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 SOUT

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:

SUPERVISORS: PROF DR NGUYỄN KIM PHI PHỤNG

PROF DR JOEL BOUSTIE

Ho Chi Minh City, 2016

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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 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

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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

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TABLE OF CONTENTS

1.1.2 Biological significance of lichen subtances 2

1.4 CHEMICAL CONSTITUENTS OF LICHENS OF ROCCELLA

2.3.3 Extraction and isolation on the lichen Parmotrema tsavoense

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

NMR Nuclear magnetic resonance

NOESY Nuclear overhauser enhancement spectroscopy

P Petroleum ether

ppm Parts per million (chemical shift value)

pTLC Preparative thin-layer chromatography

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TLC Thin-layer chromatography TMS Tetramethylsilane

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LIST OF TABLES

Page

Table 3.1: 1H NMR data of monocyclic compounds 1, 3-5, 9, and 10 40

Table 3.11: NMR data of 17, 18, and (5α,8α)- esgosterol peroxide 80

Table 3.18: % Inhibition of cytotoxic activity against four cancer cell

lines of isolated compounds

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LIST OF FIGURES AND SCHEMES

Page

Figure 1.2: Probable pathways leading to the major groups of lichen

Figure 1.7: Chemical constituents of some lichens of the Roccella genus 16

Figure 1.8: Chemical constituents of some lichens of the Parmotrema

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 &

Figure 2.4: TLC profile in order to prove the easy formation of artefacts

Figure 2.5: The preparation of acetonide derivative 47 and M1 from 44 33

Figure 3.8: Selected HMBC and NOESY correlations of 25, 32, and 33 59

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Figure 3.18: Chemical structures of the isolated sterols and triterpenes

Figure 3.21: Selected COSY, HMBC correlations and stereochemical

Figure 3.22: Selected COSY, HMBC correlations and stereochemical

Figure 3.28: The relative relationship of biological activity and chemical

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 37

Scheme 5: Proposed mechanism for the formation of acetonides 47 and

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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

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line), HeLa (cervical cancer cell line), HepG2 (liver hepacellular carcinoma), and H460 (human lung cancer cell line) by sulforhodamine B colorimetric assay method (SRB assay) [62]

NCI-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

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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]

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 )

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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]

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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]

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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: protolichesterinic

acid

HIV reverse transcriptase

(+)-Usnic acid and four orcinol depsides Epstein-Barr virus (EBV)

Emodin, 7-chloroemodin,

7-chloro-1-O-methylemodin, 5,7-dichloroemodin,

hypericin

HIV, cytomegalovirus and other viruses

Antibiotic and antifungal activities of some lichen compounds

Usnic acid and its derivatives

Gram +ve bacteria, Bacteroides spp., Clostridium

perfringens, Bacillus subtilis, Staphylococcus aureus, Staphylococcus spp., Enterococcus spp.,

Mycobacterium aurum

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

1’-Chloropannarin, pannarin Leishmania spp

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

Bis-(2,4-dihydroxy-6-n-propylphenyl)methane, divarinol, lichen

extracts from Cetraria juniperina,

Hypogymnia physodes and Letharia

Tyrosinase

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vulpina

Confluentic acid, 2β-O-methylperlatolic

acid

Monoaminoxidase B

4-O-Methylcryptochlorophaeic acid Prostataglandinsynthetase

(+)-Protolichesterinic acid 5-Lipoxygenase (HIV reverse transcriptase)

Antitumour and antimutagenic activities of some lichen compounds

Compounds Activities/cell types

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

cells, antiproliferative effects against human keratinocyte cell line

Scabrosin ester and its derivatives,

 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)

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

carotenoid

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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 meta- correlation 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 tri-

and 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

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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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)

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 1.3: Puvinic acid derivatives

Figure 1.4: Two common triterpenenoid skeletons

Figure 1.5: Some N-containing compounds

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Figure 1.6: Polyketide compounds

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Figure 1.6: Polyketide compounds

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Figure 1.6: Polyketide compounds

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Figure 1.6: Polyketide compounds

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Figure 1.6: Polyketide compounds

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1.4 CHEMICAL CONSTITUENTS OF LICHENS OF ROCCELLA GENUS

Lichens of the Roccella genus were studied over 50 years ago Phytochemical investigations on the lichens of the Roccella genus were reported including Roccella

fuciformis, R phycopis Ach., R capesis Follm , R galapagoensis, R hypomecha

Bory., R montagnei, R portentosa and R capesis Follm D-Montagnetol and D

-erythrin are major compounds, found in all studied Roccella lichens [31]

Lepraric acid (R7) was isolated from R fuciformis by Aberhart D J (1969) [1]

2-Methyl-5-hydroxy-6-hydroxymethyl-7-methoxychromone (R8) was isolated from R

fuciformis by Huneck S and Follmann G (1972)[31]

Picroroccellin (R14) was isolated by Marcuccio S M and Elix J A from R

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● Roccella portentosa

Acetylportentol (R12) and portentol (R13) were isolated from R portentosa by Aberhart D J et al (1970) [2]

1.5 CHEMICAL CONSTITUENTS OF LICHENS OF PARMOTREMA GENUS

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 [63] 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) [62] The majority of Parmeliaceae species have a

foliose, fruticose, or subfruticose growth form The family has a cosmopolitan

Figure 1.7: Chemical constituents of the Roccella lichen species

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distribution, and can be found in a wide range of habitats and climatic regions [63] Members of the Parmeliaceae can be found in most terrestrial environments

The chemical structures of compounds isolated from some lichens of

Parmotrema genus are presented in Figure 1.8

● Parmotrema conformatum

Protocetraric acid (P21), malonprotocetraric acid (P23), and (+)-(12R)-usnic acid

(P40) were isolated by Keogh M F (1977)[42]

● Parmotrema dilatum

Depside atranorin (P11), depsidones salazinic acid (P16), norstictic acid (P19),

hypostictic acid (P20), and protocetraric acid (P21) were isolated by Honda N K et

al (2009)[39]

● Pamotrema lichexanthonicum

Depside atranorin (P11), depsidone salazinic acid (P16), and xanthone

lichexanthone (P41) were isolated from the chloroform extract by Micheletti A C et

al (2009)[51]

● Parmotrema mellissii

Methyl orsellinate (P5), ethyl orsellinate (P6), n-butyl orsellinate (P7), methyl

β-orsellinate (P8), methyl haematommate (P9), ethyl chlorohaematommate (P10),

atranorin (P11), chloroatranorin (P12), α-alectoronic acid (P24), α-collatolic acid

(P25), 2'''-O-methyl-α-alectoronic acid (P26), 2'''-O-ethyl-α-alectoronic acid (P27),

dehydroalectoronic acid (P28), dehydrocollatolic acid (P29), (5'α)

In 2011, Duong T Huy et al [16] isolated 7 compounds: methyl β-orsellinate

(P8), methyl orsellinate (P5), orsellinic acid (P4), methyl haematommate (P9),

atranorin (P11), lecanoric acid (P14), and (+)-(12R)-usnic acid (P40)

● Parmotrema praesorediosum

(+)-Praesorediosic acid (P1), (+)-protopraesorediosic acid (P2), atranorin (P11)

and chloroatranorin (P12) were isolated by David F et al (1990) [15]

Lecanoric acid (P14) and stictic acid (P18) were isolated from Parmelia

praesorediosa (Nyl.) by Ramesh P et al (1994)[64]

Huỳnh B L Chi et al isolated prasoether A (P22), zeorin (P45), and

1β,3β-diacetoxyhopan-29-oic acid (P46) (2011)[29,30]

● Parmotrema reticulatum

Atranorin (P11), chloroatranorin (P12), salazinic acid (P16) and consalazinic

acid (P17) were isolated from the acetone extract by Fazio A T et al (2009)[23]

● Parmotrema saccatilobum

Atranorin (P11) and chloroatranorin (P12) were isolated from the hexane extract

by Bugni T S et al (2009)[12]

● Parmotrema sancti-angelii

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Atranorin (P11), lecanoric acid (P14), and α-collatolic acid (P25) were isolated

by Neeraj V et al (2011)[54]

Hà Xuân Phong isolated 10 compounds:

8-(2,4-Dihydroxy-6-(2-oxoheptyl)phenoxy)-6-hydroxy-3-pentyl-1H-isochromen-1-one (P39), gyrophoric acid (P13), lecanoric acid (P14), orsellinic acid (P4), methyl orsellinate (P5), methyl β-

orsellinate (P8), methyl haematomate (P9) and three new bicyclo compounds, Sancti A-C (P42-P44)[72]

● Parmotrema stuppeum

Orsellinic acid (P4), methyl orsellinate (P5), atranorin (P11), and lecanoric acid

(P14) were isolated by Javaprakasha G K et al (2000)[41]

● Parmotrema subisidiosum

Depside atranorin (P11) and two depsidones salazinic acid (P16) and

consalazinic acid (P17) were isolated from the acetone extract of P subisidiosum by

O’Donovan D G et al (1980)[57]

● Parmotrema tinctorum

Isolecanoric acid (P15) was isolated by Sakurai A et al (1987)[59]

Ethyl orsellinate (P6) was isolated by Santos L C et al (2004)[60]

Atranorin (P11) and lecanoric acid (P14) were isolated by Honda N K et al

(2010)[39]

Aliphatic acids

Figure 1.8: Chemical constituents of the Parmotrema lichen species

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Diphenyl ethers

Figure 1.8: Chemical constituents of the Parmotrema lichen species (cont.)

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Figure 1.8: Chemical constituents of the Parmotrema lichen species (cont.)

on their taxonomy but not on chemical constituents Since recent 5 years, lichens growing in Vietnam have been interested in chemical constituents, for examples, the

studies on the lichens Parmotrema prasorediosum (Huynh B L C., 2014 [73]),

Ramalina farinacea (Ly H D., 2014 [75]), Usnea aciculifera (Tuong L T., 2014

[76]), Parmotrema mellissii and Rimelia clavulifera (Le H D., 2012) Some lichens have not been studied on the chemical constituent in the past, for examples Usnea

aciculifera, Parmotrema mellissii, Rimelia clavulifera while some lichens have been

studied in the world but not in Vietnam, for examples Parmotrema prasorediosum and

Ramalina farinacea Those researches contributed considerably to the lichen

phytochemical studies in Vietnam as well as in the world with the isolation of 34 new

compounds Interestingly, Huynh B L C isolated 21 new compounds from the lichen

Parmotrema prasorediosum which was studied in the past The hot climate of Vietnam

affected much to the biosynthesis of lichen substances in Vietnam This specific climate created lots of novel compounds possessing uncommon skeletons (Huynh B

L C., 2014) Chemical studies in some lichens growing in Vietnam should improve and have more interests in order to find new compounds according to the diversity of lichens in Vietnam

From my interest in the diversity and biological activities of lichen substances, phytochemical studies on some lichens growing in Vietnam were undertaken The major goal of this thesis is to investigate the lichen substances from the lichens

collected in the highlands of Vietnam (ca 1,500 m alt.) or near the beach (ca 300 m

alt.) to isolate novel and/or bioactive compounds

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CHAPTER 2: EXPERIMENTS

2.1 MATERIALS AND INSTRUMENTS

Instruments and chemicals

The solutions of different extracts were evaporated by using rotary evaporator Buchi-111 1H-NMR, 13C-NMR and 2D-NMR spectra were acquired on Bruker Avance 500III (500 MHz for 1H-NMR and 125 MHz for 13C-NMR) and Varian Mercury-400 Plus NMR (400 MHz for 1H NMR and 100 MHz for 13C NMR) spectrometers HR-ESI-MS were recorded on Bruker micrOTOF Q-II Optical rotations were measured on a Kruss (German) polarimeter with the length of tube: d = 1 dm All instruments are in the Central Laboratory for Analysis the University of Science, National University- Ho Chi Minh City, except for Varian Mercury-400 Plus NMR spectrometer in Department of Chemistry of Chulalongkorn University, Bangkok, Thailand Thin-layer chromatography was performed on silica gel GF254 (Merck) Reagents for visualizing TLC plates included 10% solution of H2SO4 and vanillin/H2SO4 Column chromatography was performed on silica gel (Merck) Type 100 (70–230 Mesh ASTM), mostly eluted either with gradient systems of P–EA or P–EA–AcOH in different ratios Solvents: P (60-90 oC), H, C, EA, Ac, M, AcOH were from Chemsol and used as purchased

Lichen material Parmotrema sancti-angelii (Lynge) Hale (Figure 2.1)

Parmotrema sancti-angelii (Lynge) Hale was collected on the bark of tea trees Camellia sinensis (L.) O Ktze in Bao Loc city, Lam Dong province, Vietnam (August

2013 - October 2013) Its scientific name was identified by Dr Harrie J M Sipman, Botanic Garden and Botany Museum Berlin-Dahlem, Freie University, Berlin, Germany A voucher specimen (No US-B025) was deposited in the herbarium of the Department of Organic Chemistry, University of Science

Lichen material Parmotrema planatilobatum (Hale) Hale (Figure 2.1)

Parmotrema planatilobatum (Hale) Hale was collected on the bark of the Pinus dalatensis in Da Lat city, Lam Dong province (August 2009 - October 2009) Its