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
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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).