Post on 04-Nov-2019
ANALYSIS OF METHANOL EXTRACT OF SOLEN SARAWAKENSIS
Norshafida Syazarina bt Ibrahim
This project is submitted in partial fulfillment of the requirements for the degree of
Bachelor of Science with Honours
(Resource Chemistry)
Faculty of Resource Science and Technology
(UNIVERSITI MALAYSIA SARAWAK)
2008
ANALYSIS OF METHANOL EXTRACT OF SOLEN SARAWAKENSIS
Norshafida Syazarina bt Ibrahim
Bachelor of Science with Honours
(Resource Chemistry)
2008
Faculty of Resource Science and Technology
i
DECLARATION
No portion of the work referred in this dissertation has been submitted in support of an
application for another degree of qualification of this or any other university of institution of
higher learning.
_________________________
Norshafida Syazarina Ibrahim
Program of Resource Chemistry
Faculty of Resource Science and Technology
University Malaysia Sarawak
ii
ACKNOWLEDGEMENT
First and foremost, I would like to thank God because I’m able to accomplish the
report on time. Also, my sincere appreciation goes to Dr. Kamarul ‘Ain Mustafa for her
guidance and patience in guiding and helping me with relevant informations throughout this
project to ensure that it was completed successfully.
Besides, I would like to extent my gratitude to my co-supervisor Dr. Siti Akmar
Khadijah Abd. Rahim and all the lecturers for willingly shared their expertise in their field of
specialization which contributes to the timely completion of this project.
Also, not to forget, to post graduate student, Nuraqilah Othman, lab assistants and all
my fellow friends for their support and help. Finally, I would like to express my gratitude to
my parents who supported me all this while in completing the project.
Thank you so much.
iii
TABLE OF CONTENTS
Page
DECLARATION i
ACKNOWLEDGEMENT ii
TABLE OF CONTENTS iii
LIST OF ABBREVIATIONS vii
LIST OF TABLES ix
LIST OF FIGURES xi
ABSTRACT & ABSTRAK xii
CHAPTER 1: INTRODUCTION 1
CHAPTER 2: LITERATURE REVIEW 3
2.1 Classification of Razor Clam 3
2.2 Distribution of Razor Clam 4
2.3 General Characteristic of the Molluscs 4
2.4 Secondary Metabolism from Some Marine Organism 6
2.5 The Potential of Marine Organism for Drug Development 13
2.6 Previous Studies on Razor Clam (Solen spp) 17
iv
CHAPTER 3: MATERIALS AND METHOD
3.1 General Experimental Procedure 19
3.2 Sample Collection 20
3.3 Extraction
3.3.1 Extraction of Solen Shell 20
3.3.1.1 Cyclohexane Extraction 20
3.3.1.2 Methanol Extraction 20
3.3.2 Extraction of Solen flesh 21
3.3.2.1 Cyclohexane Extraction 21
3.3.2.2 Methanol Extraction 21
3.4 Isolation and Purification of Chemical Constituents
3.4.1 Thin Layer Chromatography 21
3.4.2 Iodine Vapour Test 22
3.4.3 Vanillin Dipping (Vanillin-Sulfuric Acid Reagent) 22
3.4.4 Column Chromatography 22
3.5 Analysis of Chemical Constituents
3.5.1 Gas Chromatography Mass Spectrometry 23
3.5.2 Fourier Transform Infra Red Spectrometry 23
v
3.5.3 Nuclear Magnetic Resonance Spectrometry 24
CHAPTER 4: RESULTS AND DISCUSSION
4.1 Extraction 25
4.2 Analytical TLC of Crude Extract Using Single Solvents 26
4.2.1 Analytical TLC of Crude Extract from Shell of Solen 26
Sarawakensis
4.2.1.1 Cyclohexane Crude Extract 26
4.2.1.2 MeOH Crude Extract 27
4.2.2 AnalyticaL TLC of Crude Extract from Flesh of Solen 28
Sarawakensis
4.2.2.1 Cyclohexane Crude Extract 28
4.2.2.2 MeOH Crude Extract 29
4.3 Analytical TLC of Crude Extract Using Two Solvents System 31
4.3.1 Analytical TLC of Crude Extract from Shell of Solen 31
Sarawakensis
4.3.1.1 Cyclohexane Crude Extract 31
4.3.1.2 MeOH Crude Extract 32
4.3.2 AnalyticaL TLC of Crude Extract from Flesh of Solen 33
Sarawakensis
vi
4.3.2.1 Cyclohexane Crude Extract 33
4.3.2.2 MeOH Crude Extract 34
4.4 Column Chromatography of MeOH Crude Extract 35
4.5 Gas Chromatography-Mass Spectrometry 37
4.6 Fourier Transform Infra Red Spectrometry 40
4.7 Nuclear Magnetic Resonance Spectrometry 41
CHAPTER 5: CONCLUSION AND SUGGESTIONS 45
REFERENCES 47
vii
LIST OF ABBREVIATIONS
CC Column Chromatography
cm Centimeter
CHCl3 Chloroform
CaCO3 Calcium Carbonate
DCM Dichloromethane
DEPT Distortionaless Enhancement Polarization Transfer
EtOAc Ethyl Acetate
FTIR Fourier Transform Infrared
GC-MS Gas Chromatography Mass Spectrometry
g Gram
HSV-1 Herpes Simplex Virus
HIV Human Immunodeficiency Virus
H2SO4 Sulphuric acid
I2 Iodine
IR Infrared
KBr Potassium Bromide
LD50 Lethal Dose
MeOH Methanol
m Meter
mm Milimeter
m Micrometer
mL Milliliter
g Microgram
mg Milligram
L Microliter
NMR Nuclear Magnetic Resonance Spectroscopy
ng Nanogram
viii
OBIS Ocean Biogeographic Information System
Rf Retention Factor
Rt Retention Time
sp Species
TLC Thin Layer Chromatography
VSV Vesicular Stomatitis Virus
VCAM-1 Vascular Cell Adhesion Molecule
ix
LIST OF TABLES
Page
Table 2.1
Solen sp and their distribution listed in OBIS Indo-Pacific
Molluscan Database (Reid, 2006)
5
Table 2.2 Potential therapeutic compounds from marine sources
(Kijjoa and Sawangwong, 2004)
14
Table 2.3 1998 Antitumor pharmacology of marine natural product
with determined mechanism of action
16
Table 2.4 1998 Antitumor pharmacology of marine natural product
with undetermined mechanism of action
17
Table 3.5 Solvents system used for CC
23
Table 4.6 Percentage yield from the extraction of Solen sarawakensis
25
Table 4.7 Analytical TLC of cyclohexane crude extract from shell of
Solen sarawakensis using single solvent
27
Table 4.8 Analytical TLC of MeOH crude extract from shell of Solen
sarawakensis using single solvent
28
Table 4.9 Analytical TLC of cyclohexane crude extract from flesh of
Solen sarawakensis using single solvent
29
Table 4.10 Analytical TLC of MeOH crude extract from flesh of
Solen sarawakensis using single solvent
30
Table 4.11 Analytical TLC of cyclohexane crude extract from shell of
Solen sarawakensis using combination solvent system
31
Table 4.12 Analytical TLC of MeOH crude extract from shell of Solen
sarawakensis using combination solvent system.
32
Table 4.13 Analytical TLC of cyclohexane crude extract from flesh of
Solen sarawakensis using combination solvent system.
33
Table 4.14 Analytical TLC of MeOH crude extract from flesh of Solen
sarawakensis using combination solvent system.
34
Table 4.15
Rf values of combine fractions from the fractionation of
MeOH crude extract from the flesh Solen sarawakensis
using combination solvent of DCM:EtOAc (3:1)
36
Table 4.16 1H NMR and multiplicity of fraction KMNP-S1-61-12 42
x
Table 4.17 13
C and DEPT for fraction KMNP-S1-61-12 44
xi
LIST OF FIGURES
Page
Figure 2.1 Classification of razor clam 3
Figure 2.2 Siliqua patula 6
Figure 2.3 Solen sarawakensis 6
Figure 4.4 Analytical TLC of KMNP-S1-61-12 37
Figure 4.5 Gas chromatogram obtained from GC-MS analysis for
KMNP-S1-61-12
38
Figure 4.6 Mass spectrum in the gas chromatogram for compound at
retention time 19.336 minutes
38
Figure 4.7 Mass spectrum in the gas chromatogram for compound at
retention time 21.150 minutes
38
Figure 4.8 Mass spectrum in the gas chromatogram for compound at
retention time 19.097 minutes
39
Figure 4.9 Mass spectrum in the gas chromatogram for compound at
retention time 17.218 minutes
39
Figure 4.10 Mass spectrum in the gas chromatogram for compound at
retention time 20.203 minutes
39
Figure 4.11 Mass spectrum in the gas chromatogram for compound at
retention time 20.959 minutes
40
Figure 4.12 Mass spectrum in the gas chromatogram for compound at
retention time 18.225 minutes
40
Figure 4.13 IR spectrum for fraction KMNP-S1-61-12 41
Figure 4.14 1H NMR for fraction KMNP-S1-61-12 41
Figure 4.15 13
C NMR for fraction KMNP-S1-61-12 42
Figure 4.16 DEPT for fraction KMNP-S1-61-12 43
xii
Analysis of Methanol Extract of Solen sarawakensis
Norshafida Syazarina bt Ibrahim
Resource Chemistry Programme
Faculty of Resource Science and Technology
University Malaysia Sarawak
ABSTRACT
Bivalve molluscs in Malaysia consist of a great variety of species. One bivalve species of commercial
importance is razor clam (Solen sp). Solen sp are locally known as ‘ambal ‘in Sarawak. This species
has been traditionally collected for human consumption over the past decades and is often served in
popular seafood restaurants. Razor clams can be found in the intertidal sandy beaches in Kuching and
Samarahan Division of Sarawak. Nowadays, razor clams are inadequate to meet the growing demand.
In Sarawak, razor clams number has been reduced significantly and this is especially true in
Peninsular Malaysia. In addition, there is a limited study on razor clams reported in scientific journal.
In the present, research on the potential chemical compounds present in that species is studied. The
extraction was carried out with methanol while fractionation and isolation was carried out using
column chromatography. The isolated component was analyzed using chromatographic and
spectroscopic method. Nuclear magnetic resonance (NMR) analysis of the component indicates the
presence of carbonyl group, saturated and unsaturated carbon.
Keyword: Solen spp, razor clams
ABSTRAK
Terdapat pelbagai jenis moluska yang bercangkerang di Malaysia. Salah satu spesies cangkerang
yang mempunyai nilai komersial ialah ambal (Solen sp). Ambal merupakan makanan laut yang
sangat popular di Sarawak dan menjadi sajian istimewa di restoran-restoran. Solen sp atau lebih
dikenali dengan ambal oleh penduduk di Sarawak boleh didapati di kawasan pantai sekitar Kuching
dan Samarahan. Pada masa kini, populasi ambal semakin berkurangan untuk memenuhi permintaan
yang semakin meningkat. Di Sarawak, penghasilan ambal dikatakan semakin berkurangan dan ini
dibuktikan di Semenanjung Malaysia. Dalam pada itu, kajian tentang metabolisme sekunder daripada
ambal (Solen sp) yang dilaporkan dalam jurnal saintifik adalah sangat terhad. Disebabkan itu,
kajian mengenai potensi penghasilan metabolisme sekunder daripada ambal dijalankan.
Pengekstrakan telah dijalankan menggunakan metanol sementara pemfraksian dan pemisahan telah
dijalankan menggunakan kromatografi turus. Komponen yang telah dipisahkan telah dianalisis
menggunakan kaedah kromatografi dan spektroskopi. Analisis resonans magnet nucleus (RMS)
terhadap salah satu komponen tersebut menunjukkan kehadiran kumpulan berfungsi karbonil, ikatan
karbon tepu dan tak tepu.
Kata kunci: Solen sp., ambal
1
CHAPTER 1
INTRODUCTION
The phylum mollusc is one of the largest of all animal phyla, which consist of between 50,000
and 110,000 species (Pechenik, 1991). Molluscs are unsegmented animals (Sumich and
Morrissey, 2004). Most molluscs have a hard external shell surrounding the soft body and use
a large muscular foot for locomotion, anchorage and securing food.
Bivalve molluscs in Malaysia consist of a great variety of species. Total of 185 species from
44 families have been listed and the major species of commercial importance have been
reported (Ong and Mahmood, 1989). Solen species chosen in this study are from the family of
Solenidae and are locally known as ‘ambal’ in Sarawak. It can be found on the mangrove
mudflats and intertidal sandy beaches in Peninsula Malaysia as well as on the western part of
Sarawak especially Bako, Buntal, Muara Tebas, Sambir, Sebandi, Moyan Laut, Serpan and
Asajaya Laut (Siti Akmar et al., 2006) and some were also found in Sematan, Kabong, Lundu
and Kuala Matu (Siti Akmar et al., 2006).
Information on Solen spp especially in Sarawak is still very limited. Therefore, the study on
this species is very crucial especially on the chemical properties since up to now, we could
not find any report concerning that. The shell can also be studied and not considered as waste
because there might be good properties in the shell. As what happen to the shell of shrimp or
ink-pen of squid where they found valuable material like chitin. This study will add to the
present information of Solen species and hope of bringing knowledge on the marine
aquaculture with new ideas and findings for human needs and satisfaction. The main aim of
this research is to analyze the chemical compounds from the methanol extracts of Solen sp,
fractionate the extracts and finally isolate the chemical component from the extraction. The
2
extraction is carried out using first cyclohexane and methanol and the fractionation using
column chromatography combined with TLC. The analyses of the components are determined
using spectroscopic methods such as GC-MS, FTIR and NMR.
3
CHAPTER 2
LITERATURE REVIEW
2.1 Classification of Razor Clam
The phylum mollusc is composed of seven classes that are Aplacophora, Polyplacophora,
Monoplacophora, Gastropoda, Cephalopoda, Scaphopoda and Bivalvia (Sumich and
Morrissey, 2004) (Figure 2.1). A large and diverse group of phylum mollusc make it difficult
to characterize but some common traits are observable.
The class Bivalvia contains over 25,000 species including clams, scallops, mussels and
oysters (Pechenik, 1991). Bivalves are primarily marine, but about 10 – 15 % of all species
occur in fresh water and no bivalves are terrestrial (Pechenik, 1991).
In Sarawak three different species has been reported; Solen regularis (Ambal biasa), Solen
vagina (Ambal Jernang) and Solen sarawakensis (Ambal Riong). Ambal Riong is believed to
be endemic species in Borneo (Hung, 2006). Loh (2005) reported that razor clams are
harvested seasonally in day time during low tide by local people in Sarawak from August
until March.
Figure 2.1: Classification of razor clam
4
2.2 Distribution of Razor Clam
Razor clam belongs to the species of marine bivalve molluscs of the family Solenidae
(Anonymous, 2006) (Figure 2.1). The name razor derived from their sharp razor like shell
(Loh, 2005). According to Loh (2005), razor clam can be found in intertidal sands and mud,
particularly of temperate seas. There are all over 30 species reported in the OBIS Indo-Pacific
Molluscan Database (Reid, 2006). However, only a few are listed from Malaysia which are S.
regularis, S. malaccensis, S. roseomaculatus at Indo-Malaysia, S.brevis at Borneo and S.
exiguus reported at Sarawak. Table 2.1 shows the distribution of Solen sp throughout the
Indo-Pacific.
2.3. General Characteristic of the Molluscs
Most molluscs have shells consisting primarily of calcium carbonate (CaCO3) with organic
material comprises of about 35% of the shell’s dry weight in some gastropod species and up
to about 70% of the dry weight in bivalves (Pechenik, 1991). According to Pechenik (1991),
the shells of most molluscs have thin, outer organic layer (the periostracum): a thin, innermost
calcareous middle layer (the prismatic layer) (Figure 2.2). Both the organic and inorganic
components of the shell are secreted by specialized tissue known as the mantle (Pechenik,
1991).
Razor clams for the present study are filter feeders living in the sandy beaches, with narrowed
and elongated shells (shaped like straight razors) (Figure 2.3). They have large active foot
enables them to move rapidly up and down within their burrow and retreat quickly when
disturbed (Loh, 2005). These filter feeders are species which are known to filter out food
particles from water using special filtering organ known as siphon.
5
Table 2.1: Solen sp and their distribution listed in OBIS Indo-Pacific Molluscan Database
(Reid, 2006).
Species Distribution
S. linearis South China Sea, Thailand, Central and East India Ocean
S. ceylonensis Red Sea, Sri Lanka, Aden
S. vaginoides New South Wales, Queensland, Victoria, Tasmania, India
S. corneus East China Sea, South China Sea, Yellow Sea, Taiwan, Hong Kong Hainan,
Indonesia, Gulf of Thailand, Japan , Philippines, India
S. lamarckii South China Sea, Taiwan, Philippines
S. sloanii China, South China Sea, Taiwan, Japan, Indonesia, East Africa
S. brevis Thailand, Eastern Indian Ocean, Bornoe
S. cylindraceus Red sea, Djibouti
S. strictus Japan, Gulf of Thailand
S. fonesii Northern Territory, Queensland, Western Australia
S. grandis
S. exiguous
Philippines, Thailand, East China Sea, South China Sea, Taiwan, China, Hainan,
Yellow Sea, Japan, Cheju-do, South Korea, North Korea, India
Thailand, Sarawak
S. regularis
S. malaccensis
Gulf of Thailand, Thailand, Malaysia
Thailand, Malaysia
S. luzonicus Philippines, East China Sea, Taiwan, Japan
S. lischkeanus Red Sea
S. timorensis Australia, Timor(Indonesia), Papua New Guinea
S. capensis Kuwait
S. digitalis Red Sea
S. roseomaculatus Red Sea, Central and east Indian Ocean, East Africa, Indo-China, Indo-
Malaysia, India, Hong Kong (Xianggang)
S. gordonis East China Sea, Taiwan, Japan, South China Sea
S. aureomaculatus Australia, Philippines, South China Sea
S. kajiyamai Australia
S. kurodai Queensland, Taiwan, Japan, East China Sea
S. arcuatus Shandong, East China Sea, Yellow Sea
S. canaliculatus South China Sea, East China Sea
S. crosnieri Madagascar
S. madagascariensis Madagascar
S. dactylus Persian Gulf @ Arabian Gulf, India, Pakistan
S. xishana Xisha Qundao (Spratley Islands), South China Sea
6
Figure 2.2: Siliqua patula Figure 2.3: Solen sarawakensis
2.4 Secondary Metabolism from Some Marine Organisms
Marine environment is an exceptional reservoir of secondary matabolites which exhibit
structural and chemical features not found in terrestrial natural product or chemically
synthesized (Che, 1991). Almost every class of marine organisms exhibit a variety of
molecules with unique structural features because of the physical and chemical conditions in
the marine environment (Kijjoa and Sawangwong, 2004).
According to Kijjoa and Sawangwong (2004), researchers have isolated approximately 7000
marine natural product. Of these 25% of are from algae, 33% from sponges, 18% from
coelenterates (sea whips, sea fans and soft corals),and 24% from representatives of other
invertebrate phyla such as ascidians (tunicates), opisthobranch molluscs (nudibranchs, sea
hares, etc), echinoderms (starfish, sea cucumber etc) and bryozoans (moss animals).
7
The extract of Laurencia venusta, a red algae found in Okinawan waters shows three active
compounds that are thyrsiferol (1), thysiferyl 23-acetate (2) and venustatriol (3). All the
compounds are tetracylic ethers of triterpenoid origin which active against HSV-1 and VSV
(Che, 1991).
OOH
O
OO
Br
RO
OH
1819
1 R=H 18 β-OH 19 β-Me
2 R=Ac 18 β-OH 19 β-Me
3 R=H 18 α-OH 19 α-Me
A group of sulfonic acid containing glycolipids (4-7) were extracted from the blue-green
algae (cyanobacteria) Lyngbya lagerheimii and Phormidium tenue. The different between the
ratios of fatty acid ester at R2 represent structural class of anti-HIV compounds currently
under preclinical investigation at the National Cancer Institute (Che, 1991).
8
O
HO3S
OH
OH
O
OHOR
1
OR2
R1 R2
4 16:0 Fatty acid ester 18:3 Fatty acid ester
5 16:0 18:2
6 16:0 18:1
7 16:0 16:1
Another type of marine algae, Caulerpa browni, has yielded a number of diterpenoids (8-12)
(Blunt et al., 2006).
OAc
OAc
8
9
OAc
AcO CHO
9
OAc
AcO CHO
10
OAc
CHO
11
OAc
CHO
12
Apart from that, the most studied marine organisms so far are sponge. There are 134 journals
found that discuss on natural products. The marine porifera of the genus Spongia (family
Spongidae, class Demospongiae) have proved to be rich sources of polycylic terpenes
(Grassia et al., 2001). The bioassay guided fractionations of CCl4 extract of the spongia sp.
10
collected at Vanuatu islands led to the isolation of cytotoxic metabolite spongidepsin (13)
(Grassia et al., 2001).
N
O O
O
13
Halichlorine (14) isolated from the marine sponge H. okadai Kadota; inhibits the induction of
VCAM-1 at IC50 7 μg/ml. Drugs that block VCAM-1 is useful for treating coronary artery
diseases, angina, and noncardiovascular inflammatory diseases (Kuramoto et al., 2004).
O
Cl OH
Me
O N
H
14
Another compound isolated from marine sponge Ircinia sp was ircinamine (15). It has a
unique structure with an amphibolous pyrroline ring moiety and the biological activity is
expected based on the reactivity of thioester moiety. Also this compound has moderate
activity toward P388 (LD50 24.6 μg/mL) (Kuramoto et al., 2004).