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

OH

OH

O

2

O

CH3OOH

CH3

OH

O

O

CH3

CH3

CH3

O

OH

3

CH3

OH

CH3

O

OH OH

CH3O

O

O

OCH3

4

7

O

OCH3

CH3

OH OH

5

O

OO

OHOH

CH3CH3

OH OH

6

O

O

O

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

O

O

O

O

OH OH

CH3

CH3

OH

CH3

OH

O

OH

10

O

O

OH

CH3

OH

CH3

OH O

O

OH

CH3

O

OH

OH

11

O

O

OH

CH3

OH

CH3

OH O

O

OH

OH

CH3

O

OH

12

10

O

O

OH

CH3

OH

CH3

O

O

O

OH

OH

CH3

O

CH3

13

O

O

OH

CH3

CH3

O

O

O

OH

OH

CH3

OH

O

CH3

14

O

O

CH3

CH3

O

O

O

OH

OH

CH3

OH

OH

O

CH3

15