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PHYTOCHEMICAL STUDIES AND BIOLOGICAL ACTIVITIES OF
Parmotrema sulphuratum
Soo Wai See (38904)
Bachelor of Science with Honours
(Resource Chemistry)
2015
PHYTOCHEMICAL STUDIES AND BIOLOGICAL ACTIVITIES OF
Parmotrema sulphuratum
Soo Wai See
The 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
2015
III
Declaration
I hereby declare that no portion of the work referred to in this dissertation has been submitted
in support of an application for another degree or qualification to this university or any other
institution of higher learning.
Soo Wai See
Department of Chemistry
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
IV
ACKNOWLEDGEMENT
My deep gratitude goes first to my supervisor, Professor Dr. Fasihuddin Ahmad, who
expert guided and advised me through my graduate education. Thank you for being a
committed supervisor that helps me to understand well about my Final Year Project. His
personal unwavering patience in supervising had led me gain much of chemical knowledge
which make my time at UNIMAS enjoyable and interesting. Without his guidance this work
would not have been possible.
My appreciation also extends to laboratory assistants, Leida Anthony. Puan Leida’s
mentoring and encouragement had been especially valuable, and her early insights launched
the greater part of this thesis. Thanks also go to Ismadi Rose and Haji Karni Taha, who
always there that responsible in borrowing the laboratory apparatus and chemical solvents for
us.
I am very thankful to my family, who continuous mentally and physically support me
all the time in completing this Final Year Project. And finally, I acknowledge my friends for
their help throughout the course of this work.
And most of all to God, who make me more determined to continue doing this work
in spite of the struggles that came.
V
TABLE OF CONTENTS
Title Pages
Declaration III
Acknowledgement IV
Table of Contents V
List of Abbreviations VIII
List of Tables IX
List of Figures XI
List of Appendices XIII
Abstract/ Abstrak XIV
CHAPTER 1
INTRODUCTION
1.1 Introduction 1
1.2 Objectives 2
CHAPTER 2
LITERATURE REVIEW
2.1 Description of Lichen 3
2.2 Secondary Metabolites of Lichen
2.2.1 Depsides 5
2.2.2 Tridepsides 8
2.2.3 Tetradepside 11
2.2.4 Depsidones 12
VI
2.2.5 Dibenzofurans 14
2.2.6 Naphthazarin 17
2.2.7 Isofuranonaphthoquinones 17
2.2.8 Xanthone 18
2.2.9 Fatty Acids 19
2.2.10 Sterols 24
2.2.11 Other secondary metabolites 26
2.3 Biological activities of Lichen 28
2.4 Importance or Uses of Lichen 30
CHAPTER 3
MATERIALS AND METHODS
3.1 General experimental procedure 31
3.2 Sample collection 32
3.3 Extraction 33
3.4 Isolation and purification of chemical constituents
3.4.1 Thin Layer Chromatography (TLC) 34
3.4.2 Column Chromatography (CC) 35
3.5 Analysis of chemical constituents
3.5.1 Gas chromatography-Mass Spectrometry (GC-MS) 35
3.5.2 Fourier Transform Infra-Red Spectrometer (FTIR) 36
3.5.3 Nuclear Magnetic Resonance Spectroscopy (NMR) 37
3.6 Determination of biological activities
VII
3.6.1 Toxicity to Artemia salina 38
3.6.2 Termiticidal activity test 39
CHAPTER 4
RESULTS AND DISCUSSION
4.1 Extraction 41
4.2 Analytical Thin Layer Chromatography (TLC) 42
4.3 Column chromatography of dichloromethane crude extract 44
4.4 Gas chromatography-Mass Spectrometry (GC-MS) 47
4.5 Fourier Transform Infra-Red Spectrometer (FTIR) 50
4.6 Nuclear Magnetic Resonance Spectroscopy (NMR) 52
4.7 Brine Shrimp Toxicity Test 57
4.8 Termiticidal Activity Test 60
CHAPTER 5
CONCLUSIONS AND RECOMMENDATIONS 64
REFERENCES 66-70
VIII
LIST OF ABBREVIATIONS
DCM Dichloromethane
CHCl3 Chloroform
CDCl3 Deuterated chloroform
EtOH Ethanol
TLC Thin Layer Chromatography
CC Column Chromatography
Rf Retention factor
FTIR Fourier Transform Infrared
NMR
1H-NMR
13
C-NMR
Nuclear Magnetic Resonance Spectroscopy
Proton Nuclear Magnetic Resonance
Carbon Nuclear Magnetic Resonance
GC-MS Gas Chromatography-Mass Spectrometry
GC-FID Gas Chromatography-Flame Ionization Detector
UV Ultraviolet Spectrophotometer
mg Microgram
μL Microliter
LC50 Lethal concentration
μg/mL Microgram/milliliter
ppm Part per million
min Minutes
mL Milliliter
% Percent
IX
LIST OF TABLES
Tables Pages
Table 2.1 Some common species of the lichen and their uses. 30
Table 4.1 The colours, weights and yield percentages of the different
fractions of various extracts of the Parmotrema sulphuratum
extracts.
41
Table 4.2 Solvents ratio for TLC analysis. 42
Table 4.3 Analytical TLC using different ratio of solvent system for DCM
crude extract.
43
Table 4.4 Combined fractions of the same Rf values. 44
Table 4.5 Combined fractions of the same Rf values for fraction 38-41. 45
Table 4.6 Combined fractions of the same Rf values for fraction 18-19. 46
Table 4.7 Combined fractions of the same Rf values for fraction 17-18. 46
Table 4.8 The major compound present which analyzed by MS spectra on
NIST08s.LIB library data.
49
Table 4.9 The minor compound present which analyzed by MS spectra on
NIST08s.LIB library data.
50
Table 4.10 Functional group observed. 50
Table 4.11 1H-NMR (500 MHz) and
13C-NMR (125 MHz) data in CDCl3. 53
Table 4.12 Percentage for average deaths of Artemia salina and the LC50
value.
58
Table 4.13 Percentage for average deaths of Rhinotermes sp. and the LC50
value.
60
X
Table 4.14 The average deaths of Rhinotermes sp. for crude extract of
Parmotrema sulphuratum for 3 days.
61
XI
LIST OF FIGURES
Figures Pages
Figure 3.1 Parmotrema sulphuratum. 33
Figure 3.2 Toxicity test for Artemia salina. 39
Figure 3.3 The termiticidal test for Parmotrema sulphuratum. 40
Figure 4.1 GC chromatogram for fraction 16. 45
Figure 4.2 GC chromatogram for fraction 17-18. 47
Figure 4.3 Gas chromatogram. 48
Figure 4.4 Ion fragmentation of orcinaldehyde at peak 17. 48
Figure 4.5 Ion fragmentation of atraric acid at peak 20. 49
Figure 4.6 FTIR spectrum. 51
Figure 4.7 1H-NMR spectrum of Parmotrema sulphuratum dichloromethane
extract.
54
Figure 4.8 1H-NMR signal with integration. 55
Figure 4.9 13
C-NMR spectrum of Parmotrema sulphuratum
dichloromethane extract.
56
Figure 4.10 Atraric acid. 57
Figure 4.11 Average death of Artemia salina (%) against log10 concentration
(μg/mL).
58
Figure 4.12 Percentage deaths of Rhinotermes sp. against log10 concentration
for 1 ppm concentration.
62
Figure 4.13 Percentage deaths of Rhinotermes sp. against log10 concentration
for 10 ppm concentration.
62
XII
Figure 4.14 Percentage deaths of Rhinotermes sp. against log10 concentration
for 100 ppm concentration.
63
XIII
LIST OF APPENDICES
Appendix 1: TLC plate for combined fractions 38-41 after run with smaller column
Appendix 2: The needle crystal form of combined fractions 18-19 after evaporated to
dryness
Appendix 3: TLC plate for 17-18 fractions
Appendix 4: Tiny crystal form of combined fractions 17-18 after evaporated to dryness
Appendix 5: Average death of Artemia salina as a function of concentration for the crude
extract
Appendix 6: Average death of Rhinotermes sp. as a function of concentration for the crude
extract after 24 hours
Appendix 7: Average death of Rhinotermes sp. as a function of concentration for the crude
extract after 48 hours
Appendix 8: Average death of Rhinotermes sp. as a function of concentration for the crude
extract after 72 hours
Appendix 9: GC-MS Chromatogram
XIV
ABSTRACT
The aim of this study was to determine the secondary metabolites, cytotoxic activity
and termiticidal activity of lichen Parmotrema sulphuratum, which was collected from
Borneo Highland, Sarawak. Phytochemical examination on the dichloromethane extract of
the lichen gave percentage yield of 16.66 % and led to the isolation and characterization of
atraric acid. Column Chromatography method has been developed to fractionate the
dichloromethane crude into several fractions and purify them by using the 20 cm х 20 cm
Preparative TLC plate. The pure compound was subjected to Gas Chromatography Flame
Ionization Detector to determine the purity. The structure of atraric acid was elucidated by
Gas Chromatography-Mass Spectrometry, 1H and
13C Nuclear Magnetic Resonance
Spectroscopy and Fourier Transform Infrared. The components of the lichen were identified
based on spectral data and comparison with published information. Bioassay was carried out
on Artemia salina and Rhinotermes sp. and results indicate that the all the extracts do not
exhibit cytotoxic activity and termiticidal activity.
Keyword: Parmotrema sulphuratum, atraric acid, Artemia salina, Rhinotermes sp.
ABSTRAK
Tujuan kajian ini adalah untuk menentukan metabolit sekunder, aktiviti sitotoksik dan
aktiviti termitisidal daripada liken Parmotrema sulphuratum yang dikumpul dari Borneo
Highland, Sarawak. Ekstrak diklorometana Parmotrema sulphuratum memberi peratusan
hasil 16.66 % dan membawa kepada pengasingan asid atrarik. Kaedah Kromatografi Turus
telah dibangunkan untuk memisahkan ekstrak mentah diklorometana kepada beberapa
pecahan dan ditulenkan secara KLN persediaan pada plat 20 cm х 20 cm. Ketulenan
sebatian telah dianalisis dengan menggunakan Kromatografi Gas Pengesan Pengionan
Nyala untuk memastikan ketulenan. Struktur asid atrarik telah ditentukan dengan
menggunakan kaedah Kromatografi Gas-Spektometri Jisim , 1H dan
13C Resonans Magnetik
Nuklear dan Inframerah. Komponen liken telah dikenal pasti berdasarkan data spektroskopi
dan perbandingan dengan data literatur. Bioasai dengan ujian ketosikan Artemia salina dan
termitisidal ke atas Rhinotermes sp. menunjukkan semua ekstrak tidak mempunyai aktiviti
sitotoksik dan aktiviti termitisidal.
Kata Kunci: Parmotrema sulphuratum, asid atrarik, Artemia salina, Rhinotermes sp.
1
CHAPTER 1
INTRODUCTION
1.1 Introduction
Lichens are formed from a stable symbiotic association of fungi with green algal or
cyanobacterial photobionts. According to Lange and Wagenitz (2003), 85% of lichens are
symbiotic with chlororophyta to form chlorolichens and 10% with Cyanophyta to create
cyanolichens, and the rest associated simultaneously with both groups. The green thallus
of autotrophic lichen requires sunlight for long periods. They have specific habitats such
as biological soil crusts in the environmental conditions of low temperatures, prolonged
darkness, drought and continuous light.
Joshi (2012) stated that lichen acts as air pollution indicators especially sulphur
dioxide, heavy metals and radioactive metals. Lichens play an important role in chemistry
because it is difficult to differentiate morphologically but can be distinguished by their
secondary metabolites. Lichen extracts have been used for medicinal applications
probably due to the biological activity of their endogenous secondary metabolites. Usnic
acid isolated from different lichens showed antibiotic properties like penicillin (Joshi,
2012).
Lichens produce high concentration of characteristic secondary metabolites which
known as lichen substances such as depsides, depsidones, dibenzofurans, xanthones, and
terpene derivatives that seldom found in other organisms. Lichens metabolites exhibit
biological activities especially antioxidant, antimicrobial, antibacterial, antibiotic,
antiviral, anti-protozoa, anti-proliferative, anti-inflammatory, anticancer, anti-allergenic,
2
anti-herbivoral, analgesic, antipyretic, and cytotoxic activity (Choudhary et al., 2005;
Ranković et al., 2012; Santiago et al., 2013).
Although about 8% of the terrestrial ecosystem consists of lichens, and more than 20
000 lichen species are distributed throughout the world, but their biological activities and
biologically active compounds remain unexplored in great extent (Srivastava et al., 2013).
The chemical structures of lichen metabolites compounds are almost similar and
identification is often very difficult. Until now, only a few research have proved that
lichens have cytotoxic activity (Ranković et al., 2012). Relatively few lichen substances
have been screened in detail for biological activity and therapeutic potential, due
principally to difficulties in obtaining them in quantities and purities sufficient for
structural elucidation and pharmacological testing.
With this background the aim of this study is to investigate the structural and
chemical compounds isolated from the lichen that contain termiticidal activity and
cytotoxic activities.
1.2 Objectives
The main objective for this research is to purify and elucidate the biological active
compounds of Parmotrema sulphuratum. The specific objectives are:
a. to extract, isolates and purify the secondary metabolites of Parmotrema sulphuratum.
b. to elucidate the chemical structure of the pure compounds.
c. to evaluate the termiticidal activity and cytotoxic of crude extract of Parmotrema
sulphuratum.
3
CHAPTER 2
LITERATURE REVIEW
2.1 Description of Lichen
Lichens are symbiotic organisms able to synthesize lichen metabolites that originate
from fungal. Lichens normally grow on rocks, leaves and as epiphytes on tress and leaves.
Lichens have been as a cure for stomach diseases, diabetes, dermatological diseases and
pulmonary tuberculosis for hundred years ago. These unique organisms offer many bioactive
secondary metabolites with manifold biological activities. These secondary metabolites are
aliphatic acids, pulvinic acid derivatives, hydroxybenzoic acid derivatives, depsides,
depsidones, dibenzofuran derivatives, anthraquinones, naphthoquinones and related
compounds, and epidithiopiperazinediones (Santiago et al., 2013). Lichen metabolites had
reported antibacterial, antifungal, anti-parasitic, anti-helmintic, anti-inflammatory and anti-
viral activities (Santiago et al., 2013). For example, Ramalina farinacea extracts were
reported to have antibacterial, antifungal and cytotoxic activities.
Interestingly, extracts of several species, Parmelia saxatilis, Platismatia glauca,
Ramalina pollinaria, Ramalina polymorpha and Umbilicaria nylanderiana were reported to
have activities against bacteria, fungi and yeast and possess antioxidant properties. The
antibiotic activities also showed in lichens Crocynia membranacea, Stereocaulon sp.,
Ramalina farinacea, Parmelia dactylifera, Physcia albicans, Lecanora subfusca and Usnea
species. Recently, researchers also reported inhibitory activities of the lichens Usnea baileyi,
Ramalina dendriscoides, Cladonia gracilis and Stereocaulon massartianum against Gram-
4
positive bacteria. These are just few literatures that supported the potentials of lichens as
sources of bioactive metabolites.
This study is important to make some improvements on the several questions
appeared in previous research study. First and foremost, researchers are interest about the
most abundant substances classes in the extracts examined. Researchers are interested to
search for new compounds that present in lichen. Besides, there are crucial to find out the
unique and biologically active substances in selected lichen.
5
2.2 Secondary Metabolites of Lichen
2.2.1 Depsides
Diffractic acid is belongs to depside which has been studied and isolated from Usnea
longissima ether extract (Atalay et al., 2011). According to Nishitoba et al. (1987) study,
three major chemical components have been isolated from Usnea longissima such as barbatic
acid (1) and diffractaic acid (2). In recent investigation of Usnea longissima (Ohmura, 2001)
has stated that this species contains barbatic acid (1), squamatic acid (3) and atranorin (4). In
addition, diffractic acid (2) and atranorin (4) has been reported present in Usnea orientalis.
Atranorin (4) has been isolated from Evernia punastri and Pseudoevernia furfuraceae
(Kosanić et al., 2013). Atranorin (4) has been found in Tephromela atra (Hesbacher et al.,
1996). A previous phytochemical investigation of Sticta nylanderiana has resulted in
isolation of orsellinic acid (5) and lecanoric acid (6) (Zhang et al., 2006). Lecanoric acid (6),
an orcinol depside has been identified in Stereocaulon curtatum (Hamada & Ueno, 1990).
Evernic acid (7) has been isolated from Cetrariella delisei (Narui et al., 1998).
OH
CH3
OH
CH3
O
CH3
O
OCH3
OH
O
CH3
1
6
CH3
O
CH3
OCH3
O
O
CH3
CH3
CH3
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OH
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2
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CH3OOH
CH3
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O
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3
CH3
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CH3O
O
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4
7
O
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5
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OHOH
CH3CH3
OH OH
6
O
O
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OH
OH
CH3
OH
CH3
O
CH3
7
8
2.2.2 Tridepsides
Lasallic acid (8), crustinic acid (9) and gyrophoric acid (10) has been extracted from
Lasallia asiae-orientalis (Narui et al., 1995). Eight tridepsides, crustinic acid (9), gyrophoric
acid (10), hiascic acid (11), lasallic acid (12), 4-O-methyl-gyrophoric acid (13), ovoic acid
(14), umbilicaric acid (15) and tenuiorin (16) has been isolated from Umbilicaria
cinereorufescens, Lasallia papulosa, Cetrariella delisei, Loboria sp., Melanelia tominii,
Umbilicaria polyphylla and Peltigera aphthosa (Narui et al., 1998).
O
O
O
O
OH OH
CH3
CH3
OHOH
CH3OH
O
OH
8
O
O
O
O
OH OH
CH3
CH3
OHCH3
OHOH
O
OH
9
9
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O
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O
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CH3
CH3
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CH3
OH
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10
O
O
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O
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11
O
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CH3
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12
10
O
O
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CH3
OH
CH3
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O
O
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CH3
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CH3
13
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CH3
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CH3
OH
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14
O
O
CH3
CH3
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O
O
OH
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CH3
OH
OH
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CH3
15
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