Faculty of Resource Science and Technology
Antimicrobial and Antioxidant Properties in Tissues of Mud Crab from Scylla olivacea and Scylla paramamosain
Noorasmin binti Mokhtar Ahmad
Master of Science 2022
Antimicrobial and Antioxidant Properties in Tissues of Mud Crab from Scylla olivacea and Scylla paramamosain
Noorasmin binti Mokhtar Ahmad
A thesis submitted
In fulfillment of the requirements for the degree of Master of Science (Biochemistry)
Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARAWAK
2022
i
DECLARATION
I declare that the work in this thesis was carried out in accordance with the regulations of Universiti Malaysia Sarawak. Except where due acknowledgements have been made, the work is that of the author alone. The thesis has not been accepted for any degree and is not concurrently submitted in candidature of any other degree.
………
Signature
Name: Noorasmin binti Mokhtar Ahmad
Matric No.: 18020120
Faculty of Resource Science and Technology Universiti Malaysia Sarawak
Date: 27th April 2022.
ACKNOWLEDGEMENT
First and foremost, I would like to express my gratitude and appreciation to my supervisor, Professor Dr. Awang Ahmad Sallehin bin Awang Husaini, and my co-supervisor, Pn Wan Roslina Wan Yusof and Encik Mohd Alhafiizh bin Zailani for spending much of the time for guidance, supervision, and patience to me throughout the research progress. Besides, I would like to express my special thanks to my supervisor and my co-supervisor for giving the opportunities and encouraging me to carry out my research project in a relatively new method.
I have learned a lot of unique laboratory and sampling skills throughout my research project.
Further gratitude from me to Dr. Ngieng Ngui Sing, Mrs. Zaliha Suadi, Miss Carolynna James, Miss Haifa Arghnia A. Jamal, Miss Khairun Najibah binti Mohd Said, Miss Nurul Aida Qarina binti Mohd Razali, and Miss Sharifah Nur Asyilah, who are my lab-mates and friends from Molecular Genetic Lab 2 laboratory, Faculty of Resource Science and Technology and Mrs.
Sheela Ungau, the lab assistant of Molecular Genetic Lab 2 laboratory for their assistances, supports, cares, valuable advice and concerns to me throughout my research progress in Molecular Genetic Lab 2 laboratory. They share their research experiences with me and help me a lot in the laboratory sessions during my research project.
Finally, I would like to extend my gratitude to my family members, especially my parents, for their continuous financial and moral support when I encountered problems along the way of my research project. A special thanks from me to the board of University Malaysia Sarawak for providing the necessary facilities and experiment materials for my research activities.
Thank you.
iii ABSTRACT
Mangrove crab (genus Scylla) is a kind of mud crab that has long been recognized as a rich source of protein. Several antimicrobial peptides from mud crabs have recently been identified by their antibacterial activity against Gram-positive and Gram-negative bacteria.
Meanwhile, antioxidant capabilities found in mud crabs have been linked to cell protection from free radicals. Unfortunately, to date, little is known about the antimicrobial and antioxidant activities of Scylla olivacea and Scylla paramamosain. This study investigates the natural antibacterial and non-enzymatic antioxidant properties from whole tissue extract of two mud crab species, S. olivacea, and S. paramamosain. The data from this study may offer the basis for much future research. In this study, extraction through maceration was utilized to produce tissue extract from S. olivacea and S. paramamosain.
The tissue extracts were diluted and tested against five different bacteria through microdilution analysis. These extracts were further characterized through non-enzymatic antioxidant activity. The identities of chemical constituents in the tissue extracts were then identified using Gas Chromatography-Mass Spectrometry (GC-MS). The antibacterial activities were determined using the broth microdilution method. The methanol extract of S. olivacea and S. paramamosain was found to inhibit the growth of Staphylococcus aureus, Klebsiella pneumoniea, Pseudomonas aeruginosa, Escherichia coli, and Salmonella typhi with minimum inhibitory concentration (MIC) and minimum bacteriostatic concentration (MBC) ranging from 1.56-6.25 μg/μL. Additionally, it was revealed that the highest Diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging activity was in the extracts of S. olivacea and S. paramamosain, which were 1.23 mg/mL and 1.28 mg/mL, respectively. In terms of ferric reducing antioxidant potential (FRAP), S.
paramamosain extracts in methanol, ethyl acetate, and n-hexane solvent showed better
results than the S. olivacea extracts. The FRAP value obtained was similar at 1 mg/mL, which is 0.033±0.004 Fe (II)/mg for both methanol and ethyl acetate extract of S.
paramamosain. It was observed that the highest nitric oxide scavenging activity for both species was in n-hexane extract with the EC50 values of 0.35 mg/mL and 0.36 mg/mL, respectively. The extract contained 225 compounds with up to 40 reported from previous studies to exhibit antimicrobial, antioxidant, or anti-inflammatory properties.
Keywords: Scylla olivacea, Scylla paramamosain, antibacterial, antioxidant, Gas Chromatography-Mass Spectrometry.
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Sifat Antimikrob dan Antioksidan dalam tisu Ketam Lumpur dari Scylla olivacea dan Scylla paramamosain
ABSTRAK
Ketam bakau (genus Scylla) adalah sejenis ketam lumpur yang telah lama dikenali sebagai sumber protein yang kaya. Beberapa peptida antimikroba dari ketam lumpur baru-baru ini dikenal pasti mempunyai aktiviti antibakteria terhadap bakteria Gram-positif dan Gram- negatif. Sementara itu, keupayaan antioksidan yang terdapat pada ketam lumpur telah dikaitkan dengan perlindungan sel dari radikal bebas. Namun, setakat ini, sedikit yang diketahui mengenai aktiviti antimikroba dan antioksidan Scylla olivacea dan Scylla paramamosain. Oleh itu, kajian ini menyiasat sifat antioksidan antibakteria dari ekstrak keseluruhan tisu dari dua spesies ketam lumpur, S. olivacea dan S. paramamosain. Data dari kajian ini dapat menawarkan peluang sebagai dasar bagi banyak penelitian terapan di masa depan. Dalam kajian ini, pengekstrakan melalui maserasi digunakan untuk menghasilkan ekstrak tisu dari S. olivacea dan S. paramamosain. Ekstrak tisu dicairkan dan diuji terhadap lima bakteria yang berbeza melalui analisis mikrodilusi. Ekstrak ini kemudian dicirikan melalui aktiviti antioksidan nonenzimatik. Identiti unsur kimia dalam ekstrak tisu kemudian dikenal pasti menggunakan Kromatografi Gas dan Spektometri Jisim. Kegiatan antibakteria ditentukan menggunakan kaedah mikrodilusi. Ekstrak metanol S. olivacea dan S. paramamosain didapati menghalang pertumbuhan Staphylococcus aureus, Klebsiella pneumoniea, Pseudomonas aeruginosa, Escherichia coli, dan Salmonella typhi dengan kepekatan perencatan minimum (KPM) dan kepekatan minimum bakteriostatik (KMB) mulai dari 1.56 -6.25 μg / μL. Selain itu, dinyatakan bahawa aktiviti pemuliharaan radikal bebas Diphenyl-2-picryl-hydrazyl (DPPH) tertinggi terdapat pada ekstrak S. olivacea dan S. paramamosain, masing-masing 1.23 mg / mL dan
1.28 mg / mL. Dari segi potensi pengurangan antioksidan besi (FRAP), ekstrak S.
paramamosain dalam metanol, etil asetat, dan pelarut n-heksana menunjukkan hasil yang lebih baik daripada ekstrak S. olivacea. Nilai FRAP yang diperoleh adalah serupa pada 1 mg / mL, iaitu 0,033 ± 0,004 Fe (II) / mg untuk ekstrak metanol dan etil asetat S.
paramamosain. Telah diperhatikan bahawa aktiviti pengumpulan oksida nitrat tertinggi bagi kedua-dua spesies tersebut adalah ekstrak n-heksana dengan nilai EC50 masing- masing 0.35 mg / mL dan 0.36 mg / mL. Ekstrak itu mengandungi 225 sebatian dengan hingga 40 yang dilaporkan dari kajian sebelumnya menunjukkan sifat antimikroba, antioksidan, atau anti-radang.
Kata kunci: Scylla olivacea, Scylla paramamosain, antibakteria, antioksidan, Kromatografi Gas dan Spektrometri Jisim
Kata kunci:
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TABLE OF CONTENTS
Page
DECLARATION i
ACKNOWLEDGEMENT ii
ABSTRACT iii
ABSTRAK v
TABLE OF CONTENTS vii
LIST OF TABLES xi
LIST OF FIGURES xii
LIST OF ABBREVIATIONS xv
CHAPTER 1: INTRODUCTION 1
1.1 Problem Statements 2
1.2 Objectives 3
1.3 Hypothesis 3
CHAPTER 2: LITERATURE REVIEW 4
2.1 Crustaceans 4
2.2 Mud Crab 4
2.2.1 Taxonomy of mud crab 5
2.2.2 Scylla olivacea 9
2.2.3 Scylla paramamosain 10
2.3 Bio-active Compounds 12
2.3.1 Primary and Secondary Metabolites 12
2.3.2 Functions of Secondary Metabolites in Animal 13
2.3.3 Bio-active Compound derived from Marine Invertebrate 14
2.4 Antimicrobial Compound and Pathogenic Microbes 17
2.4.1 Antimicrobial Compound 17
2.5 Free Radical 21
2.6 Oxidative Stress 22
2.7 Antioxidant Compounds 23
2.8 Biological Assays 26
2.8.1 Extraction Using Different Type of Solvents 26
2.8.2 Antimicrobial assay 28
2.8.3 Antioxidant assay 30
2.9 Chromatography Profiling 31
2.9.1 Gas Chromatography-Mass Spectrometry (GC/MS) 31
CHAPTER 3: METHODOLOGY 33
3.1 Sampling Site 33
3.2 Sample Collection and Sample Preparation 34
3.3 Sample extraction 37
3.4 Antibacterial methods 38
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3.4.1 Minimum Inhibition Concentration (MIC) 38
3.4.2 Minimum Bactericidal Concentration (MBC) 39
3.5 Non-enzymatic antioxidant activity 39
3.5.1 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) Free Radical Scavenging Activity 39
3.5.2 Ferric Reducing Antioxidant Potential (FRAP) 40
3.5.3 Nitric Oxide Scavenging Assay 41
3.6 Other Compound Analysis 42
3.6.1 Total Phenolic Content 42
3.6.2 Total Nitrate 43
3.7 Gas Chromatography/Mass Spectrometry (GC/MS) analysis 44
CHAPTER 45: RESULTS AND DISCUSSION 45
4.1 Sample extraction yield 45
4.2 Antibacterial Activity 49
4.2.1 Determination of minimum inhibitory concentration (MIC) and minimum
bactericidal concentration (MBC) 49
4.3 Non-enzymatic antioxidant properties 60
4.3.1 DPPH Free-radical scavenging activity: 60
4.3.2 Ferric Reducing Antioxidant Potential (FRAP) Activity 65
4.3.3 Nitric Oxide Scavenging Activity: 70
4.4 Other compound analysis 74
4.4.1 Total phenolic content 74
4.4.2 Total nitrate (nitrite) 77
4.5 Gas Chromatography/Mass Spectrometry (GC-MS) 79
CHAPTER 5: CONCLUSION, LIMITATION AND RECOMMENDATIONS 103
5.1 Conclusions 103
5.2 Limitations and Future Recommendations 104
REFERENCES 105
APPENDICES 131
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LIST OF TABLES
Page
Table 2.1 Taxonomy of mud crab adapted from Shelley and Lovatelli (2011) 6 Table 2.2 The most significant bioactive compound isolated from marine by
Datta et al. (2015)
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Table 4.1 Extraction yield of muscle extract from both mud crab species, S.
olivacea and S. paramamosain from maceration using methanol, ethyl acetate, and n-hexane. Data expressed as mean ± sd. (n=7)
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Table 4.2 Minimum Inhibitory Concentration (MIC) (μg/ml) and Minimum Bactericidal Concentration (MBC) (μg/ml) of S. olivacea and S.
paramamosain whole methanol extract against test organisms
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Table 4.3 DPPH scavenging activity of S. olivacea and S. paramamosain 60 Table 4.4 FRAP value of S. olivacea and S. paramamosain at the highest
concentration of 1 mg/mL
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Table 4.5 Nitric oxide scavenging activity of S. olivacea and S. paramamosain 69 Table 4.6 Compound identified by GC-MS in methanol, ethyl acetate, and
hexane extract of S. olivacea
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Table 4.7 Compound identified by GC-MS in methanol, ethyl acetate, and hexane extract of S. Paramamosain
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Table 4.8 The zoochemicals identified from methanol, ethyl acetate, and n- hexane crude extracts of mud crab species, S. olivacea, and S.
paramamosain and their biological functions.
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LIST OF FIGURES
Page
Figure 2.1a Illustrations showing diagnostic taxonomic characteristics of part of the carapace of the Scylla genus: A) S. serrata; B) S. tranquebarica;
C) S. paramamosain; D) S. olivacea. (Keenan et al., 1998).
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Figure 2.1b Drawings of right cheliped Scylla species showing diagnostic taxonomic features: A) S. serrata; B) S. tranquebarica; C) S.
paramamosain; D) S. olivacea (Keenan et al., 1998).
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Figure 2.2a Front (a) and back (b) view of Scylla olivacea 9
Figure 2.2b Front (a) and back (b) view of Scylla paramamosain 10 Figure 3.1 Location of the sampling site shown on Google map (Sungai
Asajaya).
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Figure 3.2a Morphological features of Scylla olivacea showing a pair of cheliped, 3 pair of walking leg and a pair of swimming leg.
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Figure 3.2b Morphological features of Scylla paramamosain showing a pair of cheliped, 3 pair of walking leg and a pair of swimming leg.
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Figure 3.3 Transportation of crab in tap water 33
Figure 3.4 Thermal shock in ice water 33
Figure 3.5 Destroying the nerve center through the spiking method 34 Figure 4.1 The yield of extraction against extraction species and extraction
solvent. Data expressed as mean ± sd. (n=7)
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Figure 4.2a Minimum Bactericidal Concentration (MBC) of S. olivacea whole methanol extract against (A) S. aureus, (B) S. typhi, (C) E. coli, (D) P. aeruginosa, and (E) K. pneumoniae. (R1) Replicate 1; (R2) Replicate 2; (R3) Replicate 3. The MBC values are as shown above.
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Figure 4.2b Minimum Bactericidal Concentration (MBC) of S. paramamosain whole methanol extract against (A) S. aureus, (B) S. typhi, (C) E.
coli, (D) P. aeruginosa, and (E) K. pneumoniae. (R1) Replicate 1;
(R2) Replicate 2; (R3) Replicate 3. The MBC values are as shown above.
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Figure 4.3a DPPH radical scavenging activity of different extracts of S.
olivacea. Line graph represented the mean ± sd (n = 7).
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Figure 4.3b DPPH radical scavenging activity of different extracts of S.
paramamosain. Line graph represented the mean ± sd (n = 7).
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Figure 4.4a FRAP value of three different solvent extracts of mud crab, S.
olivacea. Line graph represented the mean ± sd (n = 7).
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Figure 4.4b FRAP value of three different solvent extracts of mud crab, S.
paramamosain. Line graph represented the mean ± sd (n = 7).
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Figure 4.5a Nitric oxide scavenging activity of three different solvent extract of mud crab, S. olivacea. Line graph represented the mean ± sd (n = 7).
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Figure 4.5b Nitric oxide scavenging activity of three different solvent extract of mud crab, S. paramamosain. Line graph represented the mean ± sd (n = 7).
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Figure 4.6 Total Phenolic Content of three different solvent extracts of mud crab, S. olivacea, and S. paramamosain. Data expressed as mean ± sd. (n=7)
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Figure 4.7 Total Nitrate Content of three different solvent extracts of mud crab, S. olivacea, and S. paramamosain. Data expressed as mean ± sd. (n=7)
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Figure 4.8 GC-MS chromatogram of methanol extract of S. olivacea. 79
Figure 4.9 GC-MS chromatogram of ethyl acetate extract of S. olivacea. 80 Figure 4.10 GC-MS chromatogram of hexane extract of S. olivacea. 81 Figure 4.11 GC-MS chromatogram of methanol extract of S. paramamosain. 82 Figure 4.12 GC-MS chromatogram of ethyl acetate extract of S.
paramamosain.
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Figure 4.13 GC-MS chromatogram of hexane extract of S. paramamosain 84
xv
LIST OF ABBREVIATIONS
% Percentage
°C Degree of Celcius
µg/mL Microgram per mililitres
AMP Antimicrobial peptides
G- bacteria Gram-negative bacteria
CFU/mL Colony-forming units per milliliter
cm Centimetres
DNA Deoxyribonucleic acid
DPPH 2,2-diphenyl-1-picrylhydrazyl
EC50 Half maximal effective concentration
FeCl3 Iron(iii) chloride
g/mL Gram per milliliters
G+ bacteria Gram-positive bacteria
GC/MS Gas chromatography-mass spectroscopy
gL-1 Gram per liters
HCl Hydrochloric acid
LPS Lipopolysaccharides
LTA Lipoteichoic acid
MBC Minimum bactericidal concentration
mg/mL Miligram per mililitres
MHA Mueller Hinton agar
MHB Mueller Hinton broth
MIC Minimum inhibitory concentration
mL Milliliters
mmol Millimole
NIST National institute of standards and technology's
OH Hydroxide
PBS Phosphate-buffer saline
RNA Ribonucleic acid
RNS Reactive nitrogen species
ROS Reactive oxygen species
rpm Rotations per minutes
SPCA Society for the prevention of cruelty to animals
TPC Total phenolic content
TPTZ 2,4,6-tris(2-pyridyl)-s-triazine
w:v Weight/ volume
1 CHAPTER 1
INTRODUCTION
Mangrove crabs or mud crabs are marine organisms that live in mangrove swamps and can be found in various species and families (Azra & Ikhwanuddin, 2016). In many regions, these crabs are prepared as soup in meals because of the delectable taste. Apart from having tasty meat, it is also rich in nutrition (Paul et al., 2015). Mud crabs have been mentioned to demonstrate their potential as antioxidants and antimicrobial agents (Salaenoi et al., 2006). In Malaysia, older people prefer to eat mud crab soup as a common remedy and folk medicine to reduce dengue fever symptoms (Wan Yusof et al., 2017).
Antioxidants are critical for various nutritional effects and thus tend to be used to treat a variety of health problems. Natural antioxidants have received increased attention in recent years (Lourenço et al., 2019). These antioxidants are abundant in nature as bioactive compounds or can be extracted as bioactive peptides, and marine species are one of the valuable resources for bioactive peptides. This crab is loaded with bioactive peptides that perform critical biological functions, one of which is of interest to antioxidants (Sujeetha et al., 2015).
Antimicrobial peptides (AMPs) are a class of host immune response molecules that play a crucial role in demonstrating antimicrobial interest in fighting against invading microorganisms (Wan Yusof et al., 2017). AMPs in marine animals have piqued scientists' interest since 1982 due to their role in developing promising antibacterial remedies (Tanaka S. et al., 1982; Ohashi K. et al., 1984). Nakamura et al. (1988) characterized an AMP in crabs, Tachyplesin. Tachyplesin is a peptide traced from a combination of 18 amino acids isolated from the hemocytes of the Japanese horseshoe crab Tachypleus
tridentatus. The AMPs are thin and come in various shapes, lengths, and sequences (primarily less than 10 kDA).
Additionally, many fish and marine invertebrates are highly susceptible to bacterial infection in the water environment (Pandey et al., 2010). Thus, AMPs contribute to the innate immunity of the host against pathogens and diseases. AMPs are also thought to have antibacterial, antifungal, antiprotozoal, anti-yeast, and antiviral properties (Prakash et al., 2011). Countless antimicrobial peptides in the crab have been discovered and published over the last few years. Therefore, the present research aims to study mud crabs' antimicrobial and antioxidant properties, S. olivacea and S. paramamosain.
1.1 Problem Statements
The emergence of new diseases, resistant pathogenic microorganisms, and development of life-threatening viruses has increased the need for novel bioactive compounds to be explored (Fair & Tor, 2014). Apart from this, the search for natural antioxidants as safe alternatives has become an important aspect of the food industry because of the potential health hazards caused by synthetic antioxidants (Ngo et al., 2011).
Many recent studies have revealed that antioxidants in crustaceans play a vital role in antioxidant responses caused by oxidative stress. When living in water environments exposed to microorganisms, innate immunity plays a part in preventing infections in the host. Little research on the antimicrobial and antioxidant activity of the species S. olivacea has been reported to date. In the meantime, a few papers were written on antioxidants and antimicrobial peptides for S. paramamosain, but no study was undertaken in Sarawak, Malaysia. The findings of this study may provide a foundation for much future applied research.
3 1.2 Objectives
The objectives of this study are:
i. To establish an optimal method to extract the bioactive compound from the tissue of mud crab S. olivacea and S. paramamosain using three different solvents selected based on polarity;
ii. To characterize the antimicrobial properties of S. olivacea extracts and S.
paramamosain extracts tissue against five bacterial strains; and
iii. To characterize the antioxidant properties of S. olivacea extracts and S.
paramamosain extracts.
1.3 Hypothesis
Bioactive compound with antimicrobial and antioxidant properties is present in tissues of S. olivacea and S. paramamosain.
CHAPTER 2
LITERATURE REVIEW
2.1 Crustaceans
A diverse community of invertebrate species includes active animals such as crabs, lobsters, shrimp, krill, copepods, amphipods, and more sessile organisms such as barnacles are crustaceans (Kennedy, 2020).
Crabs belong to a group called the Brachyura and decapod crustaceans. They have a shallow "tail" projection, and their narrow abdominals are entirely concealed under the thorax. There are about 7,000 valid crab species, and there are more than 800 freshwater crabs. Water crabs, shore crabs, crayfish, and mangrove crabs are included among the crab types (Marine Education Society of Australasia, 2020).
All crabs have four pairs of walking legs and one pair of pincers (chelipeds). The first pair of crab legs are the chelipeds, which they use to catch and carry food, dig, break clecks, and repel predators. The carapace covers the internal organs of the head, thorax, and gills. The eyes are on the ends of small stalks and have a series of short legs that specialize in food processing and chewing operations that form the base of the mouth. The abdomen is thin and holds tightly under the body. Crabs have distinct sexes and are distinguished by the size of the belly (Marine Education Society of Australasia, 2020).
2.2 Mud Crab
Mud crabs are primarily found in mangrove environments. Due to its high nutritional value, it is consumed by both sea animals and humans. Mud crabs are widely
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grown in many Asian countries, including Malaysia, Indonesia, and China, due to their commercial importance (Azra & Ikwanuddin, 2016). In mangrove areas with an optimal salinity of 20 to 30 gL-1, mud crab of the genus Scylla can be commonly found (Nurdiani
& Zeng, 2007).
Four different members in the Scylla genus are Scylla paramamosain, Scylla serrata, Scylla olivacea, and Scylla transquebarica, based on genetic evidence and morphological character. The frontal lobe spines and the chelipeds are two morphological characters that help identify the genus (Keenan et al., 1998). Ikhwanuddin et al. (2011) reported on mud crab biological details and population characteristics in the Sematan mangrove forest, Malaysia, the South China Sea coastal waters, and the famous mud crab species in the South China Sea was S. olivacea and S. transquebarica.
2.2.1 Taxonomy of mud crab
The use of DNA sequencing has identified the taxonomy of mud crab, allozyme electrophoresis, and morphometrics to classify mud crab species obtained from the Red Sea to the Indo-Pacific (Keenan et al., 1998). S. serrata, also known as the giant mud crab, is the first species identified, followed by S. olivacea; orange mud crab, S.
tranquebarica; purple mud crab, and S. paramamosain; green mud crab (Ikhwanuddin et al., 2011). Table 2.1 below describe the taxonomy of mud crab adapted from Shelley and Lovatelli (2011).
Table 2.1: Taxonomy of mud crab adapted from Shelley and Lovatelli (2011)
Kingdom Animalia
Phylum Arthroposa
Subphylum Crustacea
Class Malacostraca
Order Decapoda
Infraorder Branchyura
Family Portunidae
Genus Scylla
Besides the colour, the four species of mud crab can be identified through the structure of carapace (Figure 2.1a) and cheliped (Figure 2.1b), as shown below. Based on Figure 2.1a, the carapace of Scylla serrata is observed to be in blunted point and high.
Whereas Scylla tranquebarica also has a part of the blunted carapace, it is moderate in appearance. Meanwhile, Scylla paramamosain has a rounded and low part of carapace, and Scylla olivacea has a triangular and intermediate part of the carapace.
