Test
(Toxicity
(SAE)
supplementation
in mice
activities
enzyme
cigarette
(NMR)
Aqueous
53
57
test)
on the free
exposed to
59
smoke
59
3.2.7.1
Experimental
Design
3.2.7.2
Side-stream
cigarette
3.2.7.3
Preparation
of the
3.2.7.4
Determination
of Protein
3.2.7.5
Determination
of the
(SSCS)
smoke
tissue
(Jeon
enzymes
Radical
63
et al. 2002)
(Bradford,
Concentrations
Free
60
exposure
Scavenging
1976)
Enzyme
63
65
Activities
(i) Catalase
(ii)
(CAT)
Glutathione
(Paglia
(iii)
(Marklund
3.2.8
CHAPTER
4.1
Liquid-Solid
Yield
4.2.1
extraction
of Sago
activity
66
1967)
(SOD)
& Marklund,
1974)
67
activity
68
AND
of Metroxylon
Aqueous
of antioxidant
Determination
Extract
DISCUSSIONS
sagu
69
components
(SAE)
69
69
70
activities
of antioxidant
Method (FTC)
activities
using
Ferric
70
of antioxidant
activities
using
ABTS assay
72
of antioxidant
Activity
activities
using
Metal
74
Thiocyanate
4.2.2
Determination
4.2.3
Determination
Chelating
(GSH-Px)
Dismutase
RESULTS
Determination
65
1974)
Analysis
FOUR
4.1.1
4.2
Statistical
Peroxidase
& Valentine,
Superoxide
(Aebi,
activity
vii
4.3
Isolation
(SAE)
4.4
in Sago Aqueous
Extract
and Separation
of Components
by High Performance
Liquid Chromatography
(HPLC)
Identification
of Components
(FT-IR)
spectroscopy
Infrared
4.5
Identification
of Components
Resonance
(NMR)
4.6
Brine
Shrimp
4.7
Sago Aqueous
radical
cigarette
in SAE (Peak
1) by Fourier
Transform
79
in SAE (Peak
1) by Nuclear
Magnetic
81
spectroscopy
Lethality
Extract
scavenging
76
Test
(Toxicity
(SAE)
supplementation
in mice
activities
enzyme
test)
87
on the free
exposed to side-stream
90
smoke
4.7.1
Determination
of Protein
4.7.2
Determination
of the
Concentrations
Free Radical
90
Scavenging
Enzyme
91
Activities
4.7.2.1
Determination
lung
4.7.2.2
FIVE
tissue
in the
and CAT activities
in the
94
and CAT activities
in the
96
and CAT activities
in the
98
of mice
tissue
Determination
brain
CHAPTER
of SOD, GSH-Px
Determination
kidney
4.7.2.4
tissue
and CAT activities
of mice
Determination
liver
4.7.2.3
tissue
of SOD, GSH-Px
of SOD, GSH-Px
of mice
of SOD, GSH-Px
of mice
CONCLUSIONS
101
REFERENCES
105
APPENDIX
115
viii
LIST
OF FIGURES
Page
CHAPTER
Figure
2.1
TWO
4
Formation
of Reactive Oxygen
and Zheng, 2002)
(Fang
Figure
2.2
Autooxidation
(Rice-Evan
Figure
2.3
Autoxidation
(Rice-Evan
Figure
2.4
The
Figure
2.5
Effects
Species
(ROS)
9
process by free radicals
1994)
and Burdon,
10
of PUFA in phospholipids
1994)
and Burdon,
12
membranes
airflows
of mainstream
and side-stream
smoke with the
Chemical
1980 and
constituents
of cigarette
smoke (Baker,
Elsayed & Bendich,
2001)
that
may
contribute
Figure
2.6
SOD 3-Dimensional
Structure
Figure
2.7
CAT 3-Dimensional
Structure
(www.
2.8
GSH-Px
Figure
2.9
Ascorbic
Figure
2.10
(a) a-tocopherol;
Figure
2.11
Antioxidant
Defense
Figure
2.12
Antioxidant
reactions
1997)
(Karlsson,
Figure
2.13
The
Figure
2.14
(a) The
3-Dimensional
(www.
17
com)
19
Structure
acid (Vitamin
3dchem.
de/research)
mvl. chem. tu-berlin.
Figure
15
smoke inhalation
to human diseases
of side-stream
(www.
sgc. utoronto.
ca/)
(b) a-tocotrienols
Systems
20
22
C)
reduction-oxidation
1997)
entity (Karlsson,
14
24
(Chow,
1988)
in reduction-oxidation
(red-ox)
reactions
25
(red-ox)
of quinol
sago palm; (b) Sago trunk; (c) sago pith/bole
(d) house thatch made by sago leaflets and (e) sago
pith waste which used as animal feed and fertilizer
reactions
26
28
32
ix
Figure
2.15
(a) Sago
trunks
(b)
logs chopped
Sago
drifting
(c) Sago maceration
(d)
Figure
2.16
Starch
Octadecyl
Figure
2.17
2.18
CHAPTER
The
infrared
HPLC System
(Thermo
Nicolet
Nuclear
Magnetic
ECA 600
(a) Schematic
4.3
procedure
Infrared
NEXUS
Series
53
200)
(FT-IR)
55
FT-IR)
Resonance
(NMR)
56
MHz)
diagram
of cigarette
(b) Cigarette
Experimental
smoke
62
smoke
apparatus;
mice
FOUR
69
Total
antioxidant
ascorbic
Figure
(PerkinElmer
Transform
(c) & (d)
4.2
research
44
Apparatus;
Figure
38
stationary
THREE
(JEOL
4.1
sieves
43
Fourier
Figure
cloth
of the overall
3.2
CHAPTER
through
passed
Summary
Figure
3.4
machine
functional
The
Figure
pieces
40
3.1
3.3
to small
33
river
absorption
regions with specific
K, 1995)
groups (Feinstein
Figure
Figure
into the
bonded
silane (ODS) chemically
(Braithwaite
and Smith, 1996)
phases
Figure
slurry
down
The
acid,
antioxidant
concentrations
activities
of different
BHT and SAE using the FTC method
activities
compared
to other
The
chelating
Metal
of SAE, ascorbic
of different
antioxidants
of different
acid and EDTA
4.4
The
Figure
4.5
Profile
Figure
4.6
The
Figure
4.7
1H NMR spectrum
of Tetramethylsilane
(reference
standard)
and deuterium
HPLC chromatogram
Spectrum
ABTS
SAE
SAE sample
of SAE
71
73
assay
concentrations
of 50 mg/mL
of the 50th run of the same
FT-IR
the
effect
Figure
of SAE
concentrations
using
of
75
77
78
79
(TMS)
oxide
81
(D20)
x
Figure
4.8
'H NMR
spectrum
of SAE (Peak
1)
Figure
4.9
'H NMR spectrum
of SAE (Peak
1) with
Figure
4.10
13C NMR spectrum
strength
of carbon
of SAE (Peak
NMR signals
Figure
4,11
13C NMR spectrum
of SAE (Peak
decoupled
gated NOE
Figure
4.12
Acute
LC50 of SAE determined
accumulated
Figure
4.13
Chronic
4.14
The
Standard
Bradford
deaths
Curve
integration
the
1) assigned
by plotting
values
83
relative
85
with
87
the Reed-Muench
88
and survivors
LC50 of SAE determined
accumulated
Figure
deaths
1) with
82
by plotting
the Reed-Muench
89
and survivors
for Protein
Determination
by the
91
method
xi
OF TABLES
LIST
Page
CHAPTER
Table
2.1
TWO
4
Nutrient
content
in sago
based
pith/bole
and leaf,
in Sarawak,
Malaysia
on measurements
Seram, Moluccas,
Indonesia
CHAPTER
Table
3.1
3.2
Table
4.1
1997)
44
The Total
The
Digest
as Basal
Feeding
and Ascorbic
CHAPTER
and
THREE
Pellets
Table
(Flach,
30
Nutrient
(TDN)
60
of Mouse
Diet
Groups
for
Mice used
acid Supplementation
in the SAE
Studies
69
FOUR
Total
61
antioxidant
of ascorbic
acid,
activities
of different
concentrations
BHT and SAE using the FTC method
71
Table
4.2
The
of SAE
antioxidant
activities
of different
concentrations
to other antioxidants
compared
using the ABTS assay
72
Table
4.3
The
74
Metal
chelating
of SAE, ascorbic
Table
4.4
FT-IR
Table
4.5
The
Acute
Table
4.6
The
Chronic
Table
4.7
The
absorption
acute
dichromate
effect
acid and
spectrum
of different
EDTA
concentrations
of SAE (Peak
LC50 for Sago Aqueous
LC50 for Sago Aqueous
1)
Extract
Extract
80
(SAE)
(SAE)
LC50 and chronic
LC50 of SAE and potassium
with the relative toxicity
88
89
90
X11
Table
4.8
Effects
free
radical
Table
4.9
4.10
Effects
Effects
free
mice
of
(SSCS)
smoke
95
of
of SAE and ascorbic
acid supplementation
on the
in the kidney tissue
enzyme activities
scavenging
exposed to side-stream
Effects
93
of SAE and ascorbic
radical
mice
4.11
cigarette
acid supplementation
on the
free radical scavenging
in the liver tissue
enzyme activities
to side-stream
mice exposed
cigarette
smoke (SSCS)
free
Table
acid supplementation
on the
in the lung tissue
enzyme activities
scavenging
exposed to side-stream
mice
Table
of SAE and ascorbic
cigarette
smoke
of SAE and ascorbic
radical
cigarette
smoke
of
(SSCS)
acid supplementation
in the
enzyme activities
scavenging
to side-stream
exposed
96
on the
brain tissue
98
of
(SSCS)
X111
LIST
Aqueous
OF ABBREVIATIONS
SAE
Sago
Extract
SSCS
Side-stream
FTC
Ferric
ABTS
2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonate)
UV/Vis
Ultraviolet/Visible
UV
Ultraviolet
HPLC
High
FT-IR
Fourier
NMR
Nuclear
IR
Infrared
SOD
Superoxide
dismutase
GSH-Px
Glutathione
Peroxidase
GSH
Glutathione
CAT
Catalase
Eq.
Equation
NADPH
The
NADP+
Oxidised
PUFA
Polyunsaturated
LDL
Low
HOCI
Hypochlorous
Cu, Zn-SOD
Copper,
Mn-SOD
Manganese
Fe-SOD
Iron
Cigarette
Smoke
Thiocyanate
Performance
Chromatography
Transform-Infrared
Magnetic
Resonance
form
reduced
of NADP
Nicotinamide
Density
-
Liquid
Fatty
Adenine
Dinucleotide
Phosphate
Acids
Lipoprotein
acid
Zinc - Superoxide
- Superoxide
Superoxide
Dismutase
Dismutase
Dismutase
XIV
BHT
Butylated
Hydroxy
KBr
Potassium
EDTA
Ethylene
diamine
CHD
Coronary
Heart
TMS
Tetramethyl
D20
Deuterium
LC50
Lethal
Toluene
bromide
tetra
acid
Disease
silane
oxide
Concentration
experimental
acetic
which
causes
the death
of 50%
of
animals
B
Mice fed on basal
diet only
BSm
Mice fed on basal
diet
only
BEx
Mice fed on basal
diet
+ SAE
BExSm
Mice fed on basal
diet
+ SAE + exposure
BC
Mice fed on basal
diet
+Ascorbic
BCSm
Mice fed on basal
diet
+ Ascorbic
to SSCS
+ exposure
to SSCS
acid
acid
+ exposure
to SSCS
xv
LIST
g
gravity
cm
centimeter
m
meter
ft
feet
nm
nanometer
ppm
parts
mL
mililitre
mg
milligram
M
molar
mm
milimolar
min
minute(s)
mmol
milimoles
nmol
nanomoles
pL
microlitre
pm
micrometer
Log
logarithm
0C
degree
1H
hydrogen
13C
carbon
Oz' -
superoxide
NO'
nitric
ON00-
peroxynitrite
OH- / HO'
hydroxyl
OF SYMBOLS
per million
Celsius
isotope
isotope
(tritium)
(carbon
thirteen)
radical
oxide
radical
xvi
hydrogen
H202
COO'/
R00'
peroxide
peroxyl
radical
02
oxygen
Fe3+
ferric
Cu2+
cuprous
H02,
hydroperoxyl
H2O
water
H'
hydrogen
Fe 2+
ferrous
CH2
methylene
R'
alkyl
RO'
alkoxyl
radical
ROOH
organic
hydroperoxides
ROH
organic
hydroxyl
RH
organic
substrate
%
percentage
ion
ion
or perhydroxyl
ions
ion
group
radical
xvii
CHAPTER
ONE
INTRODUCTION
1.1
Introduction
The
sago
of sago
industry
could
body
sago
properties
from
be a rich
free
in
is to
the
source
the sago
it a golden
colour
damage
can
investigate
the
starch
various
antioxidant
in the
of the
properties
search
for
a
natural,
abundant
many
studies
have
identified
are
which
and
cheap
is rich in
process
to
byproduct
this
systems
antioxidant
On exposure
colour.
compounds
production
the
example,
extraction
Hence,
rapidly.
for the
studied
for
brownish
of antioxidant
that
thoroughly
components,
from
washing
purple
radicals
has been
of other
give
a deep
to
research
pith
The
that
compounds
changes
of this
the
as "Rumbia",
are less known.
antioxidant
the
but
starch,
capacities
liquid
also known
tree,
the
of
known
body.
source
of
sago
to protect
The
aqueous
the
air,
purpose
extract
of
antioxidant
compounds.
To
cardiovascular
(Harada
factors
radicals
in lipid
diseases
et al. 2003;
that
Nicolesu
pulmonary
as side-stream
are
peroxidation
be
to
to
contributing
degenerative
known
date,
prevented
et al. 2001)
increasing
the
and
treated
and
smoke
in biological
in the various
the
incidence
cardiovascular
cigarette
generated
or
(SSCS)
systems
antioxidant
with
list
grows
and
diseases
of
cigarette
(Zhang
cause
conditions
various
smoke
et al. 2001).
oxidative
and
supplementation
continuously.
severity
is
inhalation
can
health
various
damage,
One
of the
cancers,
inhalation,
The
free
resulting
organs.
I
The
present
(negative
group)
controls)
and
or ascorbic
acid
cigarette
to increase
of free
radical
antioxidants
exposed
combined
action
Hence,
will
to
antioxidant
properties,
activities
Nakatani,
(Decker
& Welch,
capacity
decolorization
Further
establish
are
1990;
of
studies
evaluated
et
(Cano
as they
is that
of these
in
role
carried
radical
out
Metroxylon
sago
al.
as
the ferric
2003),
1999)
were
provide
levels
investigated
enzymatic
as
nonthe
via
enzymes.
was
(FTC)
thiocyanate
Yu et a!. 2004),
and metal
the absorbance
Assays
on chelating
activity.
the
SAE
compound
on
the
done
were
and based
information
and
smokers
(SAE)
by the
of the chemical
Supplementation
is indeed
well
by reading
on
reduce
if this
approaches;
out
and
out
different
carried
enzymes
true.
The
for
assays.
its
The
(Kikuzaki
method
2,2'-azinobis-(3-ethylbenzothiazoline-6-
Spectrophotometer.
radicals
radical
radical
scavenging
find
to
chemical
using
free
as side-stream
free
the
elevated
diet
elevated
enzymes.
protecting
from
such
scavenging
activities
et al. 1998,
Yen & Wu,
nature
approach
risk
various
of SAE via
the chemical
important
were
a UV/Vis
of ABTS
this
as free
as well
Rahmat
assay)
to stress
basal
SAE (experimental
with
exposed
in the
changes
fed on normal
mice
supplemented
free-radical
of the
the
when
for
reducing
using
were
using
wavelengths
antioxidant
by
to investigate
between
diet
a beneficial
extract
1993;
(ABTS
sulphonate)
have
investigations
aqueous
controls)
in lowered
result
SSCS
basal
on
activities
designed
enzymes
rationale
of antioxidants
freeze-dried
antioxidant
The
might
smokers
&
(positive
lead
levels
fed
those
(SSCS).
will
been
scavenging
smoke
levels
with
free-radical
of the
activities
has also
research
using
inhibition
HPLC,
in the extract.
presence
or
chelating
at different
values
to
activity
measure
the
of peroxidation,
FTIR
and
Those
absence
NMR
to
techniques
of
particular
I
functional
groups
the quantity
Brine
concentration
Shrimp
control
for
50%
mortality
into
the
SAE or ascorbic
At the
the
of
end
tissues
the
(Marklund
(GSH-Px)
of the
based
on the
& Marklund,
Objectives
The
main
objectives
To
extract
To assess
CAT)
with
1974),
of the
forty-nine
different
the
or carbon
nearby
nuclei.
to measure
by
male
diets
the
determining
to SAE, known
brain)
and
fed
to them,
atoms,
toxicity
of
the
lethal
as the
acute
killed
were
(Aebi,
and
and
for eight
for
and
weeks.
dislocation
used
superoxide
1974)
pellet
comprising
by cervical
isolated
were
sp. ) were
musculus
(SSCS)
smoke
enzymes,
(CAT)
catalase
(Mus
mice
cigarette
mice
radical-scavenging
& Valentine,
activities
(ii)
free
of
kidney
used
proton
of exposure).
period,
liver,
lung,
was
of exposure
to side-stream
exposed
experimental
activities
1.2
(i)
trials,
(mainly
(Paglia
24 hours
between
dichromate)
six hours
after
experimental
acid and
1993)
(potassium
LC50 (after
six groups,
of linkages
(Sam,
Test
of the
environment
and nature
Lethality
the
LC50 and the chronic
As for
electronic
present
to
relative
divided
symmetry,
of protons
The
SAE
and
and
assessment
dismutase
(SOD)
peroxidase
glutathione
1967).
Research
of this
research
the
Metroxylon
of SAE using
the activities
in the mice
SAE when
are: sagu
three
different
of free
fed on normal
exposed
components
chemical
radical-scavenging
basal
to side-stream
and
determine
the
assays.
enzymes
(SOD,
diet and mice fed on diet
cigarette
antioxidant
GSH-Px
and
supplemented
smoke.
3
CHAPTER
TWO
LITERATURE
2.1
Radicals
Free
Free radicals
are unstable
These
things.
that,
have
They
(Bagchi
reactive
& Puri,
free
"A
electron",
the
needed
also
free
production
is not tightly
level
antioxidants
radicals
of oxidative
in our
body
can
thus
controlled.
damage
system
quickly
a free
creating
in normal
of destructive
highly
caused
stress
by the
(Rice-Evans
processes
and
valence
existence".
to capture
which
free
processes,
and damaged
cells
and they
stress"
describing
the
can
radicals
& Burdon,
"oxidative
between
that
species
reactions
(Rice-Evans
stated
unpaired
compounds
is a situation
imbalance
& Burdon,
an
chain
cause
(1993)
as "any
biological
bacteria
reactive
Oxidative
radical
(Ahmad,
intermediates
Borg
with
other
normal
environment
of independent
with
during
reactive
radicals
and is incapable
react
body
electrons.
free
in living
ubiquitously
the
highly
fragment
define
in biochemical
become
from
unpaired
molecular
However,
removal
molecules
Therefore,
state
and tissues.
role in the
act as regulatory
they
2000)
without
occur
human
the
them
makes
(1995)
electrons
instability,
(Jakus,
to cells
al.
in
be introduced
which
or
that
molecules
generated
to those
et
unpaired
of their
play an important
electrons
Halliwell
electron
be destructive
are
is a molecule
one or more
reactive
can also
compared
radical
Because
they
unpaired
whereas
contains
but
1998)
highly
and
compounds
functions
physiological
1995).
REVIEW
1994).
if the
the steady
prooxidants
and
1994).
4
2.1.1
Types
of Free
Free radicals
are often
Oxygen
Reactive
superoxide
odd
(ROO'),
radical
nitric
oxide
02' - is a small
in the
inner
and
xanthine
membrane
oxidase
important
of
1&
of the
vivo (Sanchez-Moreno,
superoxide
reacts
rapidly
with
a radical
(O2'
radical
that
with
nitric
by reducing
hydrogen
peroxide
from
and
the transition
in Fenton
02
+
NADPH
+
peroxyl
them
is the
enzyme
called
of
these
pathological
may
ON00',
same
also
species.
reactive
time,
& Burdon,
are
radicals
in
processes
the
produce
Superoxide
a potent
agent
02' - triggers
Fe 3+ or Cue+) which
(either
is produced
then
of
HO'
react
1994).
oxidase
º
H+
At the
(Rice-Evans
reactions
02
+
ions
metal
other
peroxynitrite
et al. 1997).
One
anion,
complexes
for
that
dehydrogenase
of several
of metal
to produce
1994)
pathways.
by the
propagation
a precursor
(NO')
& Burdon,
to superoxide
oxygen
Autoxidation
oxide
(HO'),
radical
the
acid (HOCI).
various
to uric acid,
Xanthine
Xanthine/Hypoxanthine
(Rice-Evans
2). By reducing
becomes
are
-)
mitochondria
2002).
ROS
as
to the
refers
Major
anion.
known
are also
paragraph
hydroxyl
and hypochlorous
anion
initiation
(H202),
peroxide
(NO')
in vitro (Leeuwenburgh
LDL oxidation
production
(-)
or hypoxanthine
(Eq.
sources
while
non-polarizable
of xanthine
oxidation
(') in the following
means
They
cells.
notation
radical
Radical
in eukaryotic
The
hydrogen
-),
Superoxide
2.1.1.1
(ROS).
electron
(0z'
anion
by oxygen
generated
Species
remaining
single
Radicals
ý
Uric acid
02' -+
+
02'-
NADP+
(Eq.
1)
(Eq.
2)
5
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