Study on chemical constituents and biological activities of the lichen parmotrema Praesorediosum(NYL ) hale(parmeliaceaf)

VIETNAM NATIONAL UNIVERSITY - HO CHI MINH CITY
UNIVERSITY OF SCIENCE



HUYNH BUI LINH CHI

STUDY ON CHEMICAL
CONSTITUENTS AND BIOLOGICAL
ACTIVITIES OF THE LICHEN
PARMOTREMA PRAESOREDIOSUM
(NYL.) HALE
(PARMELIACEAE)

DOCTORAL THESIS IN CHEMISTRY

Ho Chi Minh City, 2014


VIETNAM NATIONAL UNIVERSITY - HO CHI MINH CITY
UNIVERSITY OF SCIENCE



HUYNH BUI LINH CHI

STUDY ON CHEMICAL CONSTITUENTS AND
BIOLOGICAL ACTIVITIES OF THE LICHEN
PARMOTREMA PRAESOREDIOSUM (NYL.) HALE
(PARMELIACEAE)
Subject: Organic Chemistry

Code number: 62 44 27 01

Examination Board:
Prof. Dr. Nguyen Minh Duc

(1st Reviewer)

Assoc. Prof. Dr. Tran Cong Luan

(2nd Reviewer)

Assoc. Prof. Dr. Pham Dinh Hung

(3rd Reviewer)

Assoc. Prof. Dr. Le Thi Hong Nhan

(1st Independent Reviewer)

Dr. Le Tien Dung

(2nd Independent Reviewer)

SUPERVISORS: PROF. DR. NGUYEN KIM PHI PHUNG
PROF. DR. TAKAO TANAHASHI

Ho Chi Minh City, 2014


i

SOCIALIST REPUBLIC OF VIETNAM
INDEPENDENCE-FREEDOM-HAPPINESS

DECLARATION

The work presented in this thesis was completed in the period of November
2009 to November 2013 under the co-supervision of Professor Nguyen Kim Phi
Phung of the University of Science, Vietnam National University, Ho Chi Minh
City, Vietnam and Professor Takao Tanahashi of the Kobe Pharmaceutical
University, Japan.
In compliance with the university regulations, I declare that:
1. Except where due acknowledgement has been made, the work is that of the
author alone;
2. The work has not been submitted previously, in whole or in part, to qualify for
any other academic award;
3. The content of the thesis is the result of the work which has been carried out
since the official commencement date of the approved doctoral research program;
4. Ethics procedures and guidelines have been followed.

Ho Chi Minh City, Sept 30, 2014
PhD student

HUYNH BUI LINH CHI


ii

ACKNOWLEDGEMENTS

There are many individuals without whom the work described in this thesis
might not have been possible, and to whom I am greatly indebted.
Firstly, I wish to thank my supervisor, Prof. Dr. Nguyen Kim Phi Phung for
her knowledge, support, and guidance, hundreds of meetings/emails and for always
keeping me on my toes, from the very beginning to the very end of my PhD.
I would also like to acknowledge my second supervisor, Prof. Dr. Takao
Tanahashi for his guidance, patience and who has taught me the true spirit of
research. I am deeply indebted to Dr. Yukiko Takenaka at Kobe Pharmaceutical
University, Japan for her teachings, kindness, helpful suggestion and valuable
advice in this research.
I would also like to express my sincere thanks to PhD Vo Thi Phi Giao at
University of Science, Vietnam National University, Ho Chi Minh City and Dr.
Harrie J. M. Sipman, Botanic Garden and Botany Museum Berlin-Dahlem, Freie
University, Berlin, Germany for his expertise in the identification of lichen.
I am very grateful to thank Prof. Dr. Shigeki Yamamoto, Prof. Dr. Hitoshi
Watarai at Osaka University, Japan and PhD. Do Thi My Lien for giving up their
precious time to help me with CD spectra, sample preparation and proof reading of
some isolated compounds of the thesis.
A special thanks to Dr. Le Hoang Duy for his helpful assistance and
friendship during my work at Kobe Pharmaceutical University, Japan.
I would like to acknowledge the encouragement, insightful comments of the
rest of examination board: Prof. Dr. Nguyen Cong Hao, Prof. Dr. Nguyen Minh
Duc, Assoc. Prof. Dr. Tran Cong Luan, Assoc. Prof. Dr. Pham Dinh Hung, Assoc.
Prof. Dr. Nguyen Trung Nhan, Dr. Pham Nguyen Kim Tuyen and Dr. Le Tien
Dung.


iii

Similarly, I would also like to thank my teachers, friends and students in the

Department of Organic Chemistry, Faculty of Chemistry, University of Science,
Vietnam National University-Ho Chi Minh City.
Most importantly, I would like to thank my husband, for being the most
patient and supportive witness to my academic journey over the past four years.
Without his support, love and encouragement, this study would not have been
possible.
Finally, I would like to thank my parents for believing in me and for being
proud of me. Their unconditional love and support has given me the strength and
courage while I am far from home.
THANK YOU


iv

TABLE OF CONTENTS

DECLARATION ....................................................................................................... i
ACKNOWLEDGEMENTS ...................................................................................... ii
TABLE OF CONTENTS ......................................................................................... iv
LIST OF ABBREVIATIONS.................................................................................. vi
LIST OF TABLES ................................................................................................... xi
LIST OF FIGURES ............................................................................................... xiii
LIST OF APPENDICES......................................................................................... xv
INTRODUCTION ..................................................................................................... 1
CHAPTER 1: LITERATURE REVIEW ................................................................ 3
1.1. GENERIC DESCRIPTION ............................................................................... 3
1.1.1. The lichen .......................................................................................................... 3
1.1.2. Parmotrema praesorediosum (Nyl.) Hale ......................................................... 4
1.2. CHEMICAL STUDIES ON THE LICHEN GENUS PARMOTREMA ........... 6
1.2.1. Lichen secondary metabolites ........................................................................... 6

1.2.2. Chemical studies on the lichen genus Parmotrema .......................................... 8
1.3. BIOLOGICAL ACTIVITIES ......................................................................... 14
1.3.1. The biological significance of lichen metabolites ........................................... 14
1.3.2. The biological significance of the lichen Parmotrema ................................... 15
CHAPTER 2: EXPERIMENTAL ......................................................................... 20
2.1. MATERIALS AND ANALYSIS METHODS ............................................... 20
2.2. LICHEN MATERIALS .................................................................................. 22


v

2.3. EXTRACTION AND ISOLATION PROCEDURES .................................... 22
2.3.1. Isolating compounds from the methanol precipitate ....................................... 22
2.3.2. Isolating compounds from the petroleum ether E1 extract ............................ 23
2.3.3. Isolating compounds from the petroleum ether E2 extract ............................ 23
2.3.4. Isolating compounds from the chloroform extract ......................................... 24
2.4. PREPARATION OF SOME DERIVATIVES ................................................ 28
2.4.1. Esterification of PRAES-C2 ............................................................................ 28
2.4.2. Methylation of PRAES-C25 ............................................................................ 29
2.5. BIOLOGICAL ASSAY .................................................................................. 29
2.5.1. Cytotoxicity ..................................................................................................... 29
2.5.2. In vitro acetylcholinesterase (AChE) inhibition assay .................................... 30
CHAPTER 3: RESULTS AND DISSCUSSION .................................................. 32
3.1. CHEMICAL STRUCTURE ELUCIDATION .............................................. 32
3.1.1. Chemical structure of aliphatic acids .............................................................. 33
3.1.1.1. Structure elucidation of compound PRAES-C1........................................... 33
3.1.1.2. Structure elucidation of compound PRAES-E14 ......................................... 34
3.1.1.3. Structure elucidation of compound PRAES-C10 ........................................ 35
3.1.1.4. Structure elucidation of compound PRAES-C11 ........................................ 39
3.1.1.5. Structure elucidation of compound PRAES-E19 ......................................... 40

3.1.1.6. Structure elucidation of compound PRAES-C2........................................... 41
3.1.2. Chemical structure of mononuclear phenolic compounds .............................. 45
3.1.2.1. Structure elucidation of compound PRAES-T1 ........................................... 45
3.1.2.2. Structure elucidation of compound PRAES-E1 ........................................... 46


vi

3.1.2.3. Structure elucidation of compound PRAES-T2 ........................................... 48
3.1.2.4. Structure elucidation of compound PRAES-E11 ......................................... 49
3.1.2.5. Structure elucidation of compound PRAES-T4 ........................................... 50
3.1.2.6. Structure elucidation of compound PRAES-T6 ........................................... 50
3.1.2.7. Structure elucidation of compound PRAES-E2 ........................................... 51
3.1.2.8. Structure elucidation of compound PRAES-C22 ........................................ 53
3.1.2.9. Structure elucidation of compound PRAES-C23 ........................................ 55
3.1.2.10. Structure elucidation of compound PRAES-C24 ...................................... 56
3.1.2.11. Structure elucidation of compound PRAES-C25 ...................................... 59
3.1.2.12. Structure elucidation of compound PRAES-C26 ...................................... 63
3.1.3. Chemical structure of depsides ....................................................................... 66
3.1.3.1. Structure elucidation of compound PRAES-T3 ........................................... 66
3.1.3.2. Structure elucidation of compound PRAES-C7........................................... 67
3.1.3.3. Structure elucidation of compound PRAES-E18 ......................................... 69
3.1.4. Chemical structure of depsidones ................................................................... 70
3.1.4.1. Structure elucidation of compound PRAES-C14 ........................................ 70
3.1.4.2. Structure elucidation of compound PRAES-C12 ........................................ 73
3.1.5. Chemical structure of diphenyl ethers ............................................................ 74
3.1.5.1. Structure elucidation of compound PRAES-C5........................................... 74
3.1.5.2. Structure elucidation of compound PRAES-C15 ........................................ 77
3.1.5.3. Structure elucidation of compound PRAES-C16 ........................................ 79
3.1.5.4. Structure elucidation of compound PRAES-C20 ........................................ 84

3.1.5.5. Structure elucidation of compound PRAES-C18 ........................................ 86


vii

3.1.5.6. Structure elucidation of compound PRAES-C3........................................... 89
3.1.5.7. Structure elucidation of compound PRAES-C4........................................... 91
3.1.5.8. Structure elucidation of compound PRAES-C21 ........................................ 93
3.1.6. Chemical structure of dibenzofurans .............................................................. 97
3.1.6.1. Structure elucidation of compound PRAES-E5 ........................................... 97
3.1.6.2. Structure elucidation of compound PRAES-E3 ........................................... 99
3.1.6.3. Structure elucidation of compound PRAES-C8......................................... 100
3.1.7. Chemical structure of xanthones ................................................................... 103
3.1.7.1. Structure elucidation of compound PRAES-C27 ...................................... 103
3.1.7.2. Structure elucidation of compound PRAES-C28 ...................................... 108
3.1.8. Chemical structure of triterpenoids ............................................................... 111
3.1.8.1. Structure elucidation of compound PRAES-E17 ....................................... 111
3.1.8.2. Structure elucidation of compound PRAES-E6 ......................................... 112
3.1.8.3. Structure elucidation of compound PRAES-E13 ....................................... 113
3.1.9. Chemical structure of a macrocylic compound............................................. 117
3.1.9.1. Structure elucidation of compound PRAES-E15 ....................................... 117
3.2. BIOLOGICAL ASSAY ................................................................................ 120
3.2.1. Cytotoxicity activitivy................................................................................... 120
3.2.2. Acetylcholinesterase inhibitory activity ....................................................... 121
CHAPTER 4: CONCLUSION ............................................................................. 124
4.1. CONSTITUENTS OF PARMOTREMA PRAESOREDIOSUM ................... 124
4.2. BIOLOGICAL ASSAY ................................................................................ 132
FUTURE OUTLOOK ........................................................................................... 133

viii

LIST OF PUBLICATIONS .................................................................................. 134
REFERENCES ...................................................................................................... 135
APPENDICES ....................................................................................................... 145


ix

LIST OF ABBREVIATIONS

1D

One dimensional

2D

Two dimensional

Ac

Acetone

AcOH

Acetic acid

br

Broad

C

Chloroform

calcd

Calculated

CC

Column chromatography

CD

Circular dichroism

COSY

Homonuclear shift correlation spectroscopy

CPCM

Conductor-like polarized continuum model

d

Doublet

dd

Doublet of doublets

DEPT

Distortionless enhancement by polarisation transfer

DMSO

Dimethyl sulfoxide

EA

Ethyl acetate

EI-MS

Electron-impact ionization mass spectrum

EtOH

Ethanol

H

n-Hexane

HMBC

Heteronuclear multiple bond correlation spectroscopy

HPLC

High performance liquid chromatography

HR-EIMS

High resolution electron-impact ionization mass spectrum

HR-ESIMS

High resolution electrospray ionization mass spectrum


x

HSQC

Heteronuclear single quantum correlation spectroscopy

IR

Infrared spectrophotometry

m

Multiplet

M

Methanol

MeOH

Methanol

min

Minutes

MS

Mass spectrum

NMR

Nuclear magnetic resonance

NOESY

Nuclear overhauser enhancement spectroscopy

P

Petroleum ether

ppm

Parts per million (chemical shift value)

pre TLC

Preparative thin-layer chromatography

q

Quartet

quint

Quintet

ROESY

Rotating-frame overhauser enhancement spectroscopy

s

Singlet

sext

Sextet

t

Triplet

TD-DFT

Time dependent density functional theory

TLC

Thin-layer chromatography

TMS

Tetramethylsilane

UV

Ultraviolet


xi

LIST OF TABLES

Table 1.1.

In vitro biological activities of the lichen genus Parmotrema

17

Table 3.1.

Isolated compounds from Parmotrema praesorediosum

32

Table 3.2.

1

38

Table 3.3.

13

Table 3.4.

NMR data of PRAES-T1, PRAES-E1, PRAES-T2

47

Table 3.5.

NMR data of PRAES-E11, PRAES-T4, PRAES-T6, PRAES-E2

52

Table 3.6.

NMR data of PRAES-C22, PRAES-C23, PRAES-C24

58

Table 3.7.

NMR data of PRAES-C25, PRAES-C25M, PRAES-C26

65

Table 3.8.

NMR data of PRAES-T3, PRAES-C7, PRAES-C9, PRAES-E18

68

Table 3.9.

NMR data of PRAES-C14 and PRAES-C12

72

Table 3.10.

1

H NMR data of PRAES-C5 and Lecanorol

76

Table 3.11.

1

H NMR data of PRAES-C15, PRAES-C16, PRAES-C20,

H NMR of aliphatic compounds
C NMR of aliphatic compounds

PRAES-C18, PRAES-C3 and PRAES-C4
Table 3.12

13

39

82

C NMR data of PRAES-C15, PRAES-C16, PRAES-C20,

PRAES-C18, PRAES-C3 and PRAES-C4

83

Table 3.13. NMR data of PRAES-C21

96

Table 3.14. NMR data of PRAES-E5 and PRAES-E3 (CDCl3)

98

Table 3.15. NMR data of PRAES-C8, PRAES-E5 and Usimine A

102

Table 3.16. NMR data of PRAES-C27, Blennolide G, Blennolide B and
Chromone lactone (CDCl3)
Table 3.17. NMR data of PRAES-C27 and PRAES-C28 (CDCl3)

106
110


xii

Table 3.18. NMR data of PRAES-E17, PRAES-E6, PRAES-E13 and 1β,3βDiacetoxyhopan-22-ol
Table 3.19. NMR data of PRAES-E15

115
120

Table 3.20. % Inhibition of cytotoxic activity against three cancer cell lines of
isolated compounds

122

Table 3.21. IC50 value of cytotoxic activity against three cancer cell lines of
isolated compounds

122

Table 3.22. Acetylcholinesterase inhibition of some extracts and isolated
compounds

123


xiii

LIST OF FIGURES

Figure 1.1. Types of the lichen

3

Figure 1.2. Parmotrema praesorediosum (Nyl.) Hale (Parmeliaceae)

5

Figure 1.3. Biosynthetic pathways of the major groups of lichen substances

7

Figure 2.1: Isolation of compounds from the prepicitate and petroleum ether
extracts of Parmotrema praesorediosum (Nyl.) Hale

26

Figure 2.2: Isolation of compounds from the chloroform extract of Parmotrema
praesorediosum (Nyl.) Hale

27

Figure 3.1. HMBC correlations of PRAES-C1 and PRAES-E14

36

Figure 3.2. HMBC correlations of PRAES-C10

37

Figure 3.3. HMBC correlations of PRAES-E19

41

Figure 3.4. HMBC correlations of PRAES-C2

42

Figure 3.5. Comparison of experimental CD spectrum of PRAES-C2Me and
theoretical calculated one.

44

Figure 3.6. CD spectra of isolated aliphatic compounds

45

Figure 3.7. HMBC correlations of PRAES-E1 and PRAES-T2

48

Figure 3.8. HMBC correlations of PRAES-E11, PRAES-T4 and PRAES-E2

52

Figure 3.9. HMBC and NOESY correlations of PRAES-C22

54

Figure 3.10. HMBC and NOESY correlations of PRAES-C23

56

Figure 3.11. HMBC and NOESY correlations of PRAES-C24

57

Figure 3.12. COSY, HMBC and NOESY correlations of PRAES-C25M

61

Figure 3.13. Mechanism for the methylation of PRAES-C25

62

Figure 3.14. HMBC and NOESY correlations of PRAES-C25 and PRAES-C26 63


xiv

Figure 3.15. HMBC correlations of PRAES-C9 and PRAES-C7

67

Figure 3.16. COSY and HMBC correlations of PRAES-E18

70

Figure 3.17. HMBC correlations of PRAES-C12

73

Figure 3.18. HMBC correlations of PRAES-C5

75

Figure 3.19. HMBC and NOESY correlations of PRAES-C15

78

Figure 3.20. HMBC and ROESY correlations of PRAES-C16

80

Figure 3.21. HMBC and ROESY correlations of PRAES-C20

85

Figure 3.22. 1H NMR data of PRAES-C18 and diphenyl ether

87

Figure 3.23. HMBC and ROESY correlations of PRAES-C18

88

Figure 3.24. HMBC correlations of PRAES-C3

90

Figure 3.25. HMBC correlations of PRAES-C4

93

Figure 3.26. HMBC correlations of PRAES-C21

94

Figure 3.27. ROESY correlations of PRAES-C21

95

Figure 3.28. HMBC correlations of PRAES-E5

97

Figure 3.29. HMBC correlations of PRAES-E3

99

Figure 3.30. HMBC correlations of PRAES-C8

101

Figure 3.31. The structure of Usimine A

102

Figure 3.32. COSY, HMBC and ROESY correlations of PRAES-C27

104

Figure 3.33. ROESY correlations of PRAES-C28

109

Figure 3.34. COSY and HMBC correlations of PRAES-C28

111

Figure 3.35. HMBC correlations of PRAES-E17

112

Figure 3.36. HMBC correlations of PRAES-E13

114

Figure 3.37. COSY and HMBC correlations of PRAES-E15

118

xv

LIST OF APPENDICES

Appendices 1-7: IR, MS and NMR spectra of PRAES-C1

146

Appendices 8-15: IR, MS and NMR spectra of PRAES-E14

149

Appendices 16-21: MS and NMR spectra of PRAES-C10

153

Appendices 22-26: MS and NMR spectra of PRAES-C11

156

Appendices 27-33: IR, MS and NMR spectra of PRAES-E19

159

Appendices 34-40: IR, MS and NMR spectra of PRAES-C2

162

Appendices 41-44: NMR spectra of PRAES-T1

166

Appendices 45-49: MS and NMR spectra of PRAES-E1

168

Appendices 50-54: NMR spectra of PRAES-T2

170

Appendices 55-58: NMR spectra of PRAES-E11

173

Appendices 59-62: NMR spectra of PRAES-T4

175

Appendices 63-66: MS and NMR spectra of PRAES-T6

177

Appendices 67-70: NMR spectra of PRAES-E2

179

Appendices 71-78: IR, MS and NMR spectra of PRAES-C22

181

Appendices 79-86: IR, MS and NMR spectra of PRAES-C23

185

Appendices 87-94: IR, MS and NMR spectra of PRAES-C24

189

Appendices 95-97: MS and NMR spectra of PRAES-C25

193

Appendices 98-106: IR, MS and NMR spectra of PRAES-C25M

194

Appendices 107-114: IR, MS and NMR spectra of PRAES-C26

199

Appendices 115-119: NMR spectra of PRAES-T3

203

Appendices 120-124: NMR spectra of PRAES-C7

205


xvi

Appendices 125-131: MS and NMR spectra of PRAES-E18

208

Appendices 132-136: MS and NMR spectra of PRAES-C14

211

Appendices 137-141: NMR spectra of PRAES-C12

214

Appendices 142-147: MS and NMR spectra of PRAES-C5

216

Appendices 148-155: IR, MS and NMR spectra of PRAES-C15

219

Appendices 156-163: IR, MS and NMR spectra of PRAES-C16

223

Appendices 164-172: IR, MS and NMR spectra of PRAES-C20

227

Appendices 173-180: IR, MS and NMR spectra of PRAES-C18

232

Appendices 181-186: MS and NMR spectra of PRAES-C3

236

Appendices 187-192: MS and NMR spectra of PRAES-C4

239

Appendices 193-200: IR, MS and NMR spectra of PRAES-C21

242

Appendices 201-204: NMR spectra of PRAES-E5

246

Appendices 205-207: NMR spectra of PRAES-E3

248

Appendices 208-213: MS and NMR spectra of PRAES-C8

249

Appendices 214-222: IR, MS and NMR spectra of PRAES-C27

252

Appendices 223-231: IR, MS and NMR spectra of PRAES-C28

256

Appendices 232-235: NMR spectra of PRAES-E17

261

Appendices 236-237: NMR spectra of PRAES-E6

263

Appendices 238-244: MS and NMR spectra of PRAES-E13

264

Appendices 245-259: MS and NMR spectra of PRAES-E15

268


INTRODUCTION

Lichens are by definition symbiotic organisms composed of a fungal partner
(mycobiont) and one or more photosynthetic partners (photobiont/s). The photobiont
can be either a green alga or a cyanobacterium. Morphologically lichens can be
classified into three major groups. They are foliose, fruticose and crustose. Growing
rates of lichens are extremely slow. More than twenty thousand species of lichens
have been found. They can tolerate very drastic weather conditions and are resistant

to insects and other microbial attacks. Lichens produce a variety of secondary
compounds. They play an important role in protection and maintenance of the
symbiotic relationship [1].
Many lichen secondary metabolites exhibited antibiotic, antitumour,
antimutagenic, allergenic, antifungal, antiviral, enzyme inhibitory and plant growth
inhibitory properties [5, 12]. In 2007, Balaji. P. et al. [3] indicated that
dichloromethane, ethyl acetate and acetone methanol extracts of Parmotrema
praesorediosum showed antimicrobial activity against ten bacterial (Gram + and -)
(Bacillus cereus, Corynebacterium diptheriae, Proteus mirabilis, Proteus vulgari,
Pseudomonas aeruginosa, Salmonella typhi, Shigella flexnerii, Staphylococcus
aureus, Streptococcus pyogenes and Vibrio cholera) and one fungal Candida albicans
by using standard dics diffusion method. This lichen could therefore be a potential
source in the search for pharmaceutical useful chemicals.
The primary goal of the present work was to isolate secondary metabolites
on the lichen Parmotrema praesorediosum (Nyl.) Hale. The chemical structure of
isolated compounds was characterized by spectroscopic methods (1D-, 2D-NMR,
HRMS, CD). Finally, the purified substances from this source were assayed for the
cytotoxic activities against three cell lines: MCF-7 (breast cancer cell line), HeLa
(cervical cancer cell line) and NCI-H460 (human lung cancer cell line) by

1


sulforhodamine B colorimetric assay method (SRB assay) [56] and the inhibition
against acetylcholinesterase in vitro.
Based on spectroscopic evidence and their physical properties, the chemical
structures were attributed for be forty compounds, including six aliphatic acids,
twelve mononuclear phenolic acids, three depsides, two depsidones, eight diphenyl
ethers, three dibenzofurans, two xanthones, three triterpenoids and a macrocyclic
compound. The latter twenty two compounds appeared to be new and among

eighteen known compounds, twelve compounds were known for the first time from
the genus Parmotrema. These results pointed out that the Vietnamese lichens could
be new sources of bioactive compounds with novel skeletons

2


CHAPTER 1

LITERATURE REVIEW

1.1. GENERIC DESCRIPTION
1.1.1. The lichen
Lichens are by definition symbiotic organisms, usually composed of a fungal
which is most often either a green alga or cyanobacterium. The photobionts produce
carbohydrates by photosynthesis for themselves and for their dominant fungal
counterparts (mycobionts), which provide physical protection, water and mineral
supply [73]. Overall the lichen symbiosis is a very successful one, as lichens are
found in almost all terrestrial habitats from the tropics and deserts to polar regions.
As the results of the relationship, both the fungus and algae/cyanobacterium
partners, which mostly thrive in relatively moist and moderate environments in free
living form, have expanded into many extreme terrestrial habitats, where they
would separately be rare or non-existent [52]. On the basis of their forms and
habitats, lichens are traditionally divided into three main morphological groups:
crustose, foliose and fructicose (Figure 1.1) [42].

Crustose

Foliose
Figure 1.1. Types of the lichen

3

Fructicose


The lichen symbiosis is different other than kinds of symbiosis because the
lichen takes on a new body shape that neither the fungus nor the alga had
independently [73]. About 17,000 different lichen taxa, including 16,750 lichenized
Ascomycetes, 200 Deuteromycetes, and 50 Basidiomycetes have been described
world-wide. A thallus consists of a cortex and a medulla, both made up of fungal
tissue and a photobiont layer in which the alga and cyanobacterial cells are
endeveloped by fungal hyphae.
1.1.2. Parmotrema praesorediosum (Nyl.) Hale
The Parmeliaceae is a large and diverse family of Lecanoromycetes. With
over 2000 species in roughly 87 genera, it is regarded as the largest family of lichen
forming fungi [39]. The most speciose genera in the family are the well-known
groups: Xanthoparmelia (800+ species), Usnea (500+ species), Parmotrema (350+
species), and Hypotrachyna (190+ species) [39]. Nearly all members of the family
have a symbiotic association with a green alga (most often Trebouxia spp., but
Asterochloris spp. are known to associate with some species) [73]. The majority of
Parmeliaceae species have a foliose, fruticose, or subfruticose growth form. The
family has a cosmopolitan distribution, and can be found in a wide range of habitats
and climatic regions [73]. Members of the Parmeliaceae can be found in most
terrestrial environments
Parmotrema A. Massal. (previously known as Parmelia s.lat.) is one of the
largest genera of parmelioid core in the family Parmeliaceae [39]. The Parmotrema
genus is characterized by foliose thalli forming short and broad, rarely elongated,
often ciliate lobes, a pored epicortex, cylindrical conidia and the intermediate type
of lichenan between Cetraria-type lichenan and Xanthoparmelia-type lichenan. The

lower surface of the thallus is white to black, usually sparingly rhizinate with a wide
bare marginal zone, sometimes irregularly rhizinate or finely short-rhizinate with
scattered much longer rhizines mixed without an erhizinate margin or with a very
narrow one [72].
4


The upper surface

The lower surface

Figure 1.2. Parmotrema praesorediosum (Nyl.) Hale (Parmeliaceae)
Scientific name:

Parmotrema praesorediosum (Nyl.) Hale
Parmelia praesorediosa Nyl.

Family: Parmeliaceae
Morphography: Thallus foliose, adnate to the substratum, 3~10 cm across.
Lobes round, 4~10 mm wide; margins entire or crenate, eciliate, sorediate. Upper
surface pale grey to grey, smooth, dull, emaculate, weakly rugose, lacking isidia,
sorediate. Soralia marginal, linear to crescent shaped, granular. Medulla white.
Lower surface black, minutely rugose, with shiny, mottled, ivory or brown,
erhizinate marginal zone. Rhizines sparse, simple, short. Apothecia and pycnidia is
not seen [49].
Spotest: Cortex K+ (yellow), C−, KC−, P−; medulla K−, C−, KC−, P−
TLC: atranorin, chloroatranorin, fatty acids (protopraesorediosic acid,
praesorediosic acid).

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1.2. CHEMICAL STUDIES ON THE LICHEN GENUS PARMOTREMA
1.2.1. Lichen secondary metabolites
Primary metabolites of lichens, which are intracellular, are proteins, amino
acids, polyols, carotenoids, polysaccharides and vitamins. Lichens produce a wide
array of secondary metabolites (intracellular). There are over 700 lichen substances
reported to date and many are restricted to the lichenised state. Broadly speaking,
there are three types of lichen substances based on their biosynthetic origin [43]
(Figure 1.2).
 The acetate-malonate pathway produces depsides, depsidones and
dibenzofurans. The most important of these are the esters and the oxidative
coupling products of simple phenolic units related to orcinol and 3-orcinol.
Most depsides and depsidones are colorless compounds which occure in the
medulla of the lichen. However, usnic acids, yellow cortical compounds
formed by the oxidative coupling of methylphloroacetophenone units are
found in the cortex of many lichen species. Anthraquinones, xanthones and
chromones, are all pigmented compounds which occur in the cortex. They
are also produced by the acetate-malonate pathway, but their biosynthesis
results from intramolecular condensation of long, folded polyketide units
rather than the coupling of phenolic units.
 The shikimic acid pathway produces two major groups of pigmented
compounds, which occur in the cortex: pulvinic acid derivatives and
terphenylquinones. Although most pulvinic acid derivatives lack nitrogen,
they are biosynthesized through phenylalanine. Nitrogen is strongly limited
to metabolic activities in most lichens, and nitrogen rich metabolites such as
alkaloids are unknown among lichen substances.
 The mevalonic acid pathway produces terpenoids and steroids. These
compounds are found in lichens and many of them occur in higher plants as
well.

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Alga

Fungus

glucose
erythritol
ribitol
mannitol

Usnic acids Anthraquinones
Poly saccharides
Sugars

Methylphloroacetophenone/
acetylmethylphloroglucinol

Pentose phosphate cycle

Polyketide

Malonyl-CoA

Amino acids
Mevalonic
acid

Shikimic

acid

-Orsellinic acid

Depsones
Squalenes

Geranylgeranyl-p-p

Triterpens

Phenylalanine Terphenylquinones
Diterpenes

Pulvinic acid
derivatives

Secondary
aliphatic acids,
esters and related
derivatives

Glucolysis
Acetyl CoA

Phenylpyruvic acid

Xanthones,
Chromones

Orsellinic acid
and homologues
para-

meta-Depsides
Benzyl esters

Tridepsides

Dibenzofuran

Depsidones
Diphenyl
ethers

Steroids

Carotenoids
Figure 1.3. Biosynthetic pathways of the major groups of lichen substances [43].
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