UNIVERSITI PUTRA MALAYSIA

ANTIOXIDANT AND CYTOTOXIC EFFECTS OF CRUDE EXTRACT

FROM A DIATOM, Chaetoceros calcitrans AND

GREEN ALGA, Nannochloropsis oculata

GOH SU HUA

IB 2011 32

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ANTIOXIDANT AND CYTOTOXIC EFFECTS OF CRUDE EXTRACT

FROM A DIATOM, Chaetoceros calcitrans AND

GREEN ALGA, Nannochloropsis oculata

By

GOH SU HUA

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in fulfillment of the Requirements for the Degree of Master of Science

September 2011

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment

of the requirement for the degree of Master of Science

ANTIOXIDANT AND CYTOTOXIC EFFECTS OF CRUDE EXTRACT

FROM A DIATOM, Chaetoceros calcitrans AND

GREEN ALGA, Nannochloropsis oculata

By

GOH SU HUA

September 2011

Chairman: Loh Su Peng, PhD

Faculty: Institute of Bioscience

There is a growing trend in research focusing on marine microorganisms such as

sponges, seaweeds and micro-algae as a potential source of bioactive compounds. In

Malaysia, microalgae are widely used as live feed in shrimp and fish hatcheries. The

nutrient-rich source of marine microalgae gives a promising potential to explore its

new biomedical applications. This study was carried out to investigate the

antioxidant capacity and cytotoxic effects of different polarities of crude solvent

extracts namely hexane, dichloromethane, ethyl acetate and methanol from a diatom,

Chaetoceros calcitrans and a green alga, Nannochloropsis oculata. The antioxidant

properties were determined by 1, 1-diphenyl-2-picrylhrazyl (DPPH)

radical-scavenging, ferric-reducing antioxidant power (FRAP), ferrous-ion chelating

and folin assays. Results revealed that crude ethyl acetate (EA) extract of C.

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calcitrans contained the highest total phenolic content (3775.67 ± 0.08 mg gallic acid

equivalent/g dried extract) and DPPH radical scavenging power (3722.93 ± 2.98 mg

Trolox equivalent/g dried extract). The antioxidant activities of extracts from N.

oculata were lower compared to the extract from C. calcitrans, except for EA extract

of C. calcitrans showed the highest chelating power compared to others. MTT assay

was used to determine the cytotoxic properties of crude solvent extracts from C.

calcitrans and N. oculata towards various cancer cell lines. Results showed that EA

extract of C. calcitrans significantly inhibited the growth of MDA-MB-231 cells

among the cancer cell lines tested, with IC50 60 μg/mL only after 72 hours treatment

period. Thus, EA extract of C. calcitrans was used in further assay to determine the

mode of cell death with three different concentrations : 30 (½ IC50), 60 (IC50) and

120 (2x IC50) μg/mL for 72 hours treatment. In the cell cycle with flow cytometry

analysis, EA extract of C. calcitrans arrested the cell cycle of MDA-MB-231 cells at

G2/M phase when treated with 60 μg/mL. However, when cells were treated with

low concentration (30 μg/mL) of EA extract, significant growth arrest was occurred

at G1 phase. Apoptosis was induced when the concentration was increased to 120

μg/mL. The mode of cell death was mainly apoptosis, which was proven by acridine

orange/propidium iodide (AO/PI) dual staining method, Annexin V-FITC and DNA

fragmentation (TUNEL) assays. Morphology of the treated cells which was observed

through AO/PI staining method showed the presence of blebbing cells, chromatin

condensation and DNA fragmentation as well as intake of some PI stain, proving that

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apoptosis has occurred. Early apoptosis was analysed by Annexin V-FITC apoptosis

test and DNA fragmentation test showed that DNA was cleaved into fragments.

These results indicated the presence of apoptotic cells increased with increasing

concentration of the extract. The changes of expression level of apoptotic, and

proliferative-related genes caused by EA extracts of C. calcitrans were profiled using

multiplex gene expression profiler (GeXP). Cells treated with EA extract for 6, 12

and 24 hours did not show significant changes in the expression levels of most of the

pro-apoptotic genes. The expression of pro-apoptotic genes of the cells treated for 48

hours with low concentration (30 μg/mL) of the extract was highly up-regulated.

However, the expression level was down-regulated when treated with high

concentration of EA extract (120 μg/mL). At 72 hours of treatment period, most of

the pro-apoptotic genes especially caspases related genes such as caspases-3, -4, -9,

BAK1 and p21 were found to be up-regulated. Conversely, genes that involved in

p53 network especially Bax and ING3 as well as anti-apoptotic (Bcl-2) were found to

be downregulated. These findings provided some mechanisms of EA extract of C.

calcitrans-induced apoptosis in the human breast cancer cells via the caspases

induction pathway. In conclusion, C. calcitrans is more cytotoxic compared to N.

oculata (solvent extracts). EA extract of C. calcitrans showed the best antioxidant

activities and cytotoxicity compared to others, and induced apoptosis in

MDA-MB-231 cells. The induction of apoptosis involved important pro-apoptotic

genes : p21, casp-3, -4, -9 and Bak1.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk Ijazah Master Sains

ANTIOKSIDAN DAN KESAN SITOTOKSIK EKSTRAK MENTAH

DARIPADA DIATOM, Chaetoceros calcitrans DAN

ALGA HIJAU, Nannochloropsis oculata

Oleh

GOH SU HUA

September 2011

Pengerusi: Loh Su Peng, PhD

Fakulti: Institut Biosains

Terdapat perkembangan dalam bidang penyelidikan berfokus kepada mikrorganisma

marin seperti span, rumpai laut dan mikroalga, sebagai sumber sebatian bioaktif. Di

Malaysia, mikroalga digunakan secara meluas sebagai makanan di pusat penetasan

ikan dan udang. Sumber nutrisi mikroalga yang tinggi ini menjanjikan potensi untuk

dieksploitasi bagi kegunaan baru dalam bidang bioperubatan. Kajian ini dilaksanakan

untuk menyiasat kapasiti antioksidan serta kesan sitotoksik ekstrak yang berpolariti

berbeza iaitu heksana, diklorometana, etil asetat dan metanol daripada diatom,

Chaetoceros calcitrans dan alga hijau, Nannochloropsis oculata. Ujian antioksidan

yang dijalankan adalah 1, 1-diphenyl-2-picrylhrazyl (DPPH), daya pengurangan

ferum (FRAP), ‘chelate’ ion ferus (FIC) dan kajian folin. Hasil kajian menunjukkan

bahawa ekstrak mentah etil asetat (EA) daripada C. calcitrans mengandungi

kandungan fenolik (3775.67 ± 0.08 mg setara dengan asid galik/g ekstrak kering) dan

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aktiviti penggesatan radikal DPPH (3722.93 ± 2.98 mg setara dengan Trolox/g

ekstrak kering) yang paling tinggi. Aktiviti antioksidan bagi ekstrak N. oculata

adalah rendah berbanding dengan ekstrak daripada C. calcitran, kecuali ekstrak EA

daripada N. oculata menunjukkan keupayaan pengilatan yang paling tinggi

berbanding dengan yang lain. Ujian MTT digunakan untuk menentukan sifat

ketoksikan ekstrak daripada kedua-dua C. calcitrans dan N. oculata terhadap

pelbagai jujukan sel kanser. Keputusan menunjukkan ekstrak EA daripada C.

calcitrans merencat pertumbuhan sel MDA-MB-231 secara signifikan di kalangan

sel kanser lain yang dikaji, dengan IC50 60 μg/mL selepas 72 jam rawatan. Oleh itu,

EA ekstrak daripada C. calcitrans digunakan selanjutnya untuk menentukan cara

kematian sel dengan tiga kepekatan yang berlainan: 30 (½ IC50), 60 (IC50) and 120

(2x IC50) μg/mL selama 72 jam. Dalam kitaran sel dengan analisis sitometri aliran,

ekstrak EA tersebut menyekat kitaran sel MDA-MB-231 pada fasa G2/M apabila

dirawat dengan kepekatan 60 μg/mL. Namun, ketika sel dirawat dengan kepekatan

ekstrak EA yang rendah (30 μg/mL), penyekatan pertumbuhan secara signifikan

berlaku pada fasa G1. Apoptosis diaruh apabila kepekatan ditingkatkan kepada 120

μg/mL. Mod utama kematian sel ialah apoptosis yang dibuktikan melalui kaedah

pewarnaan berganda ‘akridina jingga/propidium iodida’ (AO/PI), ujian ‘Annexin

V-FITC’ dan fragmentasi DNA (TUNEL). Morfologi sel yang dirawat yang

diperhatikan melalui kaedah pewarnaan AO/PI menunjukkan ‘blebbing’, kondensasi

kromatin dan fragmentasi DNA serta pengambilan masuk pewarna PI membuktikan

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berlakunya apoptosis. Kehadiran apoptosis awal dianalisis dengan ujian Annexin

V-FITC dan ujian fragmentasi DNA menunjukkan bahawa DNA telah difragmentasi

kepada serpihan. Keputusan ini menunjukkan bahawa bilangan sel apoptosis

bertambah dengan peningkatan kepekatan ekstrak. Perubahan dalam tahap

penzahiran gen yang berkaitan dengan apoptosis dan perebakan yang disebabkan

oleh ekstrak EA daripada C. calcitrans diprofilkan dengan ''multipleks ekspresi gen''

(GeXP). Sel yang dirawat dengan ekstrak EA selama 6, 12 dan 24 jam tidak

menunjukkan perubahan yang signifikan pada tahap penzahiran kebanyakan gen

pro-apoptotik. Penzahiran gen pro-apoptotik sel yang dirawat selama 48 jam pada

kepekatan rendah (30 μg/mL) ekstrak telah ditingkatkan. Walau bagaimanapun, tahap

penzahiran telah diturunkan apabila dirawat pada kepekatan yang tinggi ekstrak EA

(120 μg/mL). Pada 72 jam rawatan, penzahiran kebanyakan gen pro-apoptotik

terutamanya gen yang berkaitan dengan caspase seperti casp-3, -4, -9, BAK1 dan

P21 didapati ditingkatkan. Sebaliknya, penzahiran gen yang terlibat di dalam

rangkaian p53, terutamanya Bax dan ING3 serta gen anti-apoptosis (Bcl-2) didapati

diturunkan. Penemuan ini membekalkan beberapa mekanisma apoptosis yang diaruh

oleh ekstrak EA daripada C. calcitrans. Kesimpulannya, C. calcitrans lebih

sitotoksik berbanding dengan N. oculata (ekstrak pelarut). Ekstrak EA daripada C.

calcitrans menunjukkan aktiviti antioksidan dan sitotoksik yang terbaik berbanding

yang lain, dan mengaruh apoptosis dalam sel MDA-MB-231. Aruhan apoptosis

tersebut melibatkan gen pro-apoptotik yang penting: p21, casp-3, -4, -9 and Bak1.

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ACKNOWLEDGEMENTS

First and foremost, I would like to highlight my appreciation and warmest gratitude

to my supervisor, Dr Loh Su Peng for her perceptiveness, advices and

encouragement throughout the completion of my project. My deepest gratitude also

goes to Dr. Noorjahan Banu Mohamed Alitheen, Dr. Latifah Saiful Yazan and Prof.

Dr. Fatimah Md. Yusoff for their constructive suggestions and review of my works

throughout my study. I would also like to extend my highest appreciation to Mr. Yeap

Swee Keong for his generous help during the time of my study.

Not forgetting to thank all postgraduates and officers of Laboratory of Molecular

Biomedicine (LBMS) and Laboratory of Marine Science and Aquaculture

(MARSLAB) of Institute of Bioscience as well as Faculty of Biotechnology and

Biomolecular Science for their kind assistance and sharing through thick and thin.

I would also like to acknowledge and thank the financial support of Graduate

Research Fellowship from School of Graduate Studies, Universiti Putra Malaysia and

Mini Budget 2009 (Malaysia 2009 Second Stimulus Package) from the Ministry of

Higher Education.

Last but not least, I would like to express my highest appreciation and thankful to my

family. Thanks for the supports, concerns and encouragement during my study.

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I certify that a Thesis Examination Committee has met on 19 September 2011 to

conduct the final examination of Goh Su Hua on her thesis entitled ''Antioxidant

and cytotoxic effects of crude extract from a diatom, Chaetoceros calcitrans and

green algae, Nannochloropsis oculata'' in accordance with the Universities and

University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia

[P.U.(A) 106] 15 March 1998. The Committee recommends that the student be

awarded the Master of Science.

Members of the Thesis Examination Committee were as follows:

Chong Pei Pei, PhD

Professor Madya

Faculti Perubatan dan Sains Kesihatan

Universiti Putra Malaysia

(Chairman)

Asmah bt Rahmat, PhD

Professor

Faculti Perubatan dan Sains Kesihatan

Universiti Putra Malaysia

(Internal Examiner)

Latifah binti Saiful Yazan, PhD

Senior Lecturer

Faculti Perubatan dan Sains Kesihatan

Universiti Putra Malaysia

(Internal Examiner)

Nor Fadilah Rajab, PhD

Professor

Jabatan Sains Bioperubatan

Faculti Sains Kesihatan Malaysia

Universiti Kebangsaan Malaysia

(External Examiner)

________________________________

HASANAH MOHD GHAZALI, PhD

Professor/ Dean

School of Graduate Studies

Universiti Putra Malaysia

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfillment of the requirement for the degree of Master of Science. The

members of the Supervisory Committee were as follows:

Loh Su Peng, PhD

Senior Lecturer

Faculty of Medicine and Health Science

Universiti Putra Malaysia

(Chairman)

Noorjahan Banu Mohamed Alitheen, PhD

Senior Lecturer

Faculty of Biotechnology and Biomolecular Science

Universiti Putra Malaysia

(Member)

Fatimah Md Yusoff , PhD

Professor

Institute of Bioscience

Universiti Putra Malaysia

(Member)

______________________________

BUJANG BIN KIM HUAT, PhD

Professor/ Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

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DECLARATION

I declare that the thesis is my original work except for quotations and citations which

have been duly acknowledged. I also declare that it has not been previously, and is

not concurrently, submitted for any other degree at Universiti Putra Malaysia or at

any other institution.

__________________

GOH SU HUA

Date: 19 September 2011

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TABLE OF CONTENTS

Page

ABSTRACT ii

ABSTRAK v

ACKNOWLEDGEMENT viii

APPROVAL ix

DECLARATION xi

LIST OF TABLE xv

LIST OF FIGURE xvi

CHAPTER

1 INTRODUCTION

2.1 Background 1

2.2 Objectives 3

2 LITERATURE REVIEW

2.1 Algae 5

2.1.1 Macroalgae 6

2.1.2 Microalgae 6

2.1.2.1 Chaetoceros calcitrans 8

2.1.2.2 Nannochloropsis oculata 8

2.2 Antioxidant 11

2.2.1 Free radicals 11

2.2.2 Mode of antioxidant 12

2.2.3 Benefit of antioxidant 13

2.2.4 Natural product and antioxidant 14

2.2.5 Assessment of antioxidant 15

2.3 Cancer 20

2.3.1 Breast cancer 21

2.3.2 Cancer treatment 22

2.3.3 Natural product and cancer 23

2.4 Cytotoxic and cell death 24

2.4.1 Mode of cell death 24

2.4.2 Comparison between apoptosis and necrosis 26

2.4.2.1 Mechanism of apoptosis 29

2.4.2.2 Regulation of apoptosis 32

2.4.3 Assessment of apoptosis

2.4.3.1 MTT assay 38

2.4.3.2 Flow cytometric analysis 38

2.4.3.3 Acridine orange/ propidium iodide

(AO/PI) staining method

41

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2.4.3.4 Multiplexed gene expression profiler 42

3 ANTIOXIDANT PROPERTIES

3.1 Introduction 44

3.2 Methodology

3.2.1 Marine microalgae cultivation and sample

preparation

45

3.2.2 Sample extract preparation 46

3.2.3 Antioxidant evaluation

3.2.3.1 Folin-Ciocalteu assay 47

3.2.3.2 DPPH assay 47

3.2.3.3 FRAP assay 48

3.2.3.4 Metal chelating assay 48

3.3 Statistical analysis 49

3.4 Results 49

3.5 Discussion 53

3.6 Conclusion 56

4 ANTICANCER PROPERTIES

4.1 Introduction 57

4.2 Methodology

4.2.1 Cell line maintenance 58

4.2.1.1 Medium preparation 59

4.2.2 Cancer cell line screening 59

4.2.3 Elucidation of cell death mode

4.2.3.1 Cell cycle analysis 60

4.2.3.2 Acridine orange/ propidium iodide

(AO/PI) staining method

61

4.2.3.3 Annexin V-FITC staining 62

4.2.3.4 TUNEL assay 62

4.3 Statistical analysis 63

4.4 Results 64

4.5 Discussion 75

4.6 Conclusion 80

5 GENE EXPRESSION PROFILER

5.1 Introduction 81

5.2 Methodology

5.2.1 Cell treatment 82

5.2.2 RNA extraction 82

5.2.3 Primer design 83

5.2.4 cDNA synthesis 83

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5.2.5 Polymerase chain reaction (PCR) 84

5.2.6 Genomelab GeXP genetic analysis system 84

5.2.7 Fragment analysis and expression profiling

analysis

85

5.2.8 Statistical analysis 85

5.3 Results 86

5.4 Discussion 94

5.5 Conclusion 96

6 GENERAL DISCUSSION 97

7 CONCLUSION AND RECOMMENDATIONS

6.1 Conclusion 99

6.2 Recommendations 100

REFERENCES 101

APPENDIX

I Conway medium content 120

II Experimental design of the project 121

III Gene expression chromatogram profile 122

BIODATA OF STUDENT 135