Study on chemical constituents and biological activities of four lichens growing in the south of vietnam

VIETNAM NATIONAL UNIVERSITY - HO CHI MINH CITY
UNIVERSITY OF SCIENCE



DƯƠNG THÚC HUY

STUDY ON
CHEMICAL CONSTITUENTS AND
BIOLOGICAL ACTIVITIES
OF FOUR LICHENS
GROWING IN THE SOUTH OF
VIETNAM

DOCTORAL THESIS IN CHEMISTRY

Ho Chi Minh City, 2016


VIETNAM NATIONAL UNIVERSITY - HO CHI MINH CITY
UNIVERSITY OF SCIENCE



DƯƠNG THÚC HUY

STUDY ON
CHEMICAL CONSTITUENTS AND BIOLOGICAL
ACTIVITIES OF FOUR LICHENS
GROWING IN THE SOUTH OF VIETNAM
Subject: Organic Chemistry

Code number: 62 44 27 01

Examination Board:
Assoc. Prof. Dr. Tran Hung

(1st Reviewer)

Assoc. Prof. Dr. Pham Dinh Hung

(2nd Reviewer)

Assoc. Prof. Dr. Tran Cong Luan

(3rd Reviewer)

Dr. Nguyễn Trọng Tuân

(1st Independent Reviewer)

Dr. Lê Hoàng Duy

(2nd Independent Reviewer)

SUPERVISORS: PROF. DR. NGUYỄN KIM PHI PHỤNG
PROF. DR. JOEL BOUSTIE

Ho Chi Minh City, 2016


SOCIALIST REPUBLIC OF VIETNAM

INDEPENDENCE-FREEDOM-HAPPINESS

DECLARATION

The work presented in this thesis was completed in the period of September
2011 to September 2014 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 Joel Boustie of the University of Rennes 1, France.
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, March 28th, 2016
PhD student

DUONG THUC HUY

-i-


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 especially for
always keeping me on my toes, from the very beginning to the very end of my PhD.
programme.
I would also like to acknowledge my second supervisor, Prof. Dr. Joel Boustie
for his guidance, patience, and precious advice. I am deeply indebted to Prof. Dr.
Warinthorn Chavasiri at Chulalongkorn University, Thailand for his kindness, helpful
suggestion and financial support for this research. Also, I would like to thank you
Prof. Dr. Santi Tip-pyang for his scientific comments, Prof. Dr. Nongnuj Muagsin for
teaching me X-ray crystallography, and Dr. Panuwat Padungros for teaching me
organic synthesis.
I would also like to express my sincere thanks to Dr. Vo Thi Phi Giao from the
University of Science, Vietnam National University, Ho Chi Minh City, Dr. Harrie J.
M. Sipman, Botanic Garden and Botany Museum Berlin-Dahlem, Freie University,
Berlin, Germany, and Dr. Wetchasart Polysiam, Lichen Research Unit, Department of
Biology, Faculty of Science, Ramkhamhaeng University, Thailand for their expert
contribution to the identification of lichens.
In addition, I am very grateful to thank my friends Dr. Jirapast Sichaem, PhD.
students Suekanya Jarupinthusophon and Asshaima Paramita from Chulalongkorn
University,

PhD.

student

Theerapat

Luangsuphabool

from

Ramkhamhaeng

University, Dr. Huynh Bui Linh Chi, Dr. Nguyen Thi Hoai Thu, Dr. Truong Thi
Huynh Hoa, and Dr. Do Thi My Lien at University of Science, Vietnam, for their
helpful assistance and friendship during my research time at Chulalongkorn
University, Thailand as well as HCM University of Science, Vietnam.
-ii-


I would like to acknowledge the great encouragement, insightful comments,
and precious support from Dr. Duong Ba Vu, Dr. Nguyen Trung Nhan, Dr. Nguyen
Thi Anh Tuyet, Dr. Bui Xuan Hao, Msc. Vo Thi Thu Hang, and Msc. Nguyen Ngoc
Hung.
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 and the Department of Organic
Chemistry, Faculty of Chemistry, University of Pedagogy, Ho Chi Minh City,
Vietnam.
Most importantly, I would like to thank my wife for being the most patient and
supportive companion on my academic journey over the past four years. Without her
support, love and encouragement, this study would not have been possible.
Additionally, I would like to thank my children for helping me to unwind from my
stressful work.
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.

-iii-

TABLE OF CONTENTS
DECLARATION

.i

ACKNOWLEDGEMENTS

.ii

TABLE OF CONTENTS

.iv
.viii

LIST OF ABBREVIATIONS
LIST OF TABLES

.x

LIST OF FIGURES AND SCHEMES

.xi

GENERAL INTRODUCTION

.xii
1

CHAPTER 1: INTRODUCTION
1.1 INTRODUCTION

1

1.1.1 The lichen and usage of lichens

1

1.1.2 Biological significance of lichen subtances

2

1.1.3 Usage of lichens

2

1.2 BIOLOGICAL ACTIVITIES OF LICHEN SUBSTANCES

3

1.3 LICHEN SUBSTANCES

5

1.3.1 Polyketide pathways

5

1.3.1.1 Monoaromatic compounds

5

1.3.1.2 Depsides

6

1.3.1.3 Depsidone

6

1.3.1.4 Depsones

7

1.3.1.5 Diphenyl ethers

7

1.3.1.6 Dibenzofurans and usnic acid homologs

7

1.3.1.7 Aliphatic acids

8

1.3.1.8 Quinones, chromones and xanthones

8

1.3.2 Shikimic acid pathway

8

1.3.3 Mevalonic acid pathway

8

1.3.4 N-containing compounds

9

1.4 CHEMICAL CONSTITUENTS OF LICHENS OF ROCCELLA
GENUS

-iv-

15


1.5 CHEMICAL CONSTITUENTS OF LICHENS OF PARMOTREMA
GENUS
1.6 LICHENS GROWING IN VIETNAM, RESEARCH SCOPE AND
OBJECTIVES

16

23
24

CHAPTER 2: EXPERIMENTS

2.1 MATERIALS AND INSTRUMENTS

24

2.2 METHODS

25

2.3 EXPERIMENTALS

28

2.3.1 Extraction and isolation on the lichen Parmotrema sancti-angelii
(Lynge) Hale

28

2.3.2 Extraction and isolation on the lichen Parmotrema planatilobatum
(Hale) Hale

29

2.3.3 Extraction and isolation on the lichen Parmotrema tsavoense
(Krog & Swinscow) Krog & Swinscow

30

2.3.4 Extraction and isolation on the lichen Roccella sinensis (Nyl.) Hale

31

2.3.5 Experiments confirming two acetonide artefacts, 46 and 47

32

CHAPTER 3: RESULTS AND DISCUSSIONS
3.1 MONOCYCLIC COMPOUNDS

38
38

3.1.1 Methyl haematommate (1)

39

3.1.2 Methyl β-orsellinate (3)

39

3.1.3 Methyl orsellinate (4)

40

3.1.4 Orsellinic acid (5)

40

3.1.5 2,4-Dihydroxyphthalide (9)

41

3.1.6 Orcinol (10)

41

3.2 DEPSIDES

42

3.2.1 Atranorin (2)

42

3.2.2 Lecanoric acid (6)

43

3.2.3 Methyl lecanorate (8)

43

3.2.4 Lecanorin (40)

45

3.2.5 Gyrophoric acid (7)

45

3.3 DEPSIDONES

46
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3.3.1 Protocetraric acid (29)

47

3.3.2 9-O-Methylprotocetraric acid (30)

48

3.3.3 Virensic acid (36)

48

3.3.4 Parmosidone A (38)

49

3.3.4 Parmosidone B (37)

50

3.3.5 Parmosidone E (31)

51

3.4 FURFURIC ACID DERIVATIVES

52

3.4.1 Parmosidone C (34)

52

3.4.2 Parmosidone D (35)

55

3.4.3 Parmoether A (33)

56

3.4.4 Parmoether B (32)

59

3.4.5 Parmoether C (25)

60

3.5 DIPHENYL ETHERS

61

3.5.1 8-(2,4-Dihydroxy-6-(2-oxoheptyl)phenoxy)
-6-methoxy-3-pentyl-1H-isochromen-1-one (23)
3.5.2 8-(2,4-Dihydroxy-6-(2-oxoheptyl)phenoxy)

-6-hydroxy-3-pentyl-1H-isochromen-1-one (22)

61
63

3.5.3 β-Collactolic acid (21)

65

3.6 ERYTHRITOL DERIVATIVES

65

3.6.1 (+)-D-Montagnetol (43)

67

3.6.2 (+)-D-Erythrin (44)

69

3.6.3 Montagenetol B (46)

69

3.6.4 Montagnetol C (47)

71

3.6.5 Montagnetol A (41)

72

3.6.6 1-Acetylerythritol (42)

74

3.6.7 Montagnetol D (48)

74

3.6.8 Montagnetol E (45)

76

3.6.9 Montagnetol F (49)

77

3.7 STEROIDS AND TRITERPENOIDS
-vi-

78


3.7.1 (5α, 8α)-Esgosterol peroxide (17)

79

3.7.2 Brassicasterol (18)

79

3.7.3 Zeorin (11)

81

3.7.4 6-Acetoxyhopan-22-ol (12)

84

3.7.5 Leucotylin (16)

84

3.7.5 16-Acetoxyhopan-6,22-diol (14)

86

3.7.6 6-Acetoxyhopan-16,22-diol (13)

87

3.7.7 Hopan-6α,16α,22-triol (15)

87

3.8 COMPOUNDS OF OTHER TYPES

89

3.8.1 (E)-Nostodione A (39)

89

3.8.2 D-Mannitol (24)

91

3.8.3 D-Arabinitol (26)

92

3.8.4 Lichesterinic acid (20)

93

3.8.5 (+)-Prasorediosic acid (27)

93

3.8.6 (+)-Vinaprasorediosic acid A (28)

94

3.8.7 Skyrin (19)

96

3.9 CYTOTOXIC ACTIVITY AGAINST FOUR CELL LINES

97
100

CHAPTER 4: CONCLUSION
4.1. CHEMICAL CONSTITUENTS OF THE FOUR LICHENS

100

4.2. BIOLOGICAL ASSAY

105

LIST OF PUBLICATIONS
REFERENCES
APPENDICES

-vii-


LIST OF ABBREVIATIONS
1D

One dimensional

2D

Two dimensional

Ac

Acetone

AcOH

Acetic acid

br

Broad

C

Chloroform

calcd

Calculated

CC

Column chromatography

COSY

Homonuclear shift correlation spectroscopy

d

Doublet

dd

Doublet of doublets

DEPT

Distortionless enhancement by polarisation transfer

DMSO

Dimethyl sulfoxide

EA

Ethyl acetate

H

n-Hexane

HMBC

Heteronuclear multiple bond correlation spectroscopy

HPLC

High performance liquid chromatography

HR-ESI-MS

High resolution electrospray ionization mass spectrometry

HSQC

Heteronuclear single quantum correlation spectroscopy

m

Multiplet

M

Methanol

min

Minutes

MS

Mass spectrometry

NMR

Nuclear magnetic resonance

NOESY

Nuclear overhauser enhancement spectroscopy

P

Petroleum ether

ppm

Parts per million (chemical shift value)

pTLC

Preparative thin-layer chromatography

q

Quartet

quint

Quintet

s

Singlet

sext

Sextet

t

Triplet
-viii-


TLC

Thin-layer chromatography

TMS

Tetramethylsilane

UV

Ultraviolet

-ix-


LIST OF TABLES
Page
Table 1.1:

Biological activities of some lichen substances

Table 3.1:

1

Table 3.2:

NMR data of depsides 2, 6-8, and 40

44

Table 3.3:

NMR spectral data of 29-31, 37-38

48

Table 3.4:

NMR spectral data of 34-35, 38, and furfuric acid

53

Table 3.5:

NMR spectral data of 25, 32, and 33

57

Table 3.6:

NMR spectral data of 21-23

64

Table 3.7:

The coupling constants in erythritol and L-threitol

67

Table 3.8:

NMR data of 41-43, 46, and 47

69

Table 3.9:

NMR data of 44

70

Table 3.10:

NMR data of 45, 48, and 49

76

Table 3.11:

NMR data of 17, 18, and (5α,8α)- esgosterol peroxide

80

Table 3.12:

1

82

Table 3.13:

13

Table 3.14:

NMR spectral data of 39 and nostodione A

90

Table 3.15:

NMR spectral data of 24 and 26

92

Table 3.16:

NMR spectral data of 20, 27 and 28

94

Table 3.17:

NMR spectral data of 19

96

Table 3.18:

% Inhibition of cytotoxic activity against four cancer cell

97

H NMR data of monocyclic compounds 1, 3-5, 9, and 10

H NMR spectral data of 11-16
C NMR spectral data of 11-16

4
40

83

lines of isolated compounds
Table 3.19:

IC50 of cytotoxic activity against four cancer cell lines of 13,

97

32, 35, and 48
Table 4.1:

Secondary metabolites from four investigated lichens

-x-

106


LIST OF FIGURES AND SCHEMES
Page
Figure 1.1:

Growth forms of lichen

1

Figure 1.2:

Probable pathways leading to the major groups of lichen
metabolites

6

Figure 1.3:

Puvinic acid derivatives

9

Figure 1.4:

Two common triterpenenoid skeleton

9

Figure 1.5:

Some N-containing compounds

9

Figure 1.6:

Polyketide pathways

10

Figure 1.7:

Chemical constituents of some lichens of the Roccella genus

16

Figure 1.8:

Chemical constituents of some lichens of the Parmotrema
genus

19

Figure 2.1:

The lichen Parmotrema sancti-angelli (Lynge) Hale and the
lichen Parmotrema planatilobatum (Hale) Hale.

27

Figure 2.2:

The lichen Parmotrema tsavoense (Krog & Swincow) Krog &
Swincow

27

Figure 2.3:

The lichen Roccella sinensis (Nyl.) Hale

27

Figure 2.5:

TLC profile in order to prove the easy formation of artefacts
46 and 47
The preparation of acetonide derivative 47 and M1 from 44

Figure 3.1:

Chemical structures of compounds 1, 3-5, 9, and 10

39

Figure 3.2:

Chemical structures of 2, 6-8, and 40

42

Figure 3.3:

Chemical structures of 29-31, 37, and 38

46

Figure 3.4:

HMBC correlations of 29 and 30

47

Figure 3.5:

Selected HMBC correlations of 31, 37, and 38

50

Figure 3.6:

Chemical structures of 25, 32-35

52

Figure 3.7:

Selected HMBC and NOESY correlations of 34 and 35

55

Figure 3.8:

Selected HMBC and NOESY correlations of 25, 32, and 33

59

Figure 3.9:

Chemical structures of 21-23

61

Figure 3.10:

HMBC correlations of 23

63

Figure 3.11:

HMBC correlations of 22

65

Figure 3.12:

Erythritol derivatives from Roccella sinensis

66

Figure 3.13:

Selected HMBC correlations of 46 and 47

71

Figure 3.14:

Selected HMBC and NOESY correlations of 41

73

Figure 3.15:

Selected HMBC and NOESY correlations of 48

75

Figure 3.16:

Selected HMBC correlations of 45

77

Figure 3.17:

Selected HMBC and NOESY correlations of 49

78

Figure 2.4:

-xi-

33
33


Figure 3.18:

Chemical structures of the isolated sterols and triterpenes 1118

79

Figure 3.19:

Stereochemical structure of 11

84

Figure 3.20:

Stereochemical structure of 12

84

Figure 3.21:

Selected COSY, HMBC correlations and stereochemical
structures of 13, 14, and 16

85

Figure 3.22:

Selected COSY, HMBC correlations and stereochemical
structures of 15

89

Figure 3.23:

Expansion of the peak H-16 in 1H NMR spectrum

89

Figure 3.24:

HMBC correlations and the chemical structure of 39

91

Figure 3.25:

Chemical structures of 24 and 26

92

Figure 3.26:

Chemical structures of 20, 27, and 28

94

Figure 3.27:

Chemical structures of 19

95

Figure 3.28:

The relative relationship of biological activity and chemical
structure of three isolated triterpenes

98

Figure 3.29:

The relative relationship of biological activity and chemical
structure of three isolated erythritol derivatives

98

Figure 3.30:

The relative relationship of biological activity and chemical
structure of some isolated depsidones and diphenyl ethers

99

Scheme 1:

Extraction and isolation procedure for P. planatilobatum

34

Scheme 2:

Extraction and isolation procedure for P. sancti-angelli

35

Scheme 3:

Extraction and isolation procedure for Parmotrema tsavoense

36

Scheme 4:

Extraction and isolation procedure for Roccella sinensis
Proposed mechanism for the formation of acetonides 47 and

M1 from 44

37

Scheme 5:

-xii-

72


ABSTRACT
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. 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 the
protection and maintenance of the symbiotic relationship [36]. Many lichen secondary
metabolites exhibited antibiotic, antitumour, antimutagenic, allergenic, antifungal,
antiviral, enzyme inhibitory and plant growth inhibitory properties [8, 9, 53].
The lichens Parmotrema tsavoense (Krog & Swincow) Krog & Swincow and
Roccella sinenis (Nyl.) Hale have not been yet studied chemically and biologically in
the world. The lichen Parmotrema sancti-angelii (Lynge) Hale was studied by Neeraj
V. et al. (2011) [54] with report of 3 antibacterial lichen metabolites and by Ha Xuan
Phong (2012) [72] with report of three novel bicyclo compounds together with some
common lichen subtances. The lichen Parmotrema planatiobatum (Hale) Hale was
studied previously by Duong Thuc Huy (2011) with reports of common compounds.
The goal of the present work was to isolate secondary metabolites on the four
lichens Parmotrema tsavoense (Krog & Swincow) Krog & Swincow, Parmotrema

sancti-angelii (Lynge) Hale, Parmotrema planatiobatum (Hale) Hale, and Roccella
sinenis (Nyl.) Hale. The lichens were macerated in methanol or acetone to obtain the
crude methanol or acetone extracts. From the crude extract of each lichen, multi
chromatographic methods as normal phase column chromatography, reverse phase
column chromatography, preparative thin-layer chromatography, gel chromatography
were applied to isolate and purify the lichen subtances. The chemical structure of
isolated compounds was characterized by spectroscopic methods (1D-, 2D-NMR,
HRMS, CD). New compounds which were proposed to be artifacts during isolation
were confirmed their natural origin by the synthetic reactions or the characteristic TLC
method for determining lichen metabolites. Finally, the purified substances were
assayed for the cytotoxic activities against four cell lines: MCF-7 (breast cancer cell
-xiii-


line), HeLa (cervical cancer cell line), HepG2 (liver hepacellular carcinoma), and NCIH460 (human lung cancer cell line) by sulforhodamine B colorimetric assay method
(SRB assay) [62].
Based on spectroscopic evidence and their physical properties, the chemical
structures were attributed to forty seven compounds, including six mononuclear
compounds, five depsides, seven depsidones, five furfuric acid derivatives, three
diphenyl ethers, seven erythritol derivatives, eight steroid and triterpenoid compounds,
and seven compounds of other types. Thirteen new compounds were found, twelve
compounds were known for the first time from the genus Parmotrema, three
compounds were known for the first time from the genus Roccella.
Each lichens produces the specific metabolites, for instance most compounds
isolated from the lichen Parmotrema tsavoense were β-orcinol derivatives, including
various skeletons as moncyclic compounds, depside, depsidones, and diphenyl ethers.
In contrast, chemical constituents of the lichen Parmotrema sancti-angelii were
orcinol derivatives and hopane-6-ol derivatives. In the lichen Roccella sinensis, most
compounds were erythitol derivatives.
These results pointed out that the lichens growing in Vietnam possess the

number of new natural products which could be biosynthesized under a tropical
monsoon climate of Vietnam.

-xiv-


CHAPTER 1: INTRODUCTION

1.1 INTRODUCTION
1.1.1 The lichen and usage of lichens
Lichens are symbiotic organisms, usually composed of a fungal partner, the
mycobiont, and one or more photosynthetic partners, the photobiont, which is most
often either a green alga or cyanobacterium. There are about 300 genera and 18,000
species of presently recognized lichens, and they account for about 20% of all fungi.
Lichens are traditionally divided into three growth morphological forms: crustose,
foliose and fructicose (Barrington E. J. W and Willis A. J., 1974) [6].

Xanthoria sp.,
(Crustose lichen) [78]

Xanthoparmelia cf. lavicola,
(Foliose lichen) [78]

Hypogymnia cf. tubulosa,
(Fructicose lichens) [78]

Figure 1.1: Growth forms of lichen

The mycobiont of lichen lacks photosynthetic capabilities and obtains carbon
sources from the photobiont while manipulating its growth. In return, the mycobiont,

with its highly differentiated morphological structures, secures adequate illumination,
water, mineral salts and gas exchange for the photobiont. As a result of the
relationship, both the fungus and alga/cyanobacterium partners, which mostly thrive in
relatively moist and moderate environments in free-living form, have expanded into
many extreme terrestrial habitats from the tropics and deserts to polar regions and
colonized a wide range of different substrata, such as rocks, bare ground, leaves, bark,
metal, and glass, … (Le H. D., 2012 [74], Barrington E. J. W and Willis A. J., 1974 ).
-1-


1.1.2 Biological significance of lichen subtances
Some of the possible biological functions of lichen metabolites were summarized
by Huneck S. and Yoshimura I. (1996) [36] as follows:
- Antibiotic activitives, which provide protection against microorganisms.
- Photoprotective activitives – aromatic substances absorb UV light to protect algae
(photobionts) against intensive irradiation.
- Symbiotic equilibrium promotion, which affects the cell wall permeability of
photobionts.
- Chelating agents, which capture and supply important minerals from the substrate.
- Antifeedant/ antiherbivory activities – protect the lichens from the insect and animal
feedings.
- Hydrophobic properties, which prevent saturation of the medulla with water and
allow continuous gas exchange.
- Stress metabolites, metabolites secreted under extreme conditions.
1.1.3 Usage of lichens
For centuries, lichens have been used as folk medicine and their use persists to
the present day in some countries in the world. The use of lichen is especially evident
and well-documented in traditional Chinese medicine. 71 species from 17 genera (9
families) have been used for medicinal purposes in China. Lichens of the family
Parmeliaceae (17 species from 4 genera), Usneaceae (13 species from 3 genera), and

Cladionaceae (12 species from Cladonia) are the most commonly used. In other parts
of the world, Usnea species are the most utilized in India and by the Seminole tribe in
Florida, United States. A recent study of the commercial and ethnic uses of lichens in
India showed that 38 different species were sold commercially (Upreti D. K., 2003)
[71]. Most of the lichens are collected from the Western Himalayas and Central and
Western Ghats. Cetraria islandica (common name: Island moss) has also been used to
treat various lung diseases and catarrh, and is still sold in other parts of Europe (Choi
Y. H., 2008) [77].
-2-


The fragrance industry uses two species of lichens, Evernia prunastri var.
prunastri (oakmoss) and Pseudevernua furfuracea (treemoss). About 700 tons of
oakmoss are currently processed every year by French producers (Muller K., 2001)
[53].
Lichens have been used as human food although the thalli are often tasteless and
contain bitter irritating acids as well as provide little nutritional value. In Japan, the
foliose rock tripes (lichens Umbilicaria), called Iwatake, are eaten in salads as a
delicacy. The lichen Bryoria fremontii is placed into a pit oven for cooking in British
Columbia. Lichens are also food for animal. Reindeer, caribou, and deer have eaten
the lichens Cladonias and Cetrarias growing in snow during the winter in Canada. [6]
Moreover, lichens had economic importance as dyestuffs. The lichen Roccella
was used by the ancient Greeks and peoples in the Mediterranean region as a valuable
purple dye (orchid). In northern Europe the locally abundant species of the lichens
Parmelia, Evernia, and Ochrolechia were collected as brown dyes (crottal) and even
to this day support a small cottage-type dyeing industry in Scandinavia. The
amphoteric dye litmus, a familiar acid-base indicator in chemistry laboratories, is
derived from depside-containing lichens. [6]
1.2 BIOLOGICAL ACTIVITIES OF LICHEN SUBSTANCES
The biological activities as well as pharmaceutical potential of lichen metabolites

have been reviewed extensively. In general, the bioactivities of lichen metabolites
include antibiotic, antimycobacterial, antiviral, antifungal, anti-inflamatory, analgesic,
antipyretic, antiproliferative, antitumour and cytotoxic effects. The bioactivities of a
number of lichen compounds in some recent studies are summarized in Table 1.1
(Boustie J. and Grube M., 2007 [8], Boustie et al.. 2010 [9], Huneck S., 1999 [37],
Muller K., 2001 [53]). Besides the small molecule compounds traditionally
categorized as secondary metabolites or natural products, the lichen polysaccharides
have also shown various bioactivities, including antitumour, immunomodulator,
antiviral effects, and memory enhancement. The studies on various effects of lichen
polysaccharides have been covered in a recent review (Olafsdottir E. S., 2001) [58].
-3-


Table 1.1: Biological activities of some lichen substances [9] [53] [77].
Antiviral activities of some lichen compounds
Compounds

Viruses and viral enzymes

Depsidone: virensic acid and its derivatives

Human immunodeficiency virus.

Butyrolactone
acid

HIV reverse transcriptase

acid: protolichesterinic

(+)-Usnic acid and four orcinol depsides

Epstein-Barr virus (EBV)

Emodin, 7-chloroemodin, 7-chloro-1-Omethylemodin,
5,7-dichloroemodin,
hypericin

HIV, cytomegalovirus and other viruses

Antibiotic and antifungal activities of some lichen compounds
Compounds

Usnic acid and its derivatives

Organisms
Gram +ve bacteria, Bacteroides spp., Clostridium
perfringens, Bacillus subtilis, Staphylococcus aureus,
Staphylococcus
spp.,
Enterococcus
spp.,
Mycobacterium aurum

Protolichesterinic acid

Helicobacter pylori

Methyl orsellinate, ethyl orsellinate,
methyl

β-orsellinate,
methyl
haematommate

Epidermophyton floccosum, Microsporum canis, M.
gypseum, Trichophyton rubrum, T. mentagrophytes,
Verticillium achliae, Bacillus subtilis, Staphylococcus
aureus, Pseudomonas aeruginosa, Escherichia coli,
Candida albicans

Alectosarmentin

Staphylococcus aureus, Mycobacterium smegmatitis

1’-Chloropannarin, pannarin

Leishmania spp

Emodin, physcion

Bacillus brevis

Pulvinic acid and its derivatives

Drechslera rostrata, Alternaria alternata
Aerobic and anaerobic bacteria

Leprapinic acid and its derivatives

Gram +ve and –ve bacteria

Enzyme inhibitory activities of some lichen compounds
Compounds

Enzymes

Atranorin

Trypsin, Pankreaselastase, Phosphorylase

Baeomycesis acid

5-Lipoxygenase

Bis-(2,4-dihydroxy-6-npropylphenyl)methane, divarinol, lichen
extracts from Cetraria juniperina,
Hypogymnia physodes and Letharia

Tyrosinase

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vulpina
Chrysophanol

Glutathione reductase

Confluentic acid, 2β-O-methylperlatolic
acid

Monoaminoxidase B

4-O-Methylcryptochlorophaeic acid

Prostataglandinsynthetase

(+)-Protolichesterinic acid

5-Lipoxygenase (HIV reverse transcriptase)

Vulpinic acid

Phosphorylase

Norsolorinic acid

Monoamino oxidase

Physodic acid

Arginine decarboxylase

Usnic acid

Ornithine decarboxylase

Antitumour and antimutagenic activities of some lichen compounds
Compounds

Activities/cell types

(-)-Usnic acid

Antitumoral effect against Lewis Lung carcinoma,
P388 leukaemia, mitotic inhibition, apoptotic induction,
antiproliferative effect against human HaCaT
keratinocytes

Protolichesterinic acid

Antiproliferative effect against leukaemia cell K-562
and Ehrlich solid tumour

Pannarin, 1-chloropannarin, sphaerophorin

Cytotoxic effect against cell cultures of lymphocytes

Naphthazarin

Cytotoxic effect against human epidermal carcinoma
cells, antiproliferative effects against human
keratinocyte cell line

Scabrosin
euplectin

ester

and

its

derivatives,

Cytotoxic effect against murine P815 mastocytoma and
other cell lines

Hydrocarpone, salazinic acid, stictic acid

Apoptotic effect against primary culture of rat
hepatocytes

Psoromic acid, chrysophanol, emodin and
its derivatives

Antiproliferative effect against leukemia cells

1.3 LICHEN SUBTANCES
Lichen substances were classified into three main groups based on their
biosynthetic pathways as presented in Figure 1.2 (Huneck S. and Yoshimura I., 1996
[36]).
 Polyketide pathway: depsides, depsidones, quinones, xanthones, chromones,
aliphatic acids…
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 Shikimic acid pathway: terphenylquinones and derivatives of tetronic acid.
 Mevalonic acid pathway: terpenoids, steroids
1.3.1 Polyketide pathways

1.3.1.1 Monocyclic aromatic compounds
Huneck and Yoshimura (1996) [36] reviewed 32 monocyclic aromatic
compounds until the 1997 and then Huneck S. (2001) [38] reported 26 additional ones.
The most common monoaromatic compounds found in lichens are orsellinic acid,
β-orsellinic acid and its derivatives. Some of the orsellinic acid derivatives contain a
long alkyl chain at C-6 of the aromatic ring which replaces the methyl group.
Occasionally, a keto group is located at an odd-numbered carbon of the alkyl chain.
Besides, β-orsellinic acids contain an additional substituent at C-3, including a
methyl (-CH3) or an aldehyde group (-CHO) or a hydroxymethyl group (-CH2OH)
(Figure 1.6).

carotenoid

Figure 1.2: Probable pathways leading to the major groups of lichen metabolites [19].

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1.3.1.2 Depsides
Depsides consist of two basic monocyclic aromatic moieties, such as orsellinic
acid or β-orsellinic acid coupled by an ester bond. Depsides can be subdivided into
para- and meta-depsides based on the position of the second carboxyl group bonded to
the B-ring. For examples, para-depsides possess the para-correlation between the first
carboxyl group and the second one, and similarly meta-depsides possess the metacorrelation. Ortho-depsides also occur in lichens but so far only isolecanoric acid has
been known [36].
Tri- and tetra-depsides are occasionally found in lichens but most of them
incorporate orsellinic acid as the monomeric unit. The majority of aromatic units in triand tetra-depsides are joined by ester bonds at the para-positions and the simplest one
is gyrophoric acid (comprising three orsellinic acid units) (Figure 1.6).
1.3.1.3 Depsidones
Like depsides, depsidones also consist of two monoaromatic moieties with an

additional ether bond forming a specific 7-membered ring of depsidones. The
occurrence of depsides and depsidones with the same monoaromatic units has led to
the hypothesis that depsides are precursors of depsidones. However, depsides and
depsidones with the same monoaromatic units do not always occur together in the
same lichens and not all known depsidones have a corresponding depside (Choi Y. H.,
2008) [77]. The ester linkage in most depsidones is bonded at the para-position of the
B-ring, like para-depsides. Compared to the structure of known depsides, depsidones
possess the diversity of different substituents in their skeletons, such as Br atoms,
crotonyl group… (Figure 1.6).
1.3.1.4 Depsones
Depsones are rare compared to depsides and depsidones. So far only eight
depsones have been described [19]. The aromaticity of the A-ring is lost due to the
occurrence of one additional linkage between C-1 of A-ring and C-4’ of B-ring. The
best-known depsone is picrolichenic acid, found in Pertusaria amara. Two unusual

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trinuclear hydrid compounds, friesiic acid and 2’-O-methylfriesiic acid, were isolated
from the lichen Hypocenomyce friesii (Elix J. A. et al., 2004) [20] (Figure 1.6).
1.3.1.5 Diphenyl ethers
Diphenyl ethers are relatively rare in lichens compared to depsides and
depsidones. Most of the known diphenyl ethers possess the ether bond joined at the
meta-position of the B-ring and are often related to depsidones. Hence, diphenyl ethers
are proposed to be the thermal-decomposed products of depsidones (Millot M., 2008
[50]) as a result of their isolation or sometimes referred as “pseudodepsidones” due to
their apparent biosynthetic relationship (Huneck S., 2001 [38]) (Figure 1.6).
1.3.1.6 Dibenzofurans and usnic acid homologs
Dibenzofurans are the third most abundant group in lichens after depsides and
depsidones. They mostly consist of orcinol-type monoaromatic units.

Usnic acid and related compounds are not dibenzofurans due to the loss of the
aromaticity caused by the presence of the methyl group at C-12 in the second ring but
their biosynthesis is likely to involve similar mechanisms (Figure 1.6).
1.3.1.7 Aliphatic acids
Aliphatic acids found in lichen are mostly the 5-membered lactones with the
alkyl chain at C-4, such as lichesterinic acid. However, some complex aliphatic acids
were also found in lichens, for example roccelic acid (Figure 1.6) [19].
1.3.1.8 Quinones, chromones and xanthones
Quinones, chromones and xanthones possess complex structures. They can occur
in symmetric or unsymmetric dimers such as binaphthoquinone, bixanthone,
bianthraquinone. Recently, some asymmetric glycoside dimers have been isolated
from Usnea hirta (Rezanka T. & Sigler K., 2007) (Figure 1.6) [67].
1.3.2 Shikimic acid pathway
Terphenylquinones and derivatives of tetronic acids or pulvinic acid are common
examples for this pathway (Figure 1.3). They are very toxic and carcinogenic reagents.
They often contain two benzene rings with non- or mono-substituents.
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Figure 1.3: Puvinic acid derivatives

1.3.3 Mevalonic acid pathway
Triterpenes found in lichens usually possess two specific skeletons, hopane or
fern-9(11)-ene. Besides, other terpenoids, steroids or carotenoids were also isolated
from lichens, but the number of reports about them are smaller than that about
triterpenoids [19] (Figure 1.4).

Figure 1.4: Two common triterpenenoid skeletons

1.3.4 N-containing compounds

Some N-containing compounds were also found in lichens, but so far related
reports have been rare [36].

Figure 1.5: Some N-containing compounds

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