ANALYSIS OF METHANOL EXTRACT OF SOLEN SARAWAKENSIS OF METHANOL EXTRACT OF SOLEN... · CHAPTER 2:...

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


Bachelor of Science with Honours

(Resource Chemistry)

2008


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


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



4.3 Analytical TLC of Crude Extract Using Two Solvents System 31

4.3.1 Analytical TLC of Crude Extract from Shell of Solen 31

Sarawakensis



4.3.2 AnalyticaL TLC of Crude Extract from Flesh of Solen 33

Sarawakensis

vi



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


29

Table 4.10 Analytical TLC of MeOH crude extract from flesh of


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



38



39



39



39



40



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


Resource Chemistry Programme


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).

ANALYSIS OF METHANOL EXTRACT OF SOLEN SARAWAKENSIS OF METHANOL EXTRACT OF SOLEN... · CHAPTER 2:...

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