PUMS 99:1 UNlVERSITI MALAYSIA SABAH

---------------------------------------------------------------------------------

SESI PENGAJlAN: ,:) 00 6/ .:l 0 I 0

(HURUF BESAR)

tl:lengaku membenarkan tesis (LPSI Srujana/ Doktor Falsafah) ini di simpan di Perpustakaan Universiti Malaysia Sabah :iengan syarat-syarat kegunaan seperti berikut:

1. Tesis adalah hakmilik Universiti Malaysia Sabah. 2. Perpustakaan Universiti Malaysia Sabah dibenarkan membuat salinan untuk tujuan pengajian sahaja. 3. Perpustakaan dibenarkan membuat salinan tesis ini sebagai bahan pertukaran antara institusi pengajian tinggi. 4. ** Sila tandakan (I)

SULIT

TERHAD

TIDAK TERHAD

GAN PENULIS)

f:Jo..1fA 4)

~ WlOh 1:>0.1 a... , S.J 10-0 ke..ro~)

1$.<.J.o...lo. L\J.Wl r u..v-

\tikh: .d l / 5 / ~U1 0 I

l' -AT AN: * Potong yang tidak berkenaan.

(Mengandungi maklumat yang berdarjah keseJamatan . atau kepentingan Malaysia seperti yang termaktub di dalam AKT A RAHSIA RASMI 1972)

(Mengandungi maldumat ffiRHAD yang teJah ditentukakan oleh organisasilbadan di mana penyelidikan dijalankan)

Disahkan oleh

Nama Penyelia

Tarikh: .) 1 I 5 / .;;w / 0 . i

* Jika tesis ini SULIT atau TERHAD. sila lampiran surat daripada pihak berkuasalorgansasi berkenaan dengan menyatakan sekali sebab dan tempoh tesis ini perlu dikelaskan sebagai SULIT dan TERHAD.

* Tesis dimaksudkan sebagai tesis bagi Ijazah Doktor Falsafah dan Sarjana secara penyelidikan. atau disertasi bagi pengajian secara kerja kursus dan penyelidikan, atau Laporan Projek Sarjana Muda (LPSM).

ANTIOXIDANT ACTIVITIES, TOTAL PHENOLIC AND TOTAL FLAVONOID CONTENT OF THREE

SABAH TROPICAL SEAWEEDS EXTRACTS

OON XIN YAN

THESIS SUBMITTED IN FULFILLMENT FOR THE DEGREE OF FOOD SCIENCE WITH

HONORS IN FOOD SCIENCE AND NUTRITION

SCHOOL OF FOOD SCIENCE AND NUTRITION UNIVERSITY MALAYSIA SABAH

2010

DECLARATION

I hereby declare that the material in this thesis is my own except for quotations, summaries and references, which have been duly acknowledged.

8 April 2010

ii

NAME

MATRIC NO.

TITLE

DEGREE

OATE OFVlVA

1. SUPERVISOR

VERIFICATION

: OONXINYAN

: HN2006-3732

: ANTIOXIDANT ACTIVmES, TOTAL PHENOUC AND TOTAL FLAVONOID CONTENT OF THREE SABAH TROPICAL SEAWEEDS EXTRACTS

: BACHELOR OF FOOD SCIENCE HONORS

: 14 MAY 2010

CERTIFIED BY

Signature

(DR. PATRICIA MATANJUN) ~. 2. EXAMINER 1 (DR. MUHAMMAD IQBAL HASHIM I) 41if£dL, 3. EXAMINER 2 (PROF. MADYA DR. SHARIFUDIN MD. SHAARANI)

4. DEAN (PROF. MADYA DR. MOHO ISMAIL ABDULAH)

/' n_ '--' .~.

iii

ACKNOWLEDGEMENT

I would like to take this opportunity to send my greatest gratitude and appreciation to my supervisor, Dr. Patricia Matanjun on her guidance, advices and supports throughout the whole progress of my thesis. Great appreciation and gracefulness I would like to express toward Dr. Patricia on her patient and kindness on guiding me whenever I was doubt during this period.

Besides, a special thanks to Dr. Normawaty, from School of Marine Sdence that provided me some useful information and guidance in identifying my seaweed sample.

Moreover, I would like to thanks to those senior in SSMP, especially to Wong Jin Vi, Chia Kah Fei and Tin Hoe Sheng in providing me guidance and good explanation upon my confusion and doubt in conducting my experiment and teaching me on the way to operate some instruments. With their guidance and useful advises, I able to finish my experiments with least obstacles.

Further, I would like to send my appreciation toward all the lab assistance in SSMP. especially Pn. Zalnab, Encik Taipin and Cik Ireen that gave me plenty of assistances in getting instruments, equipments and chemicals. Besides, special thanks to Pn. Lucy that willing to extend her working hours even without payment sometimes to responsible on our safety in the lab.

Special thanks to all my friends that willing to give me a hand when I am facing frustration and failure during my experiment and provide me guidance and accompanies during this period. Besides never forget to thanks my beloved family members that on their concern and support throughout the days of my thesis progress.

Lastly, I shall thanks to everyone that aid me during this period with their name not mentioned above.

Oon Xin Van 15 APRIL 2010

iv

ABSTRACT

ANTIOXIDANT ACTIVITIES, TOTAL PHENOLIC AND TOTAL FLAVONOID CONTENT Of THREE SA BAH TROPICAL SEAWEEDS EXTRACTS

This study was to investigate the antioxidant activities together with total phenolic and total flavonoids content of three tropical seaweeds (Sargassum polycystum, Caulerpa lentillifera and Kappaphycus alvaerezzl) in different solvent extracts (water, ethanol, methanol, acetone, chloroform, diethyl ether and hexane). All determinations were done spectrophotometrically. Antioxidant activities were investigated by DPPH radical. scavenging activities, FRAP and beta-carotene bleaching assays. Among seven extractants, methanol shown the best extraction efficacy in S. po/ycystum whereas ethanol gave the best result in K. a/varezzi and C. /entlllifera In DPPH radical scavenging actlvlti.es, reducing power and total phenolic content. Diethyl ether worked as the best solvent in beta carotene bleaching assay for three seaweeds species. Methanollc extracts of brown, red and green seaweeds obtained highest total fl.avonoids content among all solvents. Based on the best solvent extracts, K. alvaerezzi possessed highest radical scavenging activities in DPPH assay and beta carotene bleaching assay, yet S. po/ycystum showed the greatest reducing power among three seaweeds species. Highest TPC was obtained by methanolic extract of S. polycystum and highest TFC however owned by

ethanolic extracts of K. a/vaerezzL C lentillifera generally has lowest antioxidant activities among three species with respect to their best solvent. Whereas K. alvaerezzi, can be concluded as species with highest antioxidant activities since it possessed highest activities in most tests among three species.

v

ABSTRAK

Tesis ini bertujuan untuk mengkaji aktivlti antioxidant bersama dengan jum/ah kandungan fenolik dan flavonoid bagl tiga jenls rumpai laut (5. polycystum, Caulerpa lentillifera and Kappaphycus a/vaerezzi) da/am bent uk ekstrak pelbagai jenis pelarut (air, etano~ metano~ aceton, kloroform, dietil ether dan heksana). Aktiviti antioxidant dalam rumapi laut dlkaji melalul kaedah DPPH, FRAP dan pelunturan beta carotene (BCB). Antara tujuh pelarut, ekstrak metanol merupakan pe/arut yang paling cekap bagi S. po/ycystum, manaka/a, ekstrak etano/ paling berkesan d/ C. /entillifera dan K. a/vaerezzi dalam kajian DPPH, FRAP dan jumlah kandungan fenollk. Sela/n ftu, ekstrak dletll ether paling berkesan bagl ketiga-tlga jenis rumpal /aut da/am kajian BCB. Ekstrak metanol menunjukkan kandugan flavonoid yang paling tinggi da/am rumpai /aut k/ass merah, hijau dan perang da/am ekperimen inl. Bergantung kepada pe/arut yang paling berkesan, K. alvaerezzi menunjukkan aktiviti yang paling balk dalam kajlan DPPH dan BCB, manaka/a S. po/ycystum memberi kuasa penurunan yang paling tinggi antara tiga spesis. Kandungan feno/ik yang paling tinggl dikesan di ekstrak metanal bagi S. polycystum, manakala kandungan flavonoid paling tinggi di ekstrak etanol bagi K. alvaerezzi. Antara semua, C. /entfllifera menunjukkan aktiviti antioxidan yang paling rendah dalam pelarut yang paling berkesan dalam semua kajian. Manakala, K. a/vaerezzi secara kesimpulan merupakan species yang mempunyai aktiviti antioxidant yang tertinggl memandangkan spesis ini menunjukkan aktiviti yang paling tlnngi di kebanyakan ujian antara tiga spesis.

vi

TABLE OF CONTENT

TITLE

DECLARATION

VERIFICATION

ACKNOLEDGEMENT

ABSTRACT

ABSTRAK

TABLE OF CONTENT

LIST OF TABLE

LIST OF FIGURE

LIST OF ABBREVIATION

LIST OF SYMBOL

LIST OF FORMULA

LIST OF APPENDIX

CHAPTER 1 INTRODUCTION

1.1 Background Of Study 1.2 0 bjectives

CHAPTER 2 LITERATURE REVIEW

Pages

ii

iii

iv

v

vi

vii

x

xi

xii

xiii

xiv

xv

1 7

2.1 Free Radicals and Reactive Oxygen Species (ROH) 8 2.2 Lipid Oxidation 10 2.3 Antioxidant 13

2.3.1 Synthetic Antioxidant 15 2.3.2 Natural Antioxidant 16

2.4 Factor Affecting the Antioxidant Activity 23

vii

2.5 Classification of Seaweed 23 2.5.1 Phaeophycophyta 24 2.5.2 Rhodophyceae 26 2.5.3 Chlorophyta 29

2.6 Importance of Seaweed 31 2.7 Seaweed in Malaysia 33 2.8 Seaweed Antioxidant 35

2.8.1 Phenolic Compound in Seaweed 37 2.8.2 Phenolic Compounds as Free Radical 38

Scavenger and Metal Chelator 2.8.3 Carotenoids in Seaweed 39 2.8.4 Carotenoids as Singlet Oxygen Quencher 40

and Radical Scavenger 2.9 Antioxidant activities assays 41

CHAPTER] MATERIALS AND METHODS

3.1 Materials 43 3.1.1 Instruments 43 3.1.2 Laboratory Apparatus 43 3.1.3 Chemicals and Reagents 44

3.2 Methods 45 3.2.1 Sample Collection and Preparation 45 3.2.2 Sample Extraction 45 3.2.3 DPPH Radical Scavenging Assay 46 3.2.4 Reducing Power Assay 46 3.2.5 Beta Carotene Bleaching (BCB) Assay 47 3.2.6 Total Phenolic Content 47 3.2.7 T otaJ Flavonoids Content 48

3.3 Statistical Analysis 48

CHAPTER 4 RESULT AND DISCUSSION

4.1 Extraction Yield 49 4.2 Antioxidant Activity 54

4.2.1 DPPH radical scavenging activity 54 4.2.2 Reducing Power 60 4.2.3 ~-carotene Bleaching Assay 65

4.3 Total Phenolic Content (TPC) 71 4.4 Total Flavonoids Content (TFC) 77

CHAPTERS CONCLUSION

5.1 Conclusion 81 5.2 Suggestion 83

viii

REFERENCES 84

APPENDIX 100

ix

LIST OF TABLE

Page

Table 2.1 Mechanism of antioxidant activity in different antioxidant classes 15

Table 2.2 Classification of Sargassum polycystum 27

Table 2.3 Classification of Kappaphycus alvarezi! 30

Table 2.4 Classification of Caulerpa lent/l/lrera 33

Table 4.1 Extraction yield of different solvents from S. polycystum , 49 Caulerpa lentil/irera, Kappaphycus alvaerezzi

Table 4.2 DPPH radical scavenging effect C%) of S. polycystum, Caulerpa lentil/irera, Kappaphycus alvaerezzl crude extracts 54 from different solvents

Table 4.3 Reducing power of S. polycystum , Caulerpa lentil/irera, 59 Kappaphycus alvaerezzl crude extracts from different solvents

Table 4.4 Beta carotene bleaching assay of S. polycystum, Caulerpa lentil/ifera, Kappaphycus alvaerezzi crude extracts from different 63 solvents

Table 4.5 Total phenolics content of S. polycystum, Caulerpa lentil/irera, 69 Kappaphycus alvaerezzi crude extracts from different solvents

Table 4.6 Total f1avonoids content of S. polycystum, Caulerpa lentillifera, 75 Kappaphycus alvaerezzi crude extracts from different solvents

x

-LIST OF FIGURE

Page

Figure 2.1 Mechanism of lipid autoxidation 13

Figure 2.2 Chemical Structure of BHA 16

Figure 2.3 Chemical structure of BHT 17

Figure 2.4 Chemical structure of TBHQ 17

Figure 2.5 Basic structure of flavonoid 20

Figure 2.6 Chemical structure of f1avonols 21

Figure 2.7 Chemical structure of f1avones 21

Figure 2.8 Chemical structure of f1avanones 22

Figure 2.9 Chemical structure of flavan-3-0Is 22

Figure 2.10 Chemical structure of anthocyanidins 23

Figure 2.11 Chemicals structure of Carotenoids 24

Figure 2.12 Sargassum polycystum C. Agardh 28

Figure 2.13 Kappaphycus alvarezzi (Doty) Doty ex P.C. Silva 31

Figure 2.14 Caulerpa lentillifera J. Agardh 33 . Figure 2.15 Reaction of antioxidants with radicals 41

xi

ROS

O2- ­

H02-

H20 2

OW 102

PUFA

Sens

ROOR

DPPH

FRAP

SCB

TPC

TFC

FeCb

Na2C03

mgPGE/g

mgQE/g

LIST OF ABBREVIATION

Reactive oxygen species

Superoxide anion

Perhydroxyl ·

hydrogen peroxide

Reactive hydroxyl radical

Singlet oxygen

Polyunsaturated fatty acid

Free radical

Sensitizer

Peroxyl

Metal ion

Triplet oxygen

Caroteniod radical

1, 1-diphenyl-2-picrylhydrazol

Ferric reducing antioxidant power

Beta-carotene bleaching

Total phenolic content

Total flavonoid content

Ferum (III) chloride

Sodium carbonate

milligram phloroglucinol acid equivalent per gram of extract

milligram quercetin equivalent per gram of extract

xii

LIST OF SYMBOL

"C Degree Celsius

~ Beta

j.Jm micrometer

m/z mass per charge

V gama

nm nanometer

ppm part per million

g gram

% percent

ml milliliter

gIL gram of solute in 1 liter of solution

M Mol

xiii

LIST OF FORMULA

Formula 3.1 Percentage of scavenging effect

Formula 3.2 Antioxidant activities

Formula 3.3 Total phenolic content In mgPGE/g of dry extract

xiv

Page

46

47

48

LIST OF APPENDIX

Page

Appendix A One way ANOVA for S. polycystum in DPPH in 101 different solvents

Appendix B One way ANOVA for S. polycystum in reducing power 102 in different solvents

Appendix C One way ANOVA for S. polycystum in BCB in 103 different solvents

Appendix D One way ANOVA for S. polycystum in TPC in 104 different solvents

Appendix E One way ANOVA for S. polycystum in TFC in 105 different solvents

Appendix F One way ANOVA for C. lentillifera in DPPH in 106 different solvents

Appendix G One way ANOVA for C. lentillifera in reducing power in 107 different solvents

Appendix H One way ANOVA for C. lentillifera in BCB in 108 different solvents

Appendix I One way ANOVA for C. lentillifera in TPC in 109 different solvents

Appendix J One way ANOVA for C. lentillifera in TFC in 110 different solvents

Appendix K One way ANOVA for K.alvaerezziin DPPH in 111 different solvents

Appendix L One way ANOVA for K.alvaerezziln reducing power in 112 different solvents

Appendix M One way ANOVA for K.alvaerezziin BCB in 113 different solvents

Appendix N One way ANOVA for K.alvaerezziin TPC in 114 different solvents

Appendix 0 One way ANOVA for K.alvaerezziin TFC in 115 different solvents

Appendix P DPPH in different solvent between Sargassum.spp, 116 C. lentillifera, K. alvaerezzi

xv

Appendix Q Reducing power in different solvent between 118 Sargassum.spp, C. lenti//ifera, K. alvaerezzi

Appendix R BCB in different solvent between Sargassum.spp, 120 C. lenti//ifera, K. a/vaerezzi

Appendix 5 TPC in different solvent between Sargassum.spp, 122 C. lenti//ifera, K. a/vaerezzi

Appendix T TFC in different solvent between Sargassum.spp, 124 C. lenti//ifera, K. a/vaerezzi

Appendix U Phloroglucinol standard curve for TPC and FRAP 126

Appendix V Quercetin standard curve for TFC 127

Appendix W Extraction yield and DPPH assay 128

Appendix X Ferric reducing power assay and B-carotene bleaching 129 assay

Appendix Y Total phenolic content and Total flavonoid content 130

xvi

CHAPTER 1

INTRODUCTION

1.1 Background of Study

Plant kingdom is generally divided into two different groups according to their living

platform: terrestrial or marine. As refer to marine plant, seaweeds or marine

macroalgae play the most important role in industry and received the great

attention as they are the potential renewable resource in the marine environment.

About 6000 species of seaweeds have been identified and are grouped into

different classes viz., green (Chlorophytes), brown (Pheophytes) and red

(Rhodophytes) algae (Chandini et ill., 2007).

Since ancient time, seaweeds have been used as food, fodder and fertilizer

and as sources of medicinal drugs (Sanchez-Machado et ill., 2004). In this era,

seaweeds are the raw material for global industrial production of agar, carrageenan

and alginates (McHugh, 2003), but as well continue being used in food diet, as well

as traditional remedies in Asian countries, such as in China, Japan and Korea

(Wang, 2009). Besides, seaweed also commonly being consumed in some western

country especially in Hawaii (McDermid and Stuercke, 2003) and recently has gain

their popularity among French as food or condiment (Mabeau and Fleurence, 1993).

A more recent statistical report from McHugh (2003), the seaweed industry

provides a wide variety of products that have an estimated total annual value of

US$ 5.5-6 billion. Food products for human consumption contribute about US$ 5

billiion of this. Substances extracted from seaweed's hydrocolloids account for a

large part of the remaining billion dollars, while smaller, miscellaneous uses, such

as fertilizers and animal feed additives, .make up the rest while the total global

seaweed production in the year 2004 was reported to be more than 15 million

metric tons (FAD, 2006).

Burtin (2003) has Indicated that In Asian countries, Japanese are the main

consumers of seaweed with an average of 1.4 kg (dry weight) per capita. About 25%

of all food consumed In Japan consists of seaweed prepared and served in many

forms and has become the main source of income for the fishermen. The most

popular food product from seaweed Is Haba Nori which its origin is from Petalonia

binghamiae, a brown algae as well as Nori, a red algae from Porphyra spp (Kuda et

al., 2005). In contrast, seaweed as a food In Malaysia is not as common as in

countries like Japan and China. Certain red seaweed is consumed in certain coastal

areas especially along the east coast of Peninsula Malaysia and in East Malaysia,

where it is occasionally eaten as a salad dish (Norziah and Ching, 2000).

Study has shown that seaweeds offer an excellent source of nutritious

compounds such as carotenoids, dietary fibre, protein, essential fatty acids,

vitamins and minerals (Mabeau and Fleurence, 1993). In addition, some of the

seaweeds are considered to be a rich source of antioxidants (Urn et al., 2002). For

example, some antioxidants compounds that's are structurally similar to plant

derived antioxidant such as chlorophylls, carotenoids, tocopherol derivatives such

as vitamin E, and related isoprenoids were found to be present in some marine

organisms (Takamatsu et al., 2003).

Lately, a great awareness has been turn towards the anti-tumor activity,

anticholesterolemic activity and antioxidant activity of seaweed constituents which

has lead to the blooming demand of seaweeds from the pharmaceutical industry

other than only the industry of seaweed hydrocolloids extraction (Heo et al., 2005).

The antioxidants activity of macroalgae is the main focus of pharmaceutical

industry where there is interest in anti-aging and anticarcinogenic natural bioactive

compounds, which possess health benefits (Heo et al., 2005).

Reactive oxygen species (ROS) such as superoxide anion (02-), hydroxyl

radical (HO·) and hydrogen peroxide (H202) are physiological metabolites formed

during aerobic life as a result of the metabolism of oxygen (Heo et al., 2005).

Consequently, the excessive amounts of ROS may be harmful to living organism

because they can initiate biomolecular oxidations which lead to cell injury and

2

death, and create oxidative stress (Chew et aI., 2007). ROS and oxidative stress

have been associated with the onset of a variety of chronic disease states in

humans, since DNA, cell membranes, proteins and other cellular constituents are

target site of the degradation processes where the ROS attack (Heo et aI., 2005).

The chronic diseases induced by ROS include coronary heart disease (CHO),

certain cancers, rheumatoid arthritis, muscular dystrophy, cataracts (Heo et al.,

2005), diabetes, retinopathy of prematurity, chronic inflammatory disease of the

gastrointestinal tract, as well as diseases associated with cartilage (Chauhan and

Chauhan, 2006), Alzheimer's disease (Chauhan and Chauhan, 2006), other

neurological disorders and the ageing process (Temple, 2000). On the other hand,

lipid oxidation by ROS will cause a decrease in nutritional value of lipids, as well as

reduce their safety and appearance (Kumar et al., 2008). Moreover, ROS are

predomrnant cause of qualitatrve decay of foods, which I.ead to rancidity, toxicity

and destruction of biomolecules important in physiologic metabolism (Heo et a/.,

2005).

In order to prevent the damages from ROS, complex system of antioxidant

enzymes is crucial for all living organism. For example, the thioredoxin system, are

conserved throughout evolution and are required for life. Antioxidants in biological

systems of living organism serve multiple functions, included the defending against

oxidative damage and participating in the major Signaling pathways of cells. One

major function of antioxidants in cells is to prevent damage caused by the action of

ROS (Kumar et a/., 2007).

Antioxidants are classified by the products they form during oxidation where,

these can be antioxidants themselves, inert, work with other antiOXidants as

synergist or even pro-oxidant in some cases, depend on what they can cause to the

oxidation products. In some circumstances where the oxygen pressure is high,

carotenoids may act as pro-oxidant (Jorgensen and Skibsted, 1993). Moreover, the

antioxidant may be regenerated by different antioxidants or, in the case of

"sacrifidal" antioxidants where its oxidized form may be broken down by the

organism.

3

Several synthetic antioxidants, such as butylated hydroxyanisole (BHA),

butylated hydroxytoluene (BHT) and tert-butylhydroquinone (TBHQ), are

commercially available and used as food antioxidants due to the lower cost and

more readily available characteristic. However, the usage of synthetic antioxidants

has been controlled in foods as they might possess adverse effect toward human

health (Wanasundra and Shahldi, 1998). In addition, some toxicological studies

have also concerned the use of these synthetic antioxidants in promoting the

development of cancerous ·cells in rats (Kumar et al., 2008). Thus, the development

of natural antioxidants to substitute these synthetic products has now received a

great pressure and much effort has been contributed in order to fulfill the

customer's demand (Kumar et aI., 2008).

According to Porkorny (1991),. the use af natural antioxidants Is considered

no harm to consumer because they contain no chemical contamination, is readily

acceptable and no safety test are required by the legislation since these food

components are Generally Recognized As Safe (GRAS). Therefore, a general trend

of replacing the synthetic antioxidants in food processing by natural oxidation

inhibitor or by the preferential use of ingredients that naturally acquire antioxidant

activity is established (Pokorny, 1991). Thus we can conclude that the antioxidants

are essentially desired in our body system and as well to extend the shelf life of

food because the intake of antioxidants or addition of antioxidant in food material

able to protect the body as well as foods against the damage by ROS (Ganesan et

al., 2008).

Plants are perceived to be rich in antioxidant because almost all

photosynthesizing plants including seaweeds are exposed to a combination of light

and high oxygen concentrations and consequently lead to the formation of free

radicals and other strong oxidizing agents·, However, they seldom suffer any serious

photodynamic damage during metabolism (Heo et aI., 2004). The absence of

structural damage In the seaweed leads to consider that these organisms are able

to generate the necessary compounds to protect themselves against oxidative

stress including UV radiatJon suggesting that. seaweeds possess antloxidative

mechanisms related to bioactive compounds (Heo and Jeon, 2009). Therefore,

4

seaweeds can be concluded to be a rich source of antioxidants. Recently, the

potential antioxidant compounds in seaweeds were identified as some pigments

such as fucoxanthin, astaxanthin, carotenoid (ran et aI., 1999) and polyphenols

such as phenolic acid, flavonoid, tannins (Heo et aI., 2004). Those compounds are

widely distributed in plants or seaweeds and are known to exhibit high antioxidative

activities. The activities have been reported through various methods of ROS

scavenging activity and the inhibition of lipid peroxidation (Van et aI., 1999;

Ganesan et al., 200,7; Chew et al." 20,07; Heo at el., 2004; Matanjun et al., 2008).

The most common natural antioxidants derived from plant could be referred

to phenoliC compounds where they are widely distributed among the plant kingdom

(Duan et aI., 2006). Subsequently, phenolic compounds In many cases are as well

claimed to be the major active constituent that function for the antioxidant activity

of marine algae (Shahldi and Zhong, 2007). Diverse groups of phenolic compounds

range from simple phenolics, phenolic acids, anthocyanins, hydroxycinnamic acid

derivatives, to f1avonoids are found in various plants (Bandoniene and Murkovic,

2002). Phenolic antioxidants function as free radical scavengers, redudng agent

and as welL work as metal chelator and therefore can act to inhibit lipid oxidation

(Shahidi and Zhong, 2007). The beneficial health effect of consuming plant foods

due to the presence of phenolic compounds are associated with counteracting the

risk of cardiovascular disease (CVD), cancer and cataract as well as a number of

other degenerative diseases. These are achieved by preventing lipid oxidation,

protein cross linking and DNA mutation and, at later stages, tissue damage (Shahidi

and Naczk, 2004).

Other than phenol compounds, carotenoids also possess a powerful

antioxidative characteristic. The correlation between a diet rich in carotenoids and a

diminishing risk of CVD. cancers, by ~-carotene and Iycopene, as well as

opthalmological diseases by lutein and zeaxanthin has been demonstrated in recent

studies (Burtin, 2003). Brown seaweeds are particularly rich in carotenoids

especially in fucoxanthin (Yan et al., 1999), ~-carotene, violaxanthin (Burtin, 2003).

Moreover, fucoxanthin contributes >10,% of the estimated total production of

carotenoids in nature (Jensen, 1998). On the other hand, the main carotenoids in

5

the red algae are the ~-carotene and a-carotene and their dihydroxylated

derivatives: zeaxanthin and lutein. Whereas, the carotenoid composition of the

green algae is similar to that of higher plants: the main carotenoids present are the

~-carotene, lutein, violaxanth[n, antheraxanthin, zeaxanthin and neoxanthin (Burtin,

2003).

Commercial use of seaweed in Malaysia recently has obtained an increasing

interest especially for Eucheuma, Kappaphycus and Graci/aria (Phang, 2006).

Among the Malaysian seaweeds, only Kappaphycus is cultivated in Sabah for the

extraction of semi-refined carrageenan by fishing families around Semporna which

placed at East Cost Sabah by using monofilament techniques In the reefs fringing

the island near Semporna (Phang, 2006). Several seaweeds such as the

Rhodophytes Graci/aria changll, Graef/aria tenu/spitata, Eucheuma (Kappaphycus)

species and the Chlorophytes CauJerpa /entillifera and Cau/erpa raeemosa are

collected and eaten as raw or blanced in salad by Malaysian especially by the

fishing and coastal communities. However, seaweed as a food in Malaysia is not as

common as in countries like Japan and China (Norziah and Ching, 1999). Recently,

pharmaceutical firms have switched their focus towards marine organism, including

seaweeds to be developed into new drug from natural products (Smit, 2004). Ever

since edible seaweed can post various beneficial functions to our human body and

recently has gained much attention by pharmaceutical sector, it will be a great lost

if the use of some edible seaweed in Sabah's water is only for the extraction of

carrageenan.

Since edible seaweeds possessed various beneficial functions to our body

and the usage of natural seaweed as. human food or supplement are still under

develop, study on the potential antioxidants in the local edible seaweed

(Kappaphycus a/varezz~ Cau/erpa lentilliferal Sargassum po/ycystum) in Sabah can

thus increase their usage in pharmaceutical and nutraceutical sector. Besides,

pharmaceutical sector can as well afford the higher cost to freeze dry these

seaweeds as to preserve their antioxidant compounds. In this study, water is

included in types of extraction since It is commonly used for food preparation

therefore the study of antioxidant compounds in water is vital in this case.

6

Several antioxidant properties assay on edible seaweeds in various coastal

areas had been carried out on various green, red and brown seaweeds on

antioxidant activities (ADA), total phenolic content (TPC) and reducing power. For

example, study done by Yuan and Walse (2006) on brown seaweed (kelp) and red

dulse in temperate water area has shown that red dulse contain higher reducing

power and total phenolic content than kelp In their methanolic extracts. Whereas

for seaweeds from tropical area, Padina antil/arum, Cau/erpa racemosa and

Kappaphycus a/varezzi were investigated for their TPC and antioxidant activities

using DPPH radical scavenging assay, ferric reducing power assay and beta

carotene bleaching (BCB) assay (Chew et aI., 2008). This study demonstrated that,

brown algae P. anti/arum possessed highest TPC, reducing power and scavenging

ability compared to two other species and the antioxidant activiti.es as assayed by

BCB were equivalently high (Chew et aI., 2008).

Besides, Matanjun et al. (2008) had conducted some research on

antioxidant activities in Sabah's edible seaweeds and found that green seaweeds C

/entillifera, C racemosa and brown seaweed S. po/ycystum possessed greater

scavenging and redudng power ability as well as TPC compared to red seaweed

(Matanjun et a/., 2008). However, there is no study on antioxidant activities on the

freeze dried seaweed with extracts from different solvent and water extraction from

Sabah's water.

1.2 Objectives

1. To investigate the Antioxidant activity (ADA) of K. a/varezzi, C /entil/ifera

and S. po/ycystum form Sabah water using different solvent extraction; i.e.

methanol, acetone, ethanol, chloroform, dlethyl ether, hexane and water.

2. To determine the total phenolic content (TPC) and total f1avanoids content

(TFC) of K. a/varezzi, C /entillifera and S. po/ycystum form Sabah water

using different solvent extraction; i.e. methanol, acetone, ethanol,

chloroform, diethyl ether, hexane and water.

7

REFERENCES

Alothman, M., Bhat, R. and Karim, A. A. 2009. Antioxidant capacity and phenolic content of selected tropical fruits from Malaysia, extracted with different solvents. Food Chemistry. 115(3): 785-788.

Altena, V., and Steinberg, P. D. 1992. Are differences in the responses between North American and Australasian marine herbivores to phlorotannins due to differences in phlorotannin structure? Biochemical Systematics and Ecology. 20: 493-499.

Am a rowicz, R., Naczk, M., and Shahidi, F. 2000. Antioxidant activity of various fractions of non-tannin phenolics of Canola hulls. Journal of Agricultural and Food Chemistry. 48: 2755-2759.

Ask, E., Batisbaga, A., Zertuche-Gonza'lez, J.A., and de San, M. 2003. Three decades of Kappaphycus alvarezii (Rhodophyta) introduction to non­endemic locations. Proc. Int Seaweed Symp. 17: 49-57.

Athukorala, Y., Lee, K. W., Song, C. B., Ahn, C. B., Shin, T. S., Cha, Y. J., Shahidi, F., and Jeon, Y. J. 2003. Potential antioxidant activity of marine red alga Grateloupia filidna extracts. Journal of Food Lipids. 10: 251-265.

Balasundram, N., Sundram, K., and Samman, S. 2006. Phenolic compounds next term in plants and agri-industrial by-products: Antioxidant activity, occurrence, and potential uses Food ChemiStry. 99(1): 191-203.

Bandoniene, D., and Murkovic, M. 2002. On-line HPLC-DPPH screening method for evaluation of radical scavenging phenols extracted from apples (Malus domestica L.). Journal of Agricultural and Food Chemistry. 50: 2482-2487.

Barclay, L. R. c., Vinqvist M. R., Mukai, K., Itoh, S., and Morimoto, H. 1993. Chain­breaking phenolic antioxidants: steric and electronic effect in polyalkylchromanols, tocopherol analogs, hydroquinones and superior antioxidants of polyalkylbenzochromanol and naphthofuran dass. Joumal of Organic Chem. 58: 7416-7420.

Birt, D. F., Hendrich, S. and Wang, W. 2001. Dietary agents in cancer prevention: flavonoids and isoflavonoids. Pharmacol. Therapuet 90: 157-177.

Bixler, H. J. 1996. Recent developments in manufacturing and marketing carrageenan. Hydrobiologia. 326/327: 35-37.

Blasa, M., Candiracci, M., Accorsi, A., Piacentini, M. P. and Piatti, E. 2007. Honey f1avonoids as protection agents against oxidative damage to human red blood cells. Food Chemistry. 104(4): 1635-1640.

Bohm, B. A. 1998. Introduction to flavonoids. Amsterdam: Harwood Academic Publishers.

Brand-Williams, W., Cuvelier, M. E. and Benset, C. 1995. Use of free radical method to evaluate antioxidant activity. Lebensm l#ss Techno!. 28: 25-30.

Bravo, L. 1998. Polyphenols: chemiStry, dietary sources, metabolism, and nutritional significance. Nutrition Review. 56: 317-333.

Burtin, P. 2003. Nutritional value of seaweeds. Electronic Journal of Environment, Agriculture & Food Chemistry. 2(4): 498-503.

Cahyana, A. H., Shuto, Y., and Kinoshita, Y. 1992. Pyropheophytin a as an anti oxidative substance from the marine alga, Arame (Eisenia bicyclis). Biosd BiotechnolBiochem. 56: 1533-1535.

Chandini, S. K., Ganesan,P., and Bhaskar, N. 2008. In vitro antioxidant activities of three selected brown seaweeds of India. Food ChemiStry. 107: 707-713.

Chapman, V. J., and Chapman, D.J. 1980. Sea Vegetables (algae as food for man), Seaweed and their uses. London: Chapman and Hall.

Chatterji, A. D., Sreekumar, V. K., Parameswaran, P. S., Rodrigues, R., and Kotnala, S. 2004. Anti-influenza adivity in the Indian Seaweeds - A preliminary investigation. Goa: National Institute of Oceanography.

Chauhan V and Chauhan. A. 2006. Oxidative stress in Alzheimer's disease. Pathophysiology. 13(3): 195-208.

Chen, C. W., and Ho, C.T. 1995. Antioxidants properties of polyphenols extracted from green and black teas. Journal of Food Lipids. 2: 35-36.

Chen, X., and Ahn, D.U. 1998. Antioxidant activities of six naturals phenolics against lipid oxidation induced by Fe2

+ or ultraviolet light. Journal of American Oil Chemistry Sodety. 75: 1717-1721.

85

Chew, Y. L., Lim, Y.Y., Omar, M., and Khoo, K.S. 2008. Antioxidant activity of three edible seaweeds from two areas in South East Asia. Food Science and Technology. 41: 1067-1072.

Chew, Y.-L., Goh, J.-K. and Lim, Y.-Y. 2009. Assessment of in vitro antioxidant capacity and polyphenolic composition of selected medicinal herbs from Leguminosae family in Peninsular Malaysia. Food Chemistry. 116(1): 13-18.

Chirinos, R., Rogez, H., Campos, D., Pedreschi, R. and Larondelle, Y. 2007. Optimization of extraction conditions of antioxidant phenolic compounds from mashua (Tropaeolum tuberosum Ru'lz & Pav'on) tubers. Separation and Purification Technology. 55: 217-225.

Chkhikvishili, I. D. and Ramazanov, Z. M. 2000. Phenolic substances of brown algae and their antioxidant activity App!.Biochem.Microbiol. 36: 289-291.

Cho, S. H., Kang, S. E., Cho, J. Y., Kim, A. R., Park, S. M., Hong, Y. K. and Ahn, D. H. 2007. The Antioxidant Properties of Brown Seaweed (Sargassum siliquastrum) Extracts. Journal of Medicinal Food. 10(3): 479-485.

Chu, W. L., Norazmi, M., and Phang, S. M. 2003. Fatty-acid Composition of Some Malaysian Seaweeds. Malaysian. J.5cience. 22(2): 21-27.

Dhargalkar, V. K., and Verlecar, X.N. 2009. Southern Ocean seaweeds: A resource for exploration in food and drugs: A review. Aquaculture. 287: 229-242.

Dohi, T., Anamura, 5., Shikawa, M., Okamoto, H., and Tsujimoto, A. 1991. Inhibition of lipoxygenase by phenolic compound. Japan J Pharmacol. 55: 547-550.

Dorman, H. J. D., Kosar, M., Kahlos, K., Holm, Y. and Hiltunen, R. 2003. Antioxidant properties and composition of aqueous extracts from Mentha species, hybrids, varieties, and cultivars. Joumal of Agricultural and Food ChemiStry. 51: 4563-4569.

Duan, X. J., Zhang, W.W., U, X.M., and Wang, B.G. 2006. Evaluation of antioxidant property of extract and fractions obtained from a red alga, Polysiphonia urceolata. Food Chemistry. 95: 37-43.

Dummermuth, A. L., Karsten, U., Fisch, K. M., Konig, G. M. and Wieneke, C. 2003. Responses of marine macroalgae to hydrogen-peroxide stress. Journal of Experimental Marine Bi%gy and Ecology. 289: 103-121.

Dziezak, J. C. 1986. Preservation: antioxidants. Food Technology. 9: 205-212.

86

Eide, 1., Myklestad, 5., and Melson,S. 1980. Long-term uptake and release of heavy metals by Ascophyllum nodosum (L). Le Jol. (Phaeophyceae) in situ. Environ. Pol/ut. 23: 19-28.

Encyclopedia of the Aquatic World. Vols. 6. 2004. New York: Marshall cavendish Corporation

FAO. 2006. Year book of fishery statistics. Food and Agricultural Organisation of the United Nations, Rome. 98(1&2).

Farhoosh, R., Golmovahhed, G. A. and Khodaparast, M. H. H. 2007. Antioxidant activity of various extracts of old tea leaves and black tea wastes (Camellia sinensis L.). Food Chemistry. 100(1): 231-236.

Fayaz, M., Namitha, K. K., Chidambara Murthy, K. N., Mahadeva 5wamy, M., 5arada, R., Khanam, 5., 5ubbarao, P. V., and Ravishankar, G. A. 2005. Chemical composition, iron bioavailability, and antioxidant activity of Kappaphycus alvarezzi (Doty). Joumal of Agricultural and Food Chemistry. 53: 792-797.

Ferreira, 1. C. F. R., Baptista, P., Vilas-Boas, M. and Barros, L. 2007. Free-radical scavenging capacity and reducing power of wild edible mushrooms from northeast Portugal: Individual cap and stipe activity. Food ChemiStry. 100(4): 1511-1516.

Frankel, E. N., Huang, S. W., Kanner, J. and German, J. B. 1994. Interfacial phenomena in the evaluation of antioxidants: bulk oils versus emulsions. Joumal of Agricultural and Food ChemiStry. 42: 1054-1059.

Frankel, E. N. and Meyer, A. S. 2000. The problems of using one-dimensional methods to evaluate multifunctional food and biological antioxidants. Joumal of the Science of Food and Agriculture 80: 1925-1940.

Galland-Irmouli, A.-V., Fleurence, J., Lamghari, R., Lun, M., Rouxel, C., Barbaroux, 0., Bronowicki, J.-P., Villaume, C. and Gunt, J.-L. 1999. Nutritional value of proteins from edible seaweed Palmaria palmata (dulse). The Journal of NutritionalBiochemistry. 10(6): 353-359.

Ganesan, P., Kumar, C. 5., and Bhaskar, N. 2008. Antioxidant properties of methanol extract and its solvent fractions obtained from selected Indian red seaweeds. Bioresource Technology. 99: 2717-2723.

Gerald, F, C. 2008. The Vitamin: Fundamental Aspects in Nutrition and Health New York: Elsevier.

87

Gordon, M. H. 2001. The development of oxidative rancidity in foods. In J. Pokorny, Yanishlieva, N., Gordon, M. (Ed.), Antioxidants in Food: Practical applications (pp. 7-21). Boca Raton: Woodhead Publishing.

Gray, J. J., Gomaa, E.A., and Buckley, OJ. 1996. Oxidative quality and shelf life of meats. Meat Sci. 43: 111-123.

Guiry, M. O. and Guiry, G.M. 2007. Algae base version 4.2. world-wide electronic publication, National University of Ireland, Galway. http://www.algaebase.org/search/species/ Retrieved 10th September 2009.

Haila, K. M., Uevonen, S.M., and Hainonen I.M. 1996. Effect of lutein, Iycopene, annatto and tocopherol on autoxidation of triglycerides. Joumal of Agricultural and Food Chemistry. 44: 2096-2100.

Harborne, J. B., Baxter, H., and Moss, G.P. 1999. Phytochemical dictionary.' Handbook of bioactive compounds from plants (2nd ed). London: Taylor & Francis.

Harvey, H. R., Kennicutt, M.C.1992. Selective alteration of Sargassum lipids in anoxic sediments of the Orca basin. Org. Geochem. 18: 181-187.

Hassimotto, N. M. A., Genovese, I. S. and Lajolo, F. M. 2005. Antioxidant activity of dietary fruits, vegetables, and commercial frozen fruit pulps. Journal of Agricultural and Food Chemistry. 53(8): 2928-2935.

Hayouni, E. A., Abedrabba, M., Bouix, M. and Hamdi, M. 2007. The effects of solvents and extraction method on the phenolic contents and biological activities in vitro of Tunisian Quercus coccifera L. and Juniperus phoenicea L. fruit extracts. Food Chemistry. 105(3): 1126-1134.

Hegazi, M. M., Perez-Ruzafa.A., A1mela.L., and Maria-Emilia candela. 1998. Separation and identification of chlorophylls and carotenoids from Caulerpa prolifera, Jania rubens and Padina pavonica by reversed phase high­performance liquid chromatography. Journal of Chromatography A. 829: 153-159.

Heo, S. J., Jeon, YJ., Lee, J., Kim, H.T., and Lee, K.W. 2003a. Antioxidant effect of enzymatic hydrolyzate from a Kelp, Ecklonia cava. Algae. 18: 341-347.

Heo, S. J., Lee, K.W., Song, C.B., and Jeon, YJ. 2003b. AntiOXidant activity of enzymatic extracts from brown seaweeds. Algae. 18: 71-81.

88

Heo, S. J., Park, EJ., Lee, K.W., and Jean, Y. J. 2005. Antioxidant activities of enzymatic extracts from brown seaweeds. Bioresource Technology. 96: 1613-1623.

Heo, S. J., and Jeon, Y. J. 2009. Protective effect of fucoxanthin isolated from Sargassum siliquastrum on UV-B induced cell damage. Journal of Photochemistry and Photobiology B: Biology. 95: 101-107.

Ho, S. H. 2007. Antioxidant activity and total phenolic content of, C lenti//efera and S.polycyctum from Sabah's water. School of Food Science and Nutrition, University Malaysia Sabah.

Hollman, P. C. H., and Katan, M.B. 1999. Dietary flavonoids: intake, health effects and bioavailability. Food and Chemical Toxicology. 37: 937-942.

Hosokawa, M., Wanezaki, 5., Miyauchi, K., Kurihara, H., Kohno, H., Kawabata, J., Odashima, S. and Takahashi, K. 1999. Apoptosis-inducing effect of fucoxanthin on human leukemia cell HL-60. Food Sci. Technol. Res. 5: 243-246.

Hu, J. P., Calomme, M., Lausre, A., De Bruyne, T., Peter, L., Vlietinck, A., and Van den Berghe, D.A. 1995. Structure-activity relationship of f1avonoids with superoxide scarvenging activity. BioI Trace Element Res. 47: 327-313.

Huang, H.L. and Wang, B. G. 2004. Antioxidant capacity and lipophilic content of seaweeds collected from the qingdao coastline. Journal of Agricultural and Food Chemistry. 52: 4993-4997.

Huang, D., Ou, B. and Prior, R. L. 2005. The chemistry behind antioxidant capacity assays. Journal of Agricultural and Food Chemistry. 53: 1841-1856.

Ismail, A., Tan, S.H. 2002. Antioxidant activity of selected commercial seaweeds. Mal Journal Nutrition. 8(2): 167-177."

Jabatan Perikanan Sabah. 2008. Aktiviti Pengkulturan Rumpai Laut Daerah Semporna. Sabah: Jabatan Perikanan Sabah.

Jayaprakasha, G. K., Singh, R. P. and Sakariah, K. K. 2001. Antioxidant activity of grape seed (Vitis vinifera) extracts on peroxidation models in vitro. Food Chemistry. 73: 285-290.

Jensen, S. L. 1998. Carotenoids in chemosystematics. In G. Britton, Jensen, S.L., Pfander, H. (Ed.), Carotenoids, Biosynthesis and Metabolism (Vol. 3, pp. 217-247). Switzerland: Birkhauser Basel.

89

Jime' nez-Escrig, A., Jime' nez-Jime' nez, I., Pulido, R. and Saura-Calixto, F. 2001. Antioxidant activity of fresh and processed edible seaweeds. Journal of the Science of Food and Agriculture 81: 530-534.

Jorgensen, K. and Skibsted, L. H. 1993. Carotenoid scavenging of radicals. effect of carotenoid structure and oxygen partial pressure on antioxidant activity. Z Lebensm Unyers Forsch. 196: 423-429.

Jormalainen, V., Honkanen, T., Vesakoski, O. and Koivikko, R. 2005. Polar extracts of the brown alga Fucus vesiculosus (L.) reduce assimilation efficiency but do not deter the herbivorous isopod Idotea baltica (Pallas). Journal of Experimental Marine Biology and Ecology. 317(2): 143-157.

Jovanovic, S. V., Steenken, 5., Tosic, M., Marjanovic, B., and Simic, M.G. 1994. Flavonoids as antioxidants. Journal of American Chemistry Society. 116: 4846-4851.

Jung, C.-H., Seog, H.-M., Choi, I.-W., Park, M.-W. and Cho, H.-Y. 2006. Antioxidant properties of various solvent extracts from wild ginseng leaves. LWT - Food Science and Technology. 39(3): 266-274.

Kang, H. 5., Chung, H.Y., Kim, J.Y., Son, B.W., Jung, H.A., and Choi, J.S. 2004. Inhibitory phlorotannis from the edible brown algae Ecklonia stolonifera on total reactive oxygen species (ROS) generation. Arch. Pharm. Res. 27: 194-198.

Kang, K., Park, Y., Hwang, H.J., Kim, S.H., Lee, J.G., and Shin, H.C. 2003. Antioxidative properties of brown alage polyphenolics and their perspective as chemopreventives against vascular risk factors. Arch. Pharm. Res. 26: 286-293.

Kasahara, H., Hanada, A., Kuzuyama, T., Takagi, M., Kamiya, Y., and Yamaguchi, S. 2002. Contribution of the mevalonate and methyl erythritol phosphate pathways to the biosynthesis of the gibberellins in Arabidopsis. Journal of BioI Chern. 227: 45188-45199.

Kim, S. M., Kubota, K., and Kobayashi, A. 1997. Antioxidative activity of sulfur containing flavor compounds in garlic. Biosci-Biotechnol-Biochem. 61: 1482-1485.

Koharyova, M. and Kolarova., M. 2008. Oxidative stress and thioredoxin system. General Physiological and Biophysiological. 27(2): 71-84.

90

Konig, G. M., Wright, A. D., Sticher, 0., Anghofer, C. K., and Pezutto, J. M. 1994. Biological activities of selected marine natural products. PlantiJ Med. 60: 532-537.

Konig, G. M., and Wright, A. D. 1996. Marine natural products research: current directions and future potential. PlantiJ Med. 62: 193-211.

Konishi,!., Hosokawa, M., Sashima, T., Kobayashi, H., and Miyashita, K. 2006. Halocynthiaxanthin and fucoxanthinol isolated from Halocynthia roretzi induce apoptosis in human leukemia, breast and colon cancer cells. Comput Biochem. Phys. C 142: 53-59.

Kotake-Nara, E., Asai, A., Nagao, A. 2005. Neoxanthin and fucoxanthin induce apoptosis in PC-3 human prostate cancer cells. Cancer Lett. 220: 75-84.

Koivikko, R., Loponen, J., Honkanen, T. and Jormalainen, V. 2005. Contents of soluble, cell-wall-bound and exuded phlorotannins in the brown alga Fucus vesiculosus, with implications on their ecological functions. Joumal of Chemical Ecology. 31(1): 195-212.

Kubo, 1., Himejima, M., Tsujmoto, K., Muroi, H., and Ichikawa, N. 1992. Antibacterial activity of crinitol and its potentiation. J. Nat Products Lloydia. 55: 780-785.

Kubola, J. and Siriamompun, S. 2008. Phenolic contents and antioxidant activities of bitter gourd (Momordica charantia L.) leaf, stem and fruit fraction extracts in vitro. Food Chemistry. 110: 881-890.

Kuda, T., Hishi, T., and Maekawa, S. 2006. Antioxidant properties of dried product of "haba-nori", an edible brown alga, Petalonia binghamiae (J. Agaradh) Vinogradova. Food Chemistry. 98: 545-550.

Kuda, T., Tsunekawa, M., Hishi, T., and Araki, Y. 2005. Antioxidant properties of dried 'kayamo-nori', a brown alga Scytosiphon lomentaria (Scytosiphonales, Phaeophyceae). Food Chemistry. 89: 617-622.

Kulisic, T., Radonic, A., Katalinic, V. and Milos, M. 2004. Use of different methods for testing antioxidative activity of oregano essential oil. Food Chemistry. 85: 633-640.

Kumar, K. S., Ganesan, K., and Subba Rao, P.V. 2008. Antioxidant potential of solvent extracts of Kappaphycus alvarezii (Doty) Doty - An edible seaweed. Food Chemistry. 107: 289-295.

91

Kumari, P., Kumar, M., Gupta, V., Reddy, C. R. K. and Jha, B. 2010. Tropical marine macroalgae as potential sources of nutritionally important PUFAs. Food Chemistry. 120(3): 749-757.

Kyle, D. J. 2001. The large scale production and use of a single cell oil highly enriched in docosahexaenoic acid. In F. Shahidi, Finley, J.W. (Ed.), Omega-3 Fatty adds: Chemistry, nutrition and health effects (pp. 92-107). Washington: ACS Symposium Series 788. American Chemical Society.

Lapornik, B., Prosek, M. and Wondra, A. G. 2005. Comparison of extracts prepared from plant by-products using different solvents and extraction time. Journal of Food Engineering. 71: 214-222.

Li, B. B., Smith, B. and Hossian, M. M. 2006. Extraction of phenolics from dtrus peels I. Solvent extraction method. Separation and Purification Technology. 48: 182-188.

Lim, P. E., and Phang, S. M. 2004. Gracilaria spedes (Gracilariales, Rhodophyta) of Malaysia including two new records. Malaysian J. Science. 23(2): 71-80.

Um, S. N., Cheung, P. C. K., Coi, V. E. C., and Ang, P. O. 2002. Evaluation of antioxidative activity of extracts from a brown seaweed,Surgassum siliquastrum. Journal of Agricultural and Food ChemiStry. 50: 3862-3866.

Um, Y. Y. and Quah, E. P. L. 2007. Antioxidant properties of different cultivars of portulaca oleracea. Food Chemistry. 103(3): 734-740.

Un, J.-Y. and Tang, c.-y. 2007. Determination of total phenolic and flavonoid contents in selected fruits and vegetables, as well as their stimulatory effects on mouse splenocyte proliferation. Food Chemistry. 101(1): 140-147.

Mabeau, S., and Fleurence, J. 1993. Seaweed in food products: biochemical and nutritional aspects. Trends in Food Sdence and Technology. 4(4): 103-107.

Macheix, J. J., Fleuriet, A., and Billot, J. 1990. Food phenolic. Boca Raton: CRP Press.

Marinho-Soriano, E. and Bourret, E. 2005. Polysaccharides from the red seaweed Gracilaria dura (Gracilariales, Rhodophyta). Bioresource Technology. 96(3): 379-382.

Marinova, E. M., Yanishlieva, N.V.1996. Antioxidant activity of phenolic acids on triacy!glycero!s and fatty acid methyl ester from olive oil. Food Chemistry. 56: 139-145.

92

Martinez-Valverde, I., Periago, MJ., Provan, G., and Chesson, A. 2002. Phenolic compounds, Iycopene and antioxidant activity in commerdal varieties of tomato (Lycopersicum esculentum). Joumal of the Science of Food and Agriculture. 82: 323-330.

Matanjun, P., Mohamed, S., Mustapha, N.M., Muhammad, K., and Cheng, H.M. 2008. Antioxidant activities and phenolics content of eight species of seaweeds from north Borneo. Journal of Applied Phycology. 20: 367-373.

Matsukawa, R., Dubinsky, l., Kishimoto, E., Masak, K., Masuda, Y., Takeuchi, T., Chihara, M., Yamamoto, Y., Niki, E., and Karube, 1. 1997. A comparison of screening methods for antioxidant activity in seaweeds. Journal of Applied Phycology. 9: 29-35.

McDermid, K. J., and Stuercke, B. 2003. Nutritional composition of edible Hawaiian seaweeds. Journal of Applied Phycology. 15: 513-524.

McHugh, D. J. 1987. Productjon and utilization pf products from commercial seaweeds: FAO Rsheries Technical Paper 288. Australia: FAO

McHugh, D. J. 2003. A Guide to the seaweed industry: FAO Rsheries Technical Paper 441. FAO, Rome, Italy.

Meda, A., Lamien, C. E., Romito, M., Millogo, J. and Nacoulma, O. G. 2005. Determination of the total phenolic, flavonoid and proline contents In Burkina Fasan honey, as well as their radical scavenging activity. Food Chemistry. 91(3): 571-577.

Merken, H. M., and Beecher, G.R. 2000. Measurement of food flavonoids by high­performance liquid chromatography: a review. Journal of Agricultural and Food Chemistry. 48: 577-599.

Miraliakbari, H. and Shahidi, F. 2008. Antioxidant activity of minor components of tree nut oils. Food Chemistry. 111: 412-427.

Mohd Hani Norziah, Ching, C. Y. 2000. Nutritional composition of edible seaweed Gracilaria changgi. Food Chemistry. 68: 69-76.

Mohsen, S. M. and Ammar, A. S. M. 2009. Total phenolic contents and antioxidant activity of corn tassel extracts. Food Chemistry. 112: 595-598.

Montelongo, R. G., Lobo, M. G. and Gonzalez, M. 2010. Antioxidant activity in banana pee! extracts: Testing extraction conditions and related bioactive compounds. Food ChemiStry. 119: 1030-1039.

93

Moure, A., Franco, D., Sineiro, J., Domguez, H., Nez, M. J. and Lema, J. M. 200l. Antioxidant activity of extracts from Gevuina avellana and Rosa rubiginosa defatted seeds. Food Research Intemational. 34(2-3): 103-109.

Murugadas, T. L., Phang, S.M., and Tong, S.L. 1995. Heavy metal accumulation cytotoxic principle of the brown alga Sargassum tortile. Chem. Pharm.8u//. Tokyo. 39: 2129-2131.

Nakamura, T., Nagayama, K., Uchida, K., and Tanaka, R. 1996. Antioxidant activity of phlorotannis isolated from brwon alga Eisenia bicyclis. Fish. Sci. 62: 923-926.

Nakayama, K., Iwamura, K., Shibata, T., Hirayama, 1. and Nakamura, T. 2002. Bacteridical activity of phlorotannis from the brown alga Ecklonia kurome .. J. Antimicro. Chemother. SO: 889-893.

Nakayama, Y., Takahashi, M., Fukuyama, Y., and Kinzyo, Z. 1989. An anti-plasmin inhibito, echol, isolated from brown algae Eisinia kurome OKAMURA . . Agric. BioI. Chem. 63: 3025-3030.

Nascimento, M. A., Silva, A. K., Fran, L. C. S., Quignard, E. L. J., Lez, J. A. and Almeida, M. G. 2006. Turnera ulmifolia L. (Turneraceae): Preliminary study of its antioxidant activity. 8ioresource Technology. 97(12): 1387-1391.

Nawar, W. F. 1996. Lipids. In O. R. Fennema (Ed.), Food Chemistry (pp. 254-288). New York: Marcel Dekker.

Nisizawa, K. 1988. Production and utilization of products from commercial seaweeds. In D. J. McHugh (Ed.), FAD Rsheries Technical Paper 299 (pp. 147). Rome: FAO.

Novaczek, 1. (2001). A guide to the common edible and medicinal sea plants of pacific islands., USPMarine Studies ProgrammejSPC Coastal Fisheries Programme, Training Materials for Pacific Community Fisheries.

Okuzumi, J., Takahashi, T., Yamane, T., Kitao, Y., Inagake, M., Ohya, K., Nishino, H., and Tanaka,Y. 1993. Inhibitory effects of fucoxanthin, a natural carotenoid, on N-ethyl-N'-nitro-N-nitrosoguanidineinduced mouse duodenal carcinogenesis. Cancer Lett 68: 159-168.

Omura, K. 1995. Antioxidant synergism between butylated hydroxyanisole and butylated hydroxytoluene. Joumal of American Oil Chemistry SOCiety. 72: 1565-1570.

94

Palozza, P. and Krinsky, N.I. 1992. Antioxidant effects of carotenoids in vivo and in vitro: An overview. Methods Enzymology. 213: 403-420.

Phang, S. M. 1984. Seaweed Resources of Malaysia. Wal/aceana. 38: 3-8.

Phang, S. M., and Maheswary, V. 1989. Phycocolloid Content of Some Malaysian Seaweeds. In S. M. Phang, Sasekumar, A.M., Vikeneswary, S. (Ed.), Proceedings Twelfth Annual Seminar Malaysian Society of Marine Sciences. Kuala Lumpur.

Phang, S. M. 1998. The Seaweed Resources of Malaysia. In A. T. Critchley, Chno, M. (Ed.), Seaweed Resources of The World (pp. 79-91). Yokosuka: Japan International Cooperation Agency.

Phang, S. M. 2006. Seaweed resources in Malaysia: Current status and future prospects. Aquatic Ecosystem Health and Management Society. 9(2): 185-202.

Philips, N. 1995. Biogeography of Sargassum (Phaeophyta). Proc. Pacific basin. (Vol. 5). Honolulu: International workshop on the taxonomy of economic seaweeds.

Pi etta, P. G. 2000. Flavonoids as antioxidants. Journal of Natural Products. 63: 1035-1042.

Pi mol, P., Khanidtha, M., and Prasert, P. 2008. Influence of particle size and salinity on adsorption of basic dyes by agricultural waste: dried Seagrape (Caulerpa lentillifera) Joumal of Environmental Science. 20(6): 760-768.

pokorny, J. 1991. Natural antioxidants for food use. Trends in Food Science and Technology. 2: 223-227.

pokorny, J., Yanishlieva, N., and Gordon, M. 2001. Antioxidant in Food Boca Raton: Woodhead Publishing Limited.

Ponce, N. M. A., Pujol, C. A., Damonte, E. B., Flores, M. L. and Stortz, C. A. 2003. Fucoidans from the brown seaweed Adenocystis utricularis: extraction methods, antiviral activity and structural studies. Carbohydrate Research. 338(2): 153-165.

Prior, R. L., Wu, X. and Schaich, K. 2005. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. Journal of AgricultrJral and Food Cllemist'y. 5:!: 4290-4302.

95

Rice-Evans, C. A., Sampson, J., Bramley, P. M. and Holloway, D. E. 1997. Why do we expect carotenoids to be antioxidants in vivo? Free Radical Research. 26: 381-398.

Rodriquez-Concepcion, M., and Boronat, A. 2002. Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. Plant Physiological. 130: 1079-1089.

Sa' nchez-Machado, D. I., Lo' pez-Cervantes, J., Lo' pez-Herna' ndez, J., and Paseiro-Losada, P. 2004. Fatty acids, total lipid, protein and ash contents of processed edible seaweeds. Food Chemistry. 85: 439-444.

Sakata, K. 1997. Antioxidative compounds from marine organisms. In M. Hiramatsu, Yoshikawa, T., Inoue, M. (Ed.), Foods and Free Radicals (pp. 85-100). New York: Plenum Press.

Sastry, Y. M. V. S., and Rao, G.R.K. 1995. Dioctyl phthalate, an antibacterial compound from the marine brown alga Sargassum wightii. Journal of Applied Phycology. 7: 185-186.

Satoru, K., Noboru, T., Hiroo, N., Shinji, S., and Hirishi, S. 2003. Oversulfation of fucoidan enhances its anti-angiogenic and antitumor activities. Biochem Pham. 65: 173-179.

Satue-Gracia, M., Heinonen, M., and Frankel, E.N. 1997. Antocyanins as antioxidants of human low density lipoprotein and lecithin-liposome systems Journal of Agricultural and Food Chemistry. 45: 3362-3367.

Shahidi, F. 2002. Antioxidants in plant and oliaginous seeds. In M. J. Morello, Shahidi, F. & Ho,C.T. (Ed.), Free Radicals in Food: Chemistry, Nutrition and Health Effects (pp. 162-175). Washington, D.C.: ACS Symposium Series 807. American Chemical SOCiety.

Shahidi, F., and Naczk, M. 2004. Phenolics in food and nutraceuticals. Boca Raton: CRC Press.

Shahidi, F., and Zhong, Y. 2007. Antioxidants from marine by-products. In F. Shahidl (Ed.), Maximizing the value of marine by-products (pp. 397-412). Boca Raton: Woodhead Publishing.

Shameel, M. 2001. Diversity exhibited by marine benthic algae inhabiting the coast of Balochistan. Pak. Journ81 of Marine Science. 10: 87-103.

96

Shimada, K., Fujikawa, K., Yahara, K. and Nakamura, T. 1995. Anti-oxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrln emulsion. Journal of Agricultural and Food Chemistry. 40: 945-948.

Shon, M. Y., Kim, T. H., and Sung, N. J. 2003. Antioxidants and free radical scavenging activity of Phelllnus baumll (Phellinus of Hymenochaetaceae) extracts. Food Chemistry. 82: 593-597.

Singh, N. and Rajini, P. S. 2004. Free radical scavenging activity of an aqueous extract of potato peel. Food ChemiStry. 85: 611-616.

Siriwardhana, N., Lee, K. W., Kim, S. H., Ha, J. W., and Jeon, Y. J. 2003. Antioxidant activity of Hizikia fusiformis on reactive oxygen species scavenging and lipid peroxidation inhibition. Food Sci. Tech. Int 9(5): 339-

346.

Smirnoff, N. 2005. Antioxidant and reactive oxygen species in plants. New Jersey: Blackwell Publishing.

Stahl, W., and Sies, H. 1993. Physical quenching of singlet oxygen in cis-trans isomerization of carotenoids. In K. Canfield L.M., N.I., & Olson, J.A. (Ed.), Carotenoids in Human Health. New York: New York Academy of Science.

Starvridis, c. J. 2008. Oxidation: The Cornerstone of Carcinogenesis: Oxidation and Tobacco Smoke Carcinogenesis. A Relationship Between Cause and Effect. Athen: Springer.

Swanson, A. K., and Druehl., L. D. 2002. Induction, exudation and the UV protective role of kelp phlorotannlns. Aquatic. Botany. 73: 241-253.

Tachakittirungrod, S., Okonogi, S. and Chowwanapoonpohn, S. 2007. Study on antioxidant activity of certain plants in Thailand: Mechanism of antioxidant action of guava leaf extract. Food ChemiStry. 103: 381-38S.

Takamatsu, S., Hodges, T. W., Rajbhandari, I., Gerwick, W. H., Hamann, M. T., and Nagle, D. G. 2003. Marine natural products as novel antioxidant prototypes. Journal of Natural Products. 66: 60S-60S.

Tan, S. L. (200S). Effect of different drying methods on carotenolds, 'flavanoids and vitamin C contents In edible seaweeds, Kappaphycus A varezzl, Sargassum Po/ycystum and Caulerpa Lentil/ifera., School of Food Science and Nutrition, University Malaysia Sabah.

97

Tanaka, M., Kuei, C. W., Nagashima, Y. and Taguchi, T. 1998. Application of antioxidative maillard reaction-products from histidine and glucose to sardine products. Nippon Suisan Gakkaishil. 54: 1409-1414.

Targett, N. M., and Amold, T. M. 1998. Predicting the effects of brown algal phlorotannins on marine herbivores in tropical and temperate oceans. Journal of Phycology. 34(2): 195-205.

Temple, N. 2000. Antioxidants and disease: more questions than answers. Nutrition Research. 20: 449-459.

Tutour, B. L., Benslimane, F., Gouleau, M.P., Gouygou, J.P., Saadan, B., and Quemeneur, F. 1998. Antioxidant and pro-oxidant activities of the brown algae, Laminaria digitata, Himanthalia elongata, Fucus vesiculosus, Fucus serratus and Ascophyllum nosum. Journal of Applied Phycology. 10: 121-129.

Untawale, A. G., Dhargalkar, V.K. and Agadhi, V.V. 1983. List of marine algae from India. Goa, India: N.LO. Technical Report.

USDA Database for The Flavanoid Content of Selected Foods. 2003. U.S. Department of Agriculture (USDA).

Valdebenito, H., Bittner, M., Sammes, P. G., Silva, M., and Watson, W. H. 1982. A compound with antimicrobial activity isolated from the red seaweed Laurencia chilensis. Phytochemistry. 21: 1451457.

Van Alstyne, K. L., McCarthy, J. J., Hustead, C. L. and Keards, L. J. 1999. Phlorotannin allocation among tissues of northeastern pacific kelps and rockweeds. Journal of Phycology. 35: 483-492.

Wang, B. G., Zhang, W.W., Duan, X.J., and Li, X.M. 2009a. In vitro antioxidative activities of extract and semi-purified fractions of the marine red alga, Rhodomela confervoides (Rhodomelaceae). Food Chemistry. 113: 1101-1105.

Wang, T., Jonsdottir, R. and 01 afsdottir, G. 2oo9b. Total phenolic compounds, radical scavenging and metal chelation of extracts from Icelandic seaweeds. Food Chemistry. 116: 240-248.

Waterhouse, A. L. 2002. Determination of total phenolics. In E. R. E. Wrolstad (Ed.), Current protocols In food analytical chemistry. New York: Wiley.

Waterman, P. G. and Mole, S. 1994. Analysis of Phenolic Plant Metabolites. Oxford: Blackwell Scientific.

98

Wong, C. K., OOi, V. E. C., and Ang, P. O. 2000b. Protective effects of seaweeds against liver injury caused by carbon tetrachloride in rats. Chemosphere.41: 173-176.

Wong, K. H., and Peter Cheung, C.K. 2000a. Nutritional evaluation of some subtropical red and green seaweeds Part I : proximate composition, amino acid profiles and some physico-chemical properties. Food Chemistry. 71: 475-482.

Xing, R., Uu, 5., Guo, Z., Yu, H., Wang, P. and Li, C. 2005. Relevance of molecular weight of chltosan and its derivatives and their antioxidant activities in vitro. Bioorganic and Medicinal Chemistry. 13: 1573-1577.

Xue, C. H., Fang, Y., Un, H., Chen, L., Li, Z. J. and Deng, D. 2001. Chemical characters and antioxidative properties of sulfated polysaccharides from Laminaria japonica. Journal of Applied Phycology. 13(1): 67-70.

Van, X., Nagata, T. and Fan, X. 1998. Antioxidant activities in some common seaweeds. Plant Food for Human Nutrition. 52: 253-262.

Van, X., Chuda, Y., Suzuki, M., and Nagata, T. 1999. Fucoxanthin as the major antoxidant in Hijikia fusiformis, a common edible seaweed. Biosci Blotechnol Biochem. 63: 605-607.

Yanishlieva, N. V. 1971. Autoxidation of Acyclic Olefin. Sofia: Academy of Science.

Yanishlieva, N. V. 2001. Inhibiting oxidation. In J. Pokorny, Yanishlieva, N., Gordon, M. (Ed.), Antioxidants in food: Practical applications (pp. 22-70). Boca Raton: Woodhead Publishing.

Yangthong, M., Hutadilok-Towatana, N. and Phromkunthong, W. 2009. Antioxidant Activities of Four Edible Seaweeds from the Southern Coast of Thailand. Plant Foods Hum Nutr. 64: 218-223.

Yoshie, Y., Wong, W., HSieh, Y. and Suzuki, T. 2002. Compositional difference of phenolic compounds between two seaweeds, Halimeda spp, . Journal of Tokyo University of Fisheries. 88: 21-24.

Yuan, Y. V., and Walse, N.A. 2006. Antioxidant and anti proliferative activities of extracts from a variety of edible seaweeds. Food Chemistry Toxicology. 44: 1144-1150.

Zemke-White, L. W., and Ohno, M. 1999. World seaweed utilization' an end of century summery. Journal of Applied Phycology. 11: 368-376.

99

Zhang, Q. B., Yu, P. Z., Li, Z., Zhang, H., Xu, Z. H., and Li, P. C. 2003. Antioxidant activities of sulfated polysaccharide fractions from Porphyra haitanensis. Journal of Applied Phycology. 15: 305-310.

Zhang, W. W., Duan, X. J., Huang, H. L., Zhang, Y., and Wang, B. G. 2007. Evaluation of 28 marine algae from the Qingdao coast for anti oxidative capacity and determination of antioxidant efficiency and total phenolic content of fractions and subfractions derived from Symphyocladia latiuscula (Rhodomelaceae). Journal of Applied Phycology. 19: 97-108.

Zhang, Q., Zhang, J., Shen, J., Silva, A. K., Dennis, D. A. and Barrow, C. J. 2006. A simple 96-well microplate method for estimation of total polyphenol content in seaweeds. Journal of Applied Phycology. 18: 445-450.

Zhu, Q. T., Hackman, R. M., Ensunsa, J. L., Holt, R. R. and Keen, C. L. 2002. Antioxidative activities of oolong tea. Journal of Agricultural and Food Chemistry. 50: 6929-6934.

Zvyagintseva, T. N., Shevchenko, N. M., Chizhov, A. 0., Krupnova, T. N., Sundukova, E. V. and Isakov, V. V. 2003. Water-soluble polysaccharides of some far-eastern brown seaweeds. Distribution, structure, and their dependence on the developmental conditions. Journal of Experimental Marine Biology and Ecology. 294(1): 1-13.

100