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 PRAESORE

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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

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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)

SUPERVISORS: PROF DR NGUYEN KIM PHI PHUNG

PROF DR TAKAO TANAHASHI

Ho Chi Minh City, 2014

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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

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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

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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

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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

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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

3.1.2 Chemical structure of mononuclear phenolic compounds 45

3.1.2.1 Structure elucidation of compound PRAES-T1 45

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3.1.3 Chemical structure of depsides 66

3.1.4 Chemical structure of depsidones 70

3.1.5 Chemical structure of diphenyl ethers 74

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3.1.6 Chemical structure of dibenzofurans 97

3.1.7 Chemical structure of xanthones 103

3.1.8 Chemical structure of triterpenoids 111

3.1.9 Chemical structure of a macrocylic compound 117

3.2.1 Cytotoxicity activitivy 120

3.2.2 Acetylcholinesterase inhibitory activity 121

CHAPTER 4: CONCLUSION 124

4.1 CONSTITUENTS OF PARMOTREMA PRAESOREDIOSUM 124

FUTURE OUTLOOK 133

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LIST OF PUBLICATIONS 134 REFERENCES 135 APPENDICES 145

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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

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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

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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 1H NMR of aliphatic compounds 38

Table 3.3 13C NMR of aliphatic compounds 39

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 1HNMR data of PRAES-C5 and Lecanorol 76

Table 3.11 1 H NMR data of PRAES-C15, PRAES-C16, PRAES-C20,

Table 3.12 13 C NMR data of PRAES-C15, PRAES-C16, PRAES-C20,

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

Table 3.17 NMR data of PRAES-C27 and PRAES-C28 (CDCl3) 110

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Table 3.18 NMR data of PRAES-E17, PRAES-E6, PRAES-E13 and

Table 3.19 NMR data of PRAES-E15 120

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

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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

Figure 2.2: Isolation of compounds from the chloroform extract of Parmotrema

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 3.5 Comparison of experimental CD spectrum of PRAES-C2Me and

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 3.12 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

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Figure 3.15 HMBC correlations of PRAES-C9 and PRAES-C7 67

Figure 3.16 COSY and HMBC correlations of PRAES-E18 70

Figure 3.20 HMBC and ROESY correlations of PRAES-C16 80

Figure 3.22 1H NMR data of PRAES-C18 and diphenyl ether 87

Figure 3.27 ROESY correlations of PRAES-C21 95

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 3.37 COSY and HMBC correlations of PRAES-E15 118

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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 27-33: IR, MS and NMR spectra of PRAES-E19 159

Appendices 41-44: NMR spectra of PRAES-T1 166

Appendices 45-49: MS and NMR spectra of PRAES-E1 168

Appendices 55-58: NMR spectra of PRAES-E11 173

Appendices 63-66: MS and NMR spectra of PRAES-T6 177

Appendices 98-106: IR, MS and NMR spectra of PRAES-C25M 194

Appendices 120-124: NMR spectra of PRAES-C7 205

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Appendices 137-141: NMR spectra of PRAES-C12 214

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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

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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

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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]

Figure 1.1 Types of the lichen

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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]

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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)

The upper surface The lower surface

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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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Sugars

Pentose phosphate cycle Amino acids

Shikimic acid

geranyl-p-p

Geranyl-Mevalonic acid

Squalenes Triterpens Steroids

Depsidones

Diphenylethers

Dibenzofuran Benzyl esters

meta-Depsides

Orsellinic acid and homologues

-Orsellinic acid

Polyketide

Malonyl-CoA

Secondary aliphatic acids, esters and related derivatives

Xanthones, Chromones

Anthraquinones Methylphloroacetophenone/

acetylmethylphloroglucinol

Usnic acids

Glucolysis

Tridepsides

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1.2.2 Chemical studies on the lichen genus Parmotrema

 Parmotrema praesorediosum

(+)-Praesorediosic acid (1), (+)-protopraesorediosic acid (2), atranorin (11)

and chloroatranorin (12) were isolated by David F et al (1990) [20]

Lecanoric acid (14) and stictic acid (18) were isolated from Parmelia

praesorediosa (Nyl.) by Ramesh P et al (1994) [62]

 Parmotrema sancti-angelii

Atranorin (11), lecanoric acid (14) and α-collatolic acid (25) were isolated by

Neeraj V et al (2011) [55]

 Parmotrema conformatum

Protocetraric acid (21), malonprotocetraric acid (23) and (+)-usnic acid (40)

were isolated by Keogh M F (1977) [44]

 Parmotrema dilatum

Depside atranorin (11), depsidones salazinic acid (16), norstictic acid (19),

hypostictic acid (20) and protocetraric acid (21) were isolated from Parmotrema

dilatum by Honda N K et al (2010) [32]

 Pamotrema lichexanthonicum

Depside atranorin (11), depsidone salazinic acid (16) and xanthone

lichexanthone (41) were isolated from the chloroform extract of Pamotrema

lichexanthonicum by Ana C M et al (2009) [3]

 Parmotrema mellissii

Methyl orsellinate (5), ethyl orsellinate (6), n-butyl orsellinate (7), methyl

β-orsellinate (8), methyl haematommate (9), ethyl chlorohaematommate (10), atranorin (11), chloroatranorin (12), α-alectoronic acid (24), α-collatolic acid (25),

2′′′-O-methyl-α-alectoronic acid (26), 2′′′-O-ethyl-α-alectoronic acid (27),

dehydroalectoronic acid (28), dehydrocollatolic acid (29), parmosidone A (30),

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parmosidone B (31), parmosidone C (32), isocoumarin A (33), isocoumarin B (34),

β-alectoronic acid (36), β-collatolic acid (37), 2′′′-O-methyl-β-alectoronic acid (38), 2′′′-O-ethyl-β-alectoronic acid (39), (+)-usnic acid (40) and skyrin (42) were

isolated from Parmotrema mellissii that was collected at Da Lat city, Vietnam by

Lê Hoàng Duy et al (2012) [52]

 Parmotrema nilgherrense

α-Alectoronic acid (24), α-collatolic acid (25) and dehydrocollatolic acid

(29) were isolated by Kharel M K et al (2000) [45]

Depside atranorin (11) were isolated by Neeraj V et al (2011) [55]

 Parmotrema planatilobatum

Orcinol (3), orsellinic acid (4), methyl orsellinate (5), methyl β-orsellinate

(8), methyl haematommate (9), atranorin (11), gyrophoric acid (13), lecanoric acid (14), protocetraric acid (21), 9-methylprotocetraric acid (22), methyl 2-[3-(2,6-

dihydroxy-4-methylbenzyl)-2,4-dihydroxy-6-methylphenoxy]-3-formyl-4-hydroxy-6-methylbenzoate (35) and usnic acid (40), were isolated by Duong T H et al

(2011, 2012) [22, 23]

 Parmotrema reticulatum

Atranorin (11), chloroatranorin (12), salazinic acid (16) and

consalazinic acid (17) were isolated from the acetone extract by Fazio A T et al

(2009) [25]

 Parmotrema saccatilobum

Atranorin (11) and chloroatranorin (12) were isolated from the hexane

extract of Parmotrema saccatilobum by Bugni T S et al (2009) [12]

 Parmotrema stuppeum

Orsellinic acid (4), methyl orsellinate (5), atranorin (11) and lecanoric acid

(14) were isolated by Javaprakasha G K et al (2000) [40]

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Isolecanoric acid (15) was isolated by Sakurai A et al (1987) [63]

Ethyl orsellinate (6) was isolated by Santos L C et al (2004) [64]

Atranorin (11) and lecanoric acid (14) were isolated by Honda N K et al

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11

Depsides

Depsidones

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12

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Diphenylethers

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Quinone

1 3 BIOLOGICAL ACTIVITIES

1.3.1 The biological significance of lichen metabolites

Production of secondary metabolites is costly to the organisms in terms of nutrient and energy, therefore one would expect that the plethora of metabolites produced by lichens would have biological significance to the organisms Recent field and laboratory studies have shown that many of these compounds are indeed involved in important ecological roles Some of the possible biological functions of lichen metabolites, are summarized as below [43]:

 Antibiotic activities – provide protection against microorganisms

 Photoprotective activities – aromatic substances absorb UV light to protect algae (photobionts) against intensive irradiation

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 Promote symbiotic equilibrium by affecting the cell wall permeability of photobionts

 Chelating agents – capture and supply important minerals from the substrate

 Antifeedant/ antiherbivory activities – protect the lichens from insect and animal feedings

 Hydrophobic properties – prevent saturation of the medulla with water and allow continuous gas exchange

 Stress metabolites – metabolites secreted under extreme conditions

1.3.2 The biological significance of the lichen genus Parmotrema

1.3.2.1 Antimicrobial activities

The lichen Parmotrema species were observed a marked dose dependent

inhibition of test bacteria by lichen extracts It has been found that lichens of the

genus Parmotrema are promising antimicrobial agents Balaji P et al [41] reported marked antimicrobial efficacy of dichloromethane extract of P praesorediosum collected from silicious rocks of Western Ghats of Tamil Nadu Kumar et al [50] showed the antibacterial activity of methanol extract of P pseudotinctorum from

the Western Ghats of Karnataka Sinha and Biswas [69] reported the antibacterial

efficacy of solvent extracts of P reticulatum from Sikkim, India Neeraj V et al [44] found antibacterial efficacy of solvent extracts of P nilgherrensis and P

sancti-angelii collected from Karnataka, India Chauhan and Abraham [14] showed

the inhibitory effect of methanol extract of Parmotrema sp collected from

Kodaikanal forest, India against clinical isolates of bacteria Javeria et al [3]

showed the inhibitory efficacy of solvent extracts of P nilgherrense collected from

Nainital, India against drug resistant bacteria

1.3.2.2 Antioxidant activities

Lichens have been shown promising as they possess various bioactivities including antioxidant activity The DPPH free radical scavenging assay is one of the most widely used assays to evaluate the antioxidant activity of several kinds of

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samples including lichen extracts The method is simple, rapid, sensitive and

requires small amount of samples It has been found that Parmotrema species

possess radical scavenging activity Kekuda et al [50] observed dose dependent

DPPH radical scavenging activity in the lichen P pseudotinctorum Though the

scavenging of free radicals by lichen extracts was lesser than ascorbic acid, it is evident that the extracts showed hydrogen donating ability and therefore the extracts could serve as free radical scavengers, acting possibly as primary

antioxidants Extract of P grayanum showed high scavenging activity followed by

P praesorediosum and P tinctorum as indicated by lower IC50 value [76]

Methanol and ethanol extract of P reticulatum have shown DPPH radical

scavenging activity [68] (Table 1.1)

1.3.2.3 Antitumor activities

The action of lichen-derived compounds on tumor cells has been a focus of evaluations for some decades Lichexanthone and protocetraric acid isolated from

the lichens Parmotrema dilatatum (Vain.) Hale and Parmotrema lichexanthonicum

Eliasaro & Adler were evaluated against UACC-62 and B16-F10 melanoma cells and 3T3 normal cells by Sulforhodamine B assay [7] A cytotoxicity assay was

carried out in vitro with sulforhodamine B (SRB) using HEp-2 larynx carcinoma,

MCF-7 breast carcinoma, 786-0 kidney carcinoma, and B16-F10 murine melanoma cell lines, in addition to a normal (Vero) cell line in order to calculate the selectivity

index of the compounds from the lichen Parmotrema tinctorum [8] The relationship between O-alkyl salazinic acids from Parmotrema lichexantonicum

Eliasaro and Alder and potentially cytotoxic against human colon carcinoma 8), melanoma (MDA-MB-435), and brain (SF-295) tumor cell was investigated by

(HCT-Micheletti A C et al [54]

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Table 1.1 In vitro biological activities of the lichen genus Parmotrema

The dichloromethane, ethyl acetate, acetone and methanol

extracts of P.praesorediosum

Balaji P et

al [3]

Against eight bacterial (Gram + and -) Staphylococcus aureus, S

epidermidis, Bacillus cereus, Klebsiella pneumoniae, Enterobacter aerogenes, Shigella flexneri, Salmonella typhi and Escherichia coli

by Agar well diffusion assay

The methanol extracts of P

tinctorum, P grayanum and

Agar well diffusion method The methanol extract of Parmotrema sp Chauhan R et al [14] Againts the bacterial strains Bacillus cereus, Bacillus subtilis,

Escherichia coli, Klebsiella pneumonia, Micrococcus luteus, Proteus vulgaris, Staphylococcus aureus, Streptococcus faecalis, Sarcina lutea and yeast strains Candida albicans, Cryptococcus var diffluens

The ethyl acetate extract of

Parmotrema nilgherrensis and Parmotrema sanctiangelii

Neeraj V et

al [44]

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(IC50 439.06μg/ml) by scavenging of DPPH radicals

The methanol extracts of P

tinctorum, P grayanum and P.praesorediosum

Vivek M N

et al [76]

Determined by Malondialdehyde (MDA) assay and ABTS radical quenching assay Parmotrema austrosinese and Parmotrema perforatum Vattem D A et al [75]

DPPH radical scavenging activity The ethanol and methanol

extracts of the lichen

The methanol extract of the lichen Parmotrema reticulatum

Ghate N B

et al [29]

Antioxidant activities by using DPPH, ABTS, superoxide, and hydroxyl radical scavenging assay The ethyl acetate extract of Parmotrema tinctorum Raj P S et al [61]

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Antitumor activity against malignant cell lines of erythro leukemia

Antiproliferative against capan-1 and -2, PANC-1 (parcrease), H1415 (lung cell), PC-3 (prostate), T47-D (breast), AGS (stomach), NTH: OVCAR-3 (ovaries) and JURKAT (acute promyelocytic, T-cell and erythrocell leukemina cell lines)

NCI-Parmotrema dilatatum (Vain.) Hale and Parmotrema tinctorum (Nyl.) Hale

Yousuf S et

al [77]

Toxicity test of against Artemia salina with BSLT method The dichloromethane extract and phenolic compounds of

the lichen P tinctorum

Parmotrema dilatatum (Vain.) Hale and Parmotrema lichexantonicum Eliasaro and

Alder

Brandão L

F G et al

[7]

In vitro with sulforhodamine B (SRB) using HEp-2 larynx arcinoma,

MCF7 breast carcinoma, 786-0 kidney carcinoma, and B16-F10 urine melanoma cell lines

Parmotrema tinctorum (Nyl.) Hale Bogo D et al [8]

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20

CHAPTER 2

EXPERIMENTAL

2.1 MATERIALS AND ANALYSIS METHODS

 TLC was carried out on precoated silica gel 60 F254 (Merck) and precoated Kieselgel 60F254 plates (Merck)

 Gravity column chromatography was performed with silica gel 60 (Merck) and silica gel 60 (0.040 – 0.063 mm, Himedia)

 TLC spots were detected under ultraviolet (UV254) irradiation or visualized

by spraying with a solution of 5% vanillin in ethanol, followed by heating at

100 oC

 Solvents: Hexane, diethyl ether, petroleum ether (60-90 oC), toluene, chloroform, ethyl acetate, acetone, methanol, acetic acid

 Melting points were determined on Maquenne block(a)

 The NMR experiments using residual solvent signal as internal reference:

chloroform-d H 7.24, C 77.23 and acetone-d 6 H 2.09, C 206.31, 30.6 were performed with:

 Bruker Avance 500III (500 MHz for 1H and 125 MHz for 13C-NMR(a, b)

 Varian VXR-500 spectrometers, with tetramethylsilane as internal standard(c)

 The HR–ESI–MS were recorded on

 HR–ESI–MS MicroOTOF–Q mass spectrometer(a)

 Hitachi M-4100 mass spectrometer(c)

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21

 The IR spectra were obtained with

 Bruker Vector 22 infrared spectrophotometer(a)

 Shimadzu FTIR-8200 infrared spectrophotometer(c)

 Optical rotations were measured on

 Kruss (German) digital polarimeter(a)

 Jasco DIP-370 digital polarimeter(c)

 Absorption and CD spectra were measured on

 Jasco V-570 spectrophotometer(d)

 Jasco J-820E spectropolarimeter(d)

 TD-DFT calculations of the CD spectra were optimized at the level of B3LYP/6-311++G** in vacuo and in CPCM solvent model of methanol The populations of the two stable conformers were calculated based on the relative energies with the Boltzmann distribution at 300 K The optimization under the CPCM solvent model of methanol did not change significantly these geometries or populations The electronic CD spectra of the stable conformers were calculated at the TD-DFT theory with the same basis sets as the optimizations by using Gaussian09 program, fitted by Gaussian curves with 0.30 eV line width, and then weighted-averaged based on the Boltzamann population

(a) The Center Analysis of the University of Science, National University- Ho Chi Minh City, Vietnam

(b) The Institute of Chemistry, Vietnam Academy of Science and Technology, Hanoi, Vietnam

(c) Life Science Center, Kobe Pharmaceutical University, Japan

(d) Osaka University, Japan

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22

2.2 LICHEN MATERIALS

The lichen Parmotrema praesorediosum (Nyl.) Hale was collected at Nam

Cat Tien National Forest Reserve and Intermediate Zones, Nam Cat Tien Village, Tan Phu District, Dong Nai Province, Vietnam in January-July 2009 The scientific name of the lichen was determined by MSc Vo Thi Phi Giao, Faculty of Biology, University of Science, National University – Ho Chi Minh city

A voucher specimen (No US-B020) was deposited in the Herbarium of The Department of Organic Chemistry, Faculty of Chemistry, University of Science, National University - Ho Chi Minh City-Vietnam

2.3 EXTRACTION AND ISOLATION PROCEDURES

The fresh lichen thalli (5.0 kg) were cleaned under running tap water and dried The ground powder sample (3.0 kg) was extracted with methanol at room temperature by method of maceration After filtration, the solvent was evaporated at the reduced pressure While the methanolic solution was evaporated, a precipitate occurred and was filtered off, then the solution was continued evaporated to dryness The resulting was the precipitate (9.0 g) and the crude methanolic residue (450.0 g)

The methanolic residue (450.0 g) was subjected to silica gel solid phase extraction and eluted consecutively with petroleum ether, chloroform, ethyl acetate, acetone and methanol in turn at room temperature to afford petroleum ether E1 extract (25.0 g), petroleum ether E2 extract (15.0 g), chloroform extract (105.0 g), ethyl acetate extract (50.0 g), acetone extract (45.0 g) and methanol extract (37.0 g) (Figure 2.1)

2.3.1 Isolating compounds from the methanol precipitate (Figure 2.1)

The precipitate (9.0 g) was silica gel chromatographed, eluted with petroleum ether–chloroform to give 5 fractions (symboled as fraction T1 to fraction T5)

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

The biological significance of lichen metabolites

Structure elucidation of compound PRAES-C18