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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
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