STUDY OF ANTIMICROBIAL, ANTIOXIDANT AND

CHROMATOGRAPHIC PROFILING OF GILLS AND

CARAPACES EXTRACTS OF MUD CRAB, SCYLLA

SERRATA

IZMER BIN MUSTAPHA

MASTER OF SCIENCE

2016

Study of Antimicrobial, Antioxidant and Chromatographic

Profiling of Gills and Carapaces Extracts of Mud Crab,

Scylla serrata

by

Izmer bin Mustapha

A thesis submitted in fulfilment of the requirements for degree of

Master of Science Program

Faculty of Agro-Based Industry

UNIVERSITI MALAYSIA KELANTAN

2016

i

THESIS DECLARATION

I hereby certify that the work embodied in this thesis is the result of the original research

and has not been submitted for a higher degree to any other University or Institution.

OPEN ACCESS I agree that my thesis is to be made immediately available as hardcopy or on-line open access

(full text).

EMBARGOES I agree that my thesis is to be made available as hardcopy or on-line open access (full text) for a period

approved by the Post Graduate Committee.

Dated from until

.

CONFIDENTIAL (Contain confidential information under the Official

Secret Act 1972)*

RESTRICTED (Contain restricted information as specified by the

organization where research was done)*

I acknowledge that Universiti Malaysia Kelantan reserves the right as follows.

1. The thesis is the property of University Malaysia Kelantan.

2. The library of University Malaysia Kelantan has the right to make copies for the

purpose of research only.

3. The library has the right to make copies of the thesis for academic exchange.

SIGNATURE SIGNATURE OF SUPERVISOR

IC/PASSPORT NO.

Date:

NAME OF SUPERVISOR

Date:

ii

ACKNOWLEDGEMENTS

BISMILLAHIRAHMANIRRAHIM…...In the name of Allah S.W.T, The most

gracious and His blessing and grace, I have finally completed my research and thesis after

two years of hard works, discoveries, happiness and also frustrations.

Firstly, I would like to convey my sincere gratitude toward my advisor Dr Shamsul

Muhamad for the unceasing supports on my master study and related researches, for his

persistence, inspiration, and immense knowledge. His supervisions have massively

helped especially while finalizing the research and this thesis.

Subsequently, I would like to acknowledge the rest of my thesis committee: Dr

Hasnita Che Haron and En. Shazani Sarijan, for their helpful comments and boosts,

including their constructive questions, which have eventually improvised my research in

numerous viewpoints.

My sincere acknowledgment also goes to En. Suhaimi Omar, the main assistant

of UMK Jeli campus postgraduate laboratory, who granted me the access to the various

instruments and research facilities.

Special gratitude to my fellow lab mates, especially Siti Fatimah Zahrah, for

generously assisting in various laboratory assessments. Also a sincere gratitude toward

Mohd Taufiq Jalil, Phd student in Microbiology from USM Penang campus for various

guidance and thoughts.

Finally, I would like to thank my family; my parents, brothers and sister for

being spiritually supportive throughout the research progression…THANK YOU.

iii

TABLE OF CONTENTS

PAGE

THESIS DECLARATION

ACKNOWLEDGEMENT

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATIONS

LIST OF SYMBOLS

ABSTRAK

ABSTRACT

CHAPTER 1 : INTRODUCTION

1.1. Research Background

1.2. Problem Statement

1.3. Research Objectives

1.4. Hypothesis

1.5. Scope Of Research

CHAPTER 2 : LITERATURE REVIEW

2.1. Bioactive Compounds

2.1.1. Types and Bioactivities

2.1.2. Secondary metabolites

2.1.3. Function of Secondary Metabolites in Animals

i

ii

iii

viii

x

xii

xiv

xv

xvi

1

3

3

4

4

6

7

9

12

iv

2.2. Antimicrobial Compounds and Pathogenic Microbes. 13

2.2.1. Antibacterial Agent 14

2.2.2. The Cellular Mechanism of Antimicrobial Agents 16

2.2.3. Emergence of Antibiotic Resistance Strain 17

2.3. Free Radical and Oxidative Stress 18

2.3.1. Oxidative Stress and Cancer 19

2.3.2. Antioxidant Compounds 21

2.4. Marine Invertebrates as Bioactive Compounds Sources 23

2.5. Crustacean 24

2.5.1. Bioactive Compounds in Crustaceans 26

2.5.2. Astaxanthin 28

2.6. Gills and Carapace of Crustaceans 29

2.6.1. Gills 29

2.6.2. Carapaces 30

2.7. Scylla serrata 31

2.7.1. Names and Taxonomy 31

2.7.2. Habitats and Ecological Behaviors 33

2.7.3. General Anatomy 33

2.8. Biological assays 34

2.8.1. Antimicrobial Assays 34

2.8.1.1. Kirby Bauer Test 35

2.8.2. Antioxidant Assay 36

v

2.8.2.1. DPPH Free-radicals Scavenging Assay 37

2.8.2.2. ABTS Free-radicals Scavenging Assay 37

2.8.2.3. Ferric Reduction Antioxidant Power Assay 38

2.8.3. Chromatographic Profiling 39

2.8.3.1. Thin Layer Chromatography (TLC) 40

2.8.3.2. High Performance Liquid Chromatography 41

2.8.4. Bradford Protein Assay 42

2.8.5. Brine Shrimp Lethality Test (BSLT) 43

CHAPTER 3 : MATERIALS AND METHODS

3.1. Chemicals and Instrument 44

3.2. Sample Collection 47

3.3. Sample Preparation 48

3.4. Sample Extraction 48

3.5. Antibacterial Assay 50

3.6. Anticandidal Assay 52

3.7. Antioxidant Assays 54

3.7.1. ABTS Dot Blotting Assay 54

3.7.2. TLC Bioautography 55

3.7.3. ABTS Free-radical Scavenging Assay 58

3.7.4. DPPH Free-radical Scavenging Assay 59

3.7.5. Ferric Reducing Antioxidant Power Assay 60

3.7.6. Effect of pH and Temperature on Antioxidant 61

vi

Activity

3.7.7. Total Phenolic Content (TPC)

3.7.8. Total Terpenoid Content (TTC)

3.8. Total Protein Content

3.9. Chromatographic Profiling

3.9.1. Thin Layer Chromatography (TLC)

3.9.2. High Performance Liquid Chromatography

3.10. Brine Shrimp Lethality Test (BSLT)

3.11. Statistical Analysis

CHAPTER 4 : RESULTS

4.1 Overview

4.2. Sample Preparation

4.3. Sample Extraction

4.4. Antimicrobial Assays

4.5. Antioxidant Assays

4.6 Total Protein Content

4.7. Chromatographic Profiling

4.8. Brine Shrimp Lethality Test

CHAPTER 5 : DISCUSSION

5.1. Significances of Research

5.2. Antimicrobial Activity of S.serrata Extracts

5.3. Antioxidant Activity of S.serrata Extracts

64

65

66

67

67

70

73

74

75

77

77

78

81

106

108

115

117

118

121

vii

5.4. Protein Content of S.serrata Extracts

5.5. Chromatographic Profiling of S.serrata Extracts

5.5. Toxicity of S.serrata Extracts

CHAPTER 6 : CONCLUSIONS AND FUTURE WORKS

6.1. General Summary and Conclusion

6.2. Future work

REFERENCES

APPENDIX A

APPENDIX B

APPENDIX C

APPENDIX D

129

130

134

136

138

140

162

163

168

178

viii

LIST OF TABLES

NO PAGE

2.1

2.2

2.3

2.4

3.1

3.2

4.1

4.2

4.3

4.4

4.5

4.6

Variety of naturally occurred secondary metabolites 11

The most common antioxidants and their antioxidant functions 23

Classification of crustaceans based on Martin and Davis taxonomical 26

system

Taxonomic classifications of S.serrata 32

List of crude S.serrata extracts and the designated solvents 49

HPLC setting for analysing S.serrata extracts 71

Total yield, percent yield and the colour of crude S.serrata extracts 78

Antibacterial activity of S.serrata gills and carapace extracts (5 79

mg/disc) and chloramphenicol (5 µg/disc) against five human

pathogenic bacterial species.

Anticandidal activity of S.serrata gills and carapace extracts (5 80

mg/disc) and ketoconazole (5 µg/disc) against four human pathogenic

yeast species

Antioxidant detection limit, detection time, stability and dot intensity 82

for eight S.serrata extracts, ascorbic acid and astaxanthin after 24

hours of incubation

Number of visible spots and Rf values for eight S.serrata extracts, 85

and astaxanthin after being stained with ABTS detection reagent.

Number of visible spots and Rf values for eight S.serrata extracts, 87

and astaxanthin after being stained with DPPH detection reagent.

ix

4.7 Change of ABTS free-radicals scavenging activity of S.serrata

extracts (5000 µg/ml), ascorbic acid (500 µg/ml) and astaxanthin

(500 µg/ml) at pH 1, 3, 5 and 7.

4.8 Change of ABTS free-radicals scavenging activity of S.serrata

extracts (5000 µg/ml), ascorbic acid and astaxanthin (500 µg/ml) after

heat treatment of 40, 60, 80 and 100°C.

4.9 Chemical screening of S.serrata gills and carapace extracts.

97

101

109

x

LIST OF FIGURES

NO PAGE

2.1

2.2

4.1

4.2

4.3

4.4

4.5

4.6

4.7

4.8

4.9

4.10

The skeletal formula of astaxanthin molecule.

Male adult S.serrata.

Flowchart of Research Methodologies

ABTS dot blotting images after 2 minutes (A) and 2 hours (B) of

incubation in darkness.

TLC chromatograms stained with ABTS detection reagent.

TLC chromatograms stained with DPPH detection reagent.

ABTS free-radicals scavenging activity of S.serrata extracts, ascorbic

acid and astaxanthin.

DPPH free-radicals scavenging activity) of S.serrata extracts,

ascorbic acid and astaxanthin.

Ferric reduction antioxidant potential of S.serrata extracts and

astaxanthin.

ABTS free-radicals scavenging activity of S.serrata extracts (5000

µg/ml), ascorbic acid and astaxanthin (500 µg/ml) at pH 1, 3, 5, 7, 7.7

Change of ABTS free-radicals scavenging activity of S.serrata

extracts (5000 µg/ml), ascorbic acid and astaxanthin (500 µg/ml) at

pH 1, 3, 5 and 7.

ABTS free-radicals scavenging activity for S.serrata extracts,

astaxanthin and ascorbic acid after heat treatment of 40, 60, 80 and

100°C.

28

34

76

83

85

87

90

93

95

98

99

102

xi

4.11 Change of ABTS free-radicals scavenging activity for S.serrata

extracts, astaxanthin and ascorbic acid after heat treatment of 40, 60,

80 and 100°C.

4.12 Total phenolic content for S.serrata extracts and astaxanthin

4.13 Total terpenoid content in 1 mg of crude S.serrata extracts

4.14 Total protein content in S.serrata gills and carapaces extracts

4.15 TLC chromatograms stained with iodine detection reagent.

4.16 TLC chromatograms stained with Folin-Ciocalteu detection reagent.

4.17 TLC chromatograms stained with ferric chloride detection reagent.

4.18 TLC chromatograms stained with aluminium chloride detection

reagent.

4.19 Relative astaxanthin content in S.serrata extracts (100 µg/ml) at

λ = 254 nm.

4.20 A.salina nauplii mortality percentage after 24 hours incubation in

S.serrata extracts and potassium dichromate solution.

103

105

106

107

110

110

111

111

114

116

xii

LIST OF ABBREVIATIONS

A

AA

ABTS

ABTS•+

BE

BSA

BSLT

CPNL

DF

DMSO

DPPH

FRAP

H0

HA

HCl

HPLC

GA

GE

IC50

IC100

Absorbance

Ascorbic Acid

2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid

2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid free-

radical cation

BSA Equivalent

Bovine Serum Albumin

Brine Shrimp Lethality Test

Chloramphenicol

Dilution Factor

Dimethyl Sulphoxide

1,1 diphenyl-2-picrylhydrazyl

Ferric Reducing Antioxidant Power

Null Hypothesis

Alternative Hypothesis

Hydrochloric Acid

High Performance Liquid Chromatography

Gallic Acid

Gallic Acid Equivalent

50% Inhibitory Concentration

100% Inhibitory Concentration

xiii

KCZL

LB

LC50

MP

OS

PDA

Rf

RT

ROS

SA

SAABTS

SADPPH

SD

SN

TE

TLC

TP

TPC

TPTZ

TTC

UV

Ketoconazole

Luria Broth

50% Lethality Concentration

Mortality Percentage

Oxidative Stress

Potato Dextrose Agar

Retention Value

Retention Time

Reactive Oxygen Species

Scavenging Activity

ABTS Scavenging Activity

DPPH Scavenging Activity

Standard Deviation

Serial Number

Trolox Equivalent

Thin Layer Chromatography

Total Protein Content

Total Phenolic Content

2,4,6-Tripyridyl-S-Triazine

Total Terpenoid Content

Ultra-Violet

xiv

LIST OF SYMBOLS

cm

g

l

mg

min

ml

mm

mM

mTorr

n

nm

p

rpm

s

W

µl

µg

%

°C

Å

Centimetre

Gram

Liter

Milligram

Minute

Milliliter

Millimetre

Millimol

Millithorr

Sample size

Nanometre

P-value

Rate of revolution

Second

Watt

Microliter

Microgram

Percent

Degree Celsius

Angstrom

xv

ABSTRAK

Kajian tentang Antimikrob, Antioksida dan Pemprofilan Kromatografi Ekstrak

Insang dan Karapas Ketam Nipah, Scylla Serrata

Kajian ini menfokuskan pemeriksaan untuk aktiviti antimikrob dan antioksida

dalam insang dan karapas S.serrata. Eksperimen dimulakan dengan pengekstrakan tisu-

tisu S.serrata dengan menggunakan air dan etil asetat. Ekstrak mentah kemudiannya diuji

untuk aktiviti antimicrob dan antioksida dengan menggunakan pelbagai ujian.

Pemprofilan kimia dan ujian tahap ketoksidan setiap ekstrak juga dijalankan. Berdasarkan

keputusan ujian antimikrob, tiada aktiviti direkodkan daripada kesemua lapan ekstrak ke

atas mikrob terpilih. Sementara itu untuk aktiviti antioksida, ekstrak akues menunjukkan

aktiviti antioksida yang tinggi dalam ujian ABTS (IC50 111.93 hingga 975.78 µg/ml),

ujian DPPH (IC100 3336.50 hingga 3913.39 µg/ml) dan ujian FRAP (91.57 hingga 171.65

TE). Untuk ujian kestabilan pada suhu berbeza, semua ekstrak menunjukkan sedikit

penurunan aktiviti antioksida pada suhu melebihi 80°C, dengan kadar 1.56% hingga

15.06%. Sementara untuk ujian kestabilan pada pH berbeza, semua ekstrak menunjukkan

penurunan aktiviti antioksida yang besar apabila pH diturunkan melebihi pH 7. Dalam

ujian TPC, ekstrak akues menunjukkan kandungan fenol yang lebih tinggi, dengan 51.00

hingga 58.84 GE, berbanding ekstrak etil asetat dengan 15.54 hingga 47.41 GE.

Seterusnya, ekstrak etil asetat juga menunjukkan kandungan protein yang lebih tinggi,

berbanding ekstrak akues.Dalam pemprofilan kromatografi dengan analisis TLC,

ketidakhadiran flavonoid telah disahkan di dalam kelapan-lapan ekstrak S.serrata. Dalam

pengotoran iodin, ekstrak etil asetat menunjukkan kehadiran tiga hingga empat komponen

organik utama, sementara ekstrak akues menujukkan kehadiran hanya dua komponen

organik utama. Sementara itu dalam analisis HPLC, kehadiran astaxanthin dalam semua

ekstrak akues telah disahkan. Ekstrak insang direkodkan dengan kandungan astaxanthin

yang lebih tinggi berbanding kandungan astaxanthin dalam ekstrak karapas. Akhir sekali

dalam ujian BLST, semua ekstrak akues disahkan tidak toksik (LC50 1219.93 hingga

2797.96 µg/ml) sementara ekstrak etil asetat insang disahkan toksik (LC50 642.00 hingga

886.36 µg/ml). Dengan itu, kajian ini mengesahkan bahawa insang dan karapas S.serrata

mengandungi aktiviti antioksida yang stabil dan berpotensi tinggi. Namun kesemua

ekstrak disahkan tidak mengandungi aktiviti antimikrob terhadap mikrob penyebab

penyakit yang terpilih.

1

ABSTRACT

Study of Antimicrobial, Antioxidant and Chromatographic Profiling of Gills and

Carapace Extracts from Mud crab, Scylla serrata

This research focused on screening for antimicrobial and antioxidant activities

from S.serrata gills and carapaces. The experimental steps started with tissue extraction

in ethyl acetate and water. The extracts were subsequently assayed for antimicrobial

properties toward human pathogenic bacteria and yeasts. Whereas for antioxidant

property, the extracts were tested with ABTS, DPPH and FRAP scavenging activity

assays. The chemical profile and toxicity of each extract was also determined. For the

result, in antimicrobial assays, there was no activity recorded in all eight extracts on

selected human pathogenic microbes. Whereas for antioxidant activity, aqueous extracts

showed significant degree of antioxidant activity in ABTS (IC50 of 111.93 to 975.78

µg/ml), DPPH (IC100 of 3336.50 to 3913.39 µg/ml) and FRAP assays [91.57 to 171.65

TE (mg of Trolox/mg of extract)]. For stability test, all extracts showed slight decrement

of antioxidant activity at temperature higher than 80°C, with 1.56% to 15.06% decrement.

While for pH stability, all extracts showed significant decrement of antioxidant activity

as the pH dropped lower than pH 7. In TPC, aqueous extracts showed significant amount

of phenolic content with 51.00 to 58.84 GE (mg of gallic acid/mg of extract), compared

to ethyl acetate extracts with 15.54 to 47.41 GE (mg of gallic acid/mg of extract).In

protein content assay, ethyl acetate extracts were determined with considerably higher

protein contents than the protein content of aqueous extracts. In chromatographic

profiling with TLC analysis, the absence of flavonoid was confirmed in all S.serrata

extracts. In iodine staining, ethyl acetate extracts were determined with three to four major

organic components, while aqueous extracts were observed with two major organic

components. Whereas in HPLC analysis, the presence of astaxanthin, a xanthophyll

carotenoid, was confirmed in all four aqueous S.serrata extracts. Gills extract were

recorded with slightly higher content of astaxanthin, compared to carapace extracts.

Lastly for BSLT, all aqueous extracts were determined to be non-toxic (LC50 of 1219.93

to 2797.96 µg/ml) while ethyl acetate gills extract were determined to be toxic (LC50

642.00 to 886.36 µg/ml). As a conclusion, this research concluded that S.serrata gills and

carapace extracts carry promising potential of antioxidant property, with notable stability

toward heat exposure. Unfortunately, both extracts do not contain antimicrobial property

toward selected human pathogenic microbes.

CHAPTER 1

2

INTRODUCTION

1.1 Research Background

Recently, crustaceans are being progressively studied for their bioactive

proteins and compounds (Ibañez et al., 2012). In Vellar Estuary, India, a study

was carried with the aim to identify various antimicrobial proteins from the

haemolymph Charybdis lucifera . The investigation was based on the notion of

humeral immunity in marine invertebrates is sustained by numerous antimicrobial

agents in the plasma and blood cells, as well as actions such as haemolymph

coagulation crab (Rameshkumar et al., 2009). Several years later, an investigation

on Scylla traqueberica was completed and resulted with promising discoveries.

The study disclosed the presence of promising unknown antibacterial compound

in the haemoplymph of mud crab, Scylla traqueberica, particularly towards Gram-

negative Vibrio cholerea (Veeruraj et al., 2008).

Crustaceans are known for the superiority in thriving hazardous oxidative

stress induced by surrounding reactive oxygen species (ROS) and pollutants in

their habitats (Lawniczak et al., 2013). For instance, most exoskeletons of marine

crustaceans contain with astaxanthin, a xanthophyll carotenoid with potent

antioxidant activity (Dalei & Sahoo, 2015). Whereas marine extremophile, such

as red alga, was also determined with bioactive antioxidants

3

compounds, but in a form of densely brominated substituted mono- bis- phenols.

These compounds were tested with DPPH free-radicals scavenging assay and

determined with considerably high antioxidant activity, even significantly higher

than synthetic positive control, butylated hydroxytoluene (BHT) (Giddings &

Newman, 2015). Generally, marine bioactive compounds are embedded with

hydroxyl side chains, which have greatly contributed toward the superiority of

their scavenging activity (Sunassee & Davies-Coleman, 2012). Also, marine

microorganism such as psychrophilic Antarctic eubacterium Pseudoalteromonas

halaplanktis was also determined with potent antioxidant compounds, which

appeared to be novel diketopiperazine and several linear peptides (Giddings &

Newman, 2015).

In estuaries of tropical Indo-Pacific regions, mud crab or scientifically

known as Scylla serrata is renowned for its prominent role in culinary (Hamad,

2012). It is also highlighted as one of the tastiest crab species, due to its

palatability, nutritive value and distinctiveness (Finkl & Makowski, 2014). In

Malaysia, S. serrata is occasionally used as remedies for treating cold and dengue

fever (Wong & AbuBakar, 2013). The wide-spread craze and application of S

serrata parts in therapeutic purposes has grown the interest on isolating and

elucidating the potential bioactive substances, which can be perchance used as

antibiotics and may be effective against infectious diseases such as HIV-1;

conditions of multiple bacterial infections (penicillin, cephalosporines,

streptomycin, and vancomycin); or neural tube defects and neuropsychiatric

(Newman & Cragg, 2004). As yet, the number of investigations on crustacean

4

haemolymph and flesh are intensifying rapidly, yet other body parts, such as back

carapace and gills are remained underutilized (Fredrick & Ravichandran, 2012;

Gagneten et al., 2012) .

1.2 Problem Statement

In eastern Malaysia, mud crab or Scylla serrata has been one of the major

components in traditional medication. The decoction of whole crabs are rumoured

to be effective in comforting colds and fever, however there is no clinical study,

or even scientific evidence published regarding this matter. Hence, this research

is aiming to screen for bioactivity, specifically antimicrobial, antioxidant activities

from S. serrata gill and carapace extracts.

1.3. Research Objectives

i. To determine antimicrobial and antioxidant properties in gills and

carapace extracts of S.serrata.

ii. To determine the effect of temperature and pH on antimicrobial and

antioxidant properties in gills and carapace extracts of S.serrata.

iii. To determine the chromatographic profiles of gills and carapace

extracts of S.serrata.

iv. To determine the toxicity of gills and carapace extracts of S.serrata.

5

1.4. Hypothesis

i. The null hypothesis (H0) states S.serrata gills and carapace extracts

have noticeable antioxidant activity. Whereas the alternative

hypothesis (HA) states that S.serrata book gill and carapace extracts do

not have any antioxidant activity.

ii. The null hypothesis (H0) states S.serrata gills and carapace extracts

have noticeable antimicrobial activity. Whereas the alternative

hypothesis (HA) states that S.serrata book gill and carapace extracts do

not have any antimicrobial activity.

1.5. Scope of Research

This research revolved on the determination of antioxidant and

antimicrobial properties from gills and carapace of male and female mud crab,

Scylla serrata. The selected tissues were segregated and extracted with ethyl

acetate and distilled water. For antimicrobial assays, disc diffusion antibacterial

and anticandidal test were performed. Whereas for antioxidant assays, the extracts

were tested with ABTS-free radicals scavenging test, DPPH free- radicals

scavenging test and ferric reduction antioxidant potential test (FRAP).Meanwhile

for antioxidant stability test, the extracts were assayed with modified ABTS free-

radicals scavenging tests, with the aim of monitoring the

6

effect of two variables; pH and temperature. Subsequently,the extracts were

assayed with total phenolic content (TPC), total terpenoid content (TTC), total

protein content (TP), thin layer chromatography (TLC) and ended with HPLC

analysis. The research was concluded with toxicity assay on S.serrata gills and

carapaces extracts using brine shrimp lethality test (BLST).

7

CHAPTER 2

LITERATURE REVIEW

2.1. Bioactive Compounds

Bioactive compounds or nutraceuticals are natural constituents in food and

carry noticeable effect on living creatures, especially at cellular level. These

substances are usually found in small quantities in foods and not essential to living

organism since the cells can operate and thrive normally without them (Kris-

Etherton et al., 2002). For humans, foreign bioactive compound could possibly

convey noticeable influences, positively or negatively (Liu, 2013).For instance,

carotenoids such as lycopene and beta-carotene are highly suggested for healthy

dietary due to the ability in decreasing the risk of getting certain cancers (Nile &

Park, 2014). Unfortunately, consuming large doses of carotenoid might be

harmful to individual with lung cancer (Bjelakovic et al., 2014).

Bioactive compounds are usually harvested from almost every living

creature, especially plants and certain animals (Kris-Etherton et al., 2002). With

the discovery and elucidation of various beneficial bioactive compounds,

researchers have been synthetically manufacturing the analogues and the tests on

human are being selectively ventured (Bonilla et al., 2015). Lucrative bioactive

8

compounds such as flavonoids and phytoestrogens can be exclusively found in

plants while certain phenolic compounds with unique side chains can also be

found in animals (Cos et al., 2003).

The production of various synthetic bioactive compounds is expanding

rapidly as production costs can be significantly minimized with the introduction

of various cheaper substrates and precursors during manufacturing process

(Mitchell, 2011). The application of genetically modified organisms, particularly

bacteria and fungus, in bioactive compounds production has greatly expanded the

prospect of pharmaceutical industry (Cragg & Newman, 2013). Unfortunately, the

inadequacy on research regarding safety and long-term effects is getting a major

concern from scientific community. Certain bioactive compounds can eventually

cause harmful effect, or in certain cases indirectly react with other prescript drugs

and cause chelating and amplifying effects. The optimum of bioactive compounds

intake for every person is slightly different as a proper prescription should be

issued by the doctors (Grienke et al., 2014).

2.1.1. Types and Activities of Natural Bioactive Compounds

Bioactive compounds are naturally occurred in various chemical structures

and carry various functions (Cragg & Newman, 2013). Most bioactive compounds

in plants are phenolic-structured (Martins et al., 2011). Compounds such as

flavonoids and terpenoids can be found in almost plant-based food such as cereals,

legumes, nuts, essential oils, fruits and wines (Bittner et al., 2013).