Chemistry of the lichen type common in southern Vietnam pptx

Doctoral Thesis CHEMICAL STUDY OF COMMON LICHENS IN THE SOUTH OF VIETNAM Le Hoang Duy Department of Organic Chemistry Graduate School of Kobe Pharmaceutical University Kobe Pharmaceutical University March 2012 Content page List of abbreviations i List of figures iv List of photos v List of tables v Chapter 1: General introduction 1.1 The lichen and usage of lichens 1.2 Lichen substances 1.3 Cultivation of lichen mycobionts 1.4 Vietnamese lichen 1.5 Research scope and objectives Chapter 2: Lichen substances from the lichen thalli of Parmotrema mellissii and Rimelia clavulifera 2.1 Chemical investigation of the lichen thalli of P mellissii 2.1.1 Mono-aromatic compounds 2.1.2 Depsides 10 2.1.3 Depsidones and Isocoumarin derivatives 11 2.1.4 Other lichen substances 28 2.2 Chemical investigation of the lichen thalli of R clavulifera 30 Chapter 3: Secondary metabolites from the cultured lichen mycobionts 33 3.1 Chemical investigation of the cultured mycobionts of Graphis vestitoides 33 3.2 Chemical investigation of the cultured mycobionts of Sacographa tricosa 44 3.3 Chemical investigation of the cultured mycobionts of Pyrenula sp 58 Chapter 4: Biological activity of isolated compounds 77 4.1 Inhibitory effect on mammalian DNA polymerase activity 77 4.2 Inhibitory effect on cancer cell growth 81 Chapter 5: Conclusions 82 Acknowledgment 86 Experimental section 87 References 129 List of compounds List of abbreviations 1D one dimensional 2D two dimensional Ac acetyl alt altitude aq aqueous ax axial br broad calcd calculated CC silica gel column chromatography CD circular dichroism COSY homonuclear shift correlation spectroscopy d doublet dd doublet of doublets ddd doublet of doublets of doublets dddd doublet of doublets of doublets of doublets dec decomposed DEPT distortionless enhancement by polarisation transfer DMF N,N-dimethyl formamide DMSO dimethyl sulfoxide DNA deoxyribonucleic acid dq doublet of quartets dt doublet of triplets dtd doublet of triplets of doublets dTTP 2'-deoxythymidine 5'-triphosphate EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDTA ethylenediaminetetraacetic acid eq equatorial EI-MS electron-impact ionization mass spectrum HMBC heteronuclear multiple bond correlation spectroscopy HOBT 1-hydroxybenzotriazole -i- HPLC high performance liquid chromatography hr hour HR-APCIMS high resolution atmospheric pressure chemical ionization mass spectrum HR-EIMS high resolution electron-impact ionization mass spectrum HR-ESIMS high resolution electrospray ionization mass spectrum HR-SIMS high resolution secondary ion mass spectrum HSQC heteronuclear single quantum correlation spectroscopy IR infrared spectrophotometry lit literature m multiplet Me methyl minutes mp melting point MPA methoxyphenylacetic acid MS mass spectrum MTPA 2-methoxy-2-trifluoromethylphenylacetic acid NMR nuclear magnetic resonance NOESY nuclear Overhauser enhancement spectroscopy PGME phenylglycine methyl ester ppm parts per million (chemical shift value) Prep HPLC preparative high performance liquid chromatography Prep TLC preparative thin-layer chromatography PyBOP benzotriazolyloxytri(pyrrolidinyl)phosphonium hexafluorophosphate rel int relative intensity rt room temperature q quartet qd quartet of doublets quint quintet ROESY rotating-frame Overhauser enhancement spectroscopy s singlet sh shoulder -ii- SIMS secondary ion mass spectrum t triplet td triplet of doublets tdd triplet of doublets of doublets tdt triplet of doublets of triplets TLC thin-layer chromatography TMS tetramethylsilane UV ultraviolet -iii- List of figures Page Fig The structure of first known lichen substances .2 Fig Characteristic lichen substances .3 Fig Ahmadjian’s method for isolating lichen mycobionts by means of spores .3 Fig Selected metabolites isolated from the cultured lichen mycobionts .4 Fig Selected bioactive lichen substances isolated from Parmotrema species .6 Fig Extraction and isolation procedure for P mellissii Fig Tautomeric interchange of α-alectoronic acid (11) 13 Fig Chiral HPLC of 18 18 Fig Proposed stereochemistry of spiro-ring system of 22 .25 Fig 10 Extraction and isolation procedure for R clavulifera 30 Fig 11 Selected secondary metabolites from the thalli and cultured mycobionts of Graphis species 33 Fig 12 Extraction and isolation procedure for cultured mycobionts of G vestitoides 35 Fig 13 MTPA ester of 38 38 Fig 14 PGME method for determination of absolute configuration of carboxylic acid 41 Fig 15 Extraction and isolation procedure for cultured mycobionts of S tricosa 45 Fig 16 Determination of absolute configuration by MPA esters 48 Fig 17 Configuration of compound 48 .50 Fig 18 Metabolites from thalli and cultured mycobionts of Pyrenula species 58 Fig 19 Extraction and isolation procedure for cultured mycobionts of Pyrenula sp 59 Fig 20 Determination of absolute configuration of 66 .69 Fig 21 Determination of absolute configuration of 69 .73 Fig 22 Proposed biosynthesis of pyrenulic acids and related compounds 75 Fig 23 Structure of reported bioactive metabolites 77 Fig 24 Selected compounds for bio-assay 78 Fig 25 Inhibitory effects of isolated compounds on calf DNA polymerase α 79 Fig 26 Inhibitory effects of isolated compounds on rat DNA polymerase β 80 Fig 27 Inhibitory effects of isolated compounds on human DNA polymerase κ .80 Fig 28 Inhibitory effects of isolated compounds on HCT116 cultured cell growth 81 -iv- List of photos Page Photo Growth forms of lichen Photo The thalli of foliose lichens P mellissii and R clavulifera Photo G vestitoides thalli and its cultured mycobionts 34 Photo S tricosa thalli and its cultured mycobionts 44 Photo Pyrenula sp thalli and its cultured mycobionts 59 List of tables Page 13 Table H- and C-NMR spectroscopic data of 11, 11a and 11b in CDCl3 14 Table 1H- and 13C-NMR spectroscopic data of 12 and 12a in CDCl3 15 Table 1H- and 13C-NMR spectroscopic data of 15-17 in CDCl3 19 Table 1H-NMR spectroscopic data of 19-22 in CDCl3 21 Table 13C-NMR spectroscopic data of 19-25 in CDCl3 22 Table 1H-NMR spectroscopic data of 23-25 in CDCl3 27 Table 1H- and 13C-NMR spectroscopic data of 42 and 42m 43 Table 13C-NMR spectroscopic data of 47, 48, 52-54 and 58 in CDCl3 51 Table 1H-NMR spectroscopic data of 48, 52-54 in CDCl3 52 Table 10 1H-NMR spectroscopic data of 63-66 in CDCl3 64 Table 11 13C-NMR spectroscopic data of 63, 64 and related compounds in CDCl3 65 Table 12 13C-NMR spectroscopic data of 65-70 in CDCl3 70 Table 13 1H-NMR spectroscopic data of 67-70 74 -v- Chapter 1: General introduction 1.1 The lichens and usage of lichens The lichens are symbiotic organisms, usually composed of a fungal partner (mycobiont) and one or more photosynthetic partners (photobionts), which is most often either a green alga or cyanobacterium About 17,000 different lichen taxa, including 16,750 lichenized Ascomycetes, 200 Deuteromycetes, and 50 Basidiomycetes have been described world-wide The photobionts produce carbohydrates by photosynthesis for themselves and for their dominant fungal counterparts (mycobionts), which provide physical protection, water and mineral supply Based on this association, lichens have adapted to extreme ecological conditions, being dominant at high altitudes, in Arctic boreal and also tropical habitats, and colonized a wide range of different substrata, such as rocks, bare ground, leaves, bark, metal, glass Lichens are traditionally divided into three growth morphological forms: these are the crustose, foliose and fruticose types (Photo 1).1-3) Crustose Foliose Fruticose Photo Growth forms of lichen Lichens have been used by humans for centuries as food,4) as source of dye,5) as raw materials in perfumery and for therapeutic properties in folk medicine The fragrance industry uses two species of lichen Evernia prunastri var prunastri (oakmoss) and Pseudevernia furfuracea (treemoss) About 700 tons of oakmoss are currently processed every year by French producers.6,7) Several lichen extracts have been used for various remedies in folk medicine, such as Lobaria pulmonaria for lung troubles, Xanthoria parientina for jaundice, Usnea spp for strengthening hair, Cetraria islandica (Iceland moss) for tuberculosis, chronic bronchitis and diarrhea.4,8) The screening tests with lichens have indicated the frequent occurrence of metabolites with antioxidant, antibiotic, antimicrobial, antiviral, antitumor, analgestic and antipyretic properties.9-11) -1- These usages of lichen are limited to folk medicine, perfume and dying industry, although manifold biological activities of lichen metabolites have been recognized with potential applications in medicine, agriculture and cosmetics industry.11,12) 1.2 Lichen substances Lichens are one of the most important sources of biologically active compounds other than plants The chemistry of lichen was attractive the chemists from the early time of organic chemistry The chemical aspect of lichen substances was published by Zopf in early 19th century The lichen substances first known in their structure were vulpinic acid (1) and lecanoric acid (2) (Fig 1) The structure of most lichen substances remained unknown till the studies of Asahina and Shibata in early 20th century The development of TLC and HPLC in 1960s, together with modern spectroscopic methods led to the isolation and identification of many new lichen substances.13) Fig The structure of first known lichen substances Recently, over 800 lichen substances were isolated and classified in many classes: aliphatic acids, γ-, δ- and macrocyclic lactones, monocyclic aromatic compounds, quinones, chromones, xanthones, dibenzofurans, depsides, depsidones, depsones, terpenoids, steroids, carotenoids and diphenyl ethers.3,13,14) Among them, depsides, depsidones and dibenzofurans are unique to lichens (Fig 2) Depsides are formed by condensation of two or more hydroxybenzoic acids whereby the carboxyl group of one molecule is esterified with a phenolic hydroxyl group of a second molecule Depsidones have an ether linkage in addition to the ester linkage of the depsides, resulting in a rigrid polycyclic system.12) The main natural roles of lichen substances, although they are not all well understood yet, include: protection against a large spectrum of viral, baterial and protozoan parasites, against animal predators such as insects and nematodes and against -2- MPA-OCH3), 3.380 (1H, m, H-22eq), 3.479 (1H, d, J=6.0 Hz, H-19), 3.601 (1H, td, J=13.0, 2.0 Hz, H-22ax), 3.760 (3H, s, COOCH3), 4.245 (1H, dd, J=10.0, 6.0 Hz, H-6), 4.654 (1H, s, MPA-CH), 5.354 (1H, br s, H-15), 5.400 (1H, br d, J=5.0 Hz, H-11), 5.442 (1H, dd, J=10.0, 2.0 Hz, H-7), 5.604 (1H, d, J=15.0 Hz, H-2), 5.652 (1H, dd, J=15.5, 6.0 Hz, H-5), 5.787 (1H, dd, J=15.5, 11.0 Hz, H-4), 6.914 (1H, dd, J=15.5, 11.0 Hz, H-3), 7.320-7.352 (5H, m, MPA-Ph) C-NMR (CDCl3): δ 13.5 (C-23), 16.1 (C-24), 23.3 (C-26), 25.6 (C-25), 31.8 (C-21), 13 32.3 (C-20), 32.9 (C-10), 33.5 (C-9), 37.3 (C-13), 42.0 (C-14), 46.4 (C-8), 51.6 (COOCH3), 54.5 (C-22), 55.6 (C-17), 57.4 (MPA-OCH3), 74.7 (C-19), 75.2 (C-6), 76.3 (C-7), 79.6 (C-18), 82.7 (MPA-CH), 121.4 (C-11), 121.7 (C-2), 127.2 (MPA-Ph), 128.0 (C-15), 128.7, 128.9 (MPA-Ph), 129.9 (C-4), 134.5 (C-12), 135.5 (C-16), 135.8 (MPAPh), 139.3 (C-5), 143.6 (C-3), 167.1 (C-1), 169.4 (MPA-CO) NOESY: H-2/H-4, H-3/H-5, H-6/H-10eq, H-7/H-17, H-7/H-19, H-9/H3-24, H-17/H-19, H-22ax/H3-24, H3-23/H3-24 HMBC: COOCH3→C-1; H-2→C-1, 4; H-3→C-1; H-4→C-5; H-5→C-3, 4; H-6→C-4, 7, 19; H-7→C-5, 6, 9, MPA-CO; H-11→C-9, 26; H-15→C-9, 17, 25; H-17→C-15, 16, 18, 24, 25; H-19→C-6, 17, 18, 20, 23, 24; H-20→C-19, 23; H2-21→C-19, 22, 23; H22eq→C-18, 20; H3-23→C-19, 20, 21; H3-24→C-17, 18, 19; H3-25→C-15, 16, 17; H326→C-11, 12, 13 HR-ESIMS m/z: 613.3135 (calcd for C36H46O7Na: 613.3143 [M+Na]+) New polyketide pyrenulic acid E (67) Colorless solid ROESY: H-2/H-4, H-3/H-5, H-5/H-7, H-6/H-10eq, H-7/H-17, H-7/H-19, H-9/H3-24, H17/H-19 H-NMR: Table 13 13 C-NMR: Table 12 HR-ESIMS m/z: 429.2660 (calcd for C26H37O5: 429.2643 [M-H]-) New polyketide pyrenulic acid F (68) Colorless solid -124- [α]D24 -11.2o (c=0.85, CHCl3) UV λmax (EtOH) nm (log ε): 252.5 (4.38) IR (KBr) νmax cm-1: 3439, 2962, 2927, 1694, 1644, 1620, 1434, 1380, 1269 H-NMR: Table 13 13 C-NMR: Table 12 ROESY: H-2/H-4, H-3/H-5, H-5/H-7, H-6/H-10eq, H-7/H-17, H-7/H-19, H-9/H3-24, H17/H-19 HR-ESIMS m/z: 445.2612 (calcd for C26H37O6: 445.2592 [M-H]-) New polyketide pyrenulic acid G (69) Colorless solid [α]D23 -8.2o (c=0.60, CHCl3) UV λmax (EtOH) nm (log ε): 256.5 (4.37) IR (KBr) νmax cm-1: 3420, 2964, 1692, 1639, 1614, 1434, 1380, 1237 H-NMR: Table 13 13 C-NMR: Table 12 NOESY: H-2/H-4, H-3/H-5, H-7/H-10eq, H-7/H-17, H-7/H-19, H-17/H-19 HR-ESIMS m/z: 411.2548 (calcd for C26H35O4: 411.2537 [M-H]-) Preparation of (R)- and (S)-MPA esters of 69 Compound 69 (8.2 mg) was methylated by TMS-CHN2 as described above to yield a methyl ester (6.4 mg) Portion of methyl ester of 69 (3.2 mg) and (2.3 mg) was esterified to (R)-MPA ester 69a (1.7 mg) and (S)-MPA ester 69b (1.2 mg), respectively Compound 69a H-NMR (CDCl3): δ 0.812 (3H, d, J=6.5 Hz, H3-23), 0.878 (3H, t, J=7.0 Hz, H3-22), 1.089 (1H, m, H-21), 1.150 (1H, m, H-10), 1.590 (3H, br s, H3-25), 1.609 (3H, br s, H326), 1.671 (1H, m, H-13), 1.750 (1H, m, H-9), 1.750 (1H, m, H-20), 1.770 (1H, m, H10), 1.770 (1H, m, H-14), 1.781 (1H, m, H-21), 1.854 (1H, m, H-8), 1.891 (1H, m, H13), 2.763 (1H, d, J=5.0 Hz, H-17), 3.351 (3H, s, MPA-OCH3), 3.667 (1H, d, J=9.0 Hz, H-19), 3.768 (3H, s, COOCH3), 4.333 (1H, m, H-6), 4.602 (1H, s, MPA-CH), 5.041 -125- (1H, s, H-24), 5.188 (1H, br s, H-11), 5.245 (1H, s, H-24), 5.330 (1H, br s, H-15), 5.417 (1H, br d, J=8.0 Hz, H-7), 5.852 (1H, d, J=15.5 Hz, H-2), 5.982 (1H, dd, J=15.5, 7.0 Hz, H-5), 6.245 (1H, dd, J=15.5, 11.0 Hz, H-4), 7.158 (1H, dd, J=15.5, 11.0 Hz, H-3), 7.312-7.348 (5H, m, MPA-Ph) C-NMR (CDCl3): δ 10.9 (C-22), 16.2 (C-23), 22.1 (C-25), 23.3 (C-26), 25.3 (C-21), 13 30.8 (C-10), 32.0 (C-9), 37.1 (C-20), 37.6 (C-13), 39.2 (C-14), 43.7 (C-8), 51.0 (C-17), 51.6 (COOCH3), 57.5 (MPA-OCH3), 74.3 (C-6), 76.1 (C-7), 82.3 (C-19), 82.9 (MPACH), 117.0 (C-24), 121.45 (C-11), 121.50 (C-2), 127.4 (MPA-Ph), 128.0 (C-15), 128.6, 128.9 (MPA-Ph), 129.9 (C-4), 132.8 (C-16), 133.2 (C-12), 135.6 (MPA-Ph), 139.7 (C5), 143.9 (C-3), 145.6 (C-18), 167.3 (C-1), 169.4 (MPA-CO) NOESY: H-2/H-4, H-3/H-5, H-6/H-10eq, H-7/H-17, H-7/H-19, H-17/H-19 HMBC: COOCH3→C-1; H-2→C-1, 4; H-3→C-1; H-5→C-3; H-15→C-9, 14, 17, 25; H-17→C-16, 18; H-19→C-17, 18, 20, 23, 24; H2-21→C-19, 20, 22, 23; H3-22→C-20, 21; H3-23→C-19, 20, 21; H2-24→C-17, 18, 19; H3-25→C-15, 16, 17; H3-26→C-11, 12, 13 HR-ESIMS m/z: 597.3187 (calcd for C36H46O6Na: 597.3194 [M+Na]+) Compound 69b H-NMR (CDCl3): δ 0.802 (3H, d, J=6.5 Hz, H3-23), 0.849 (3H, t, J=7.0 Hz, H3-22), 1.040 (1H, m, H-21), 1.617 (3H, br s, H3-25), 1.669 (3H, br s, H3-26), 1.726 (1H, m, H21), 1.738 (1H, m, H-20), 1.760 (1H, m, H-13), 1.800 (1H, m, H-10), 1.850 (1H, m, H9), 1.980 (1H, m, H-14), 1.990 (1H, br d, J=14.0 Hz, H-13), 2.080 (1H, br d, J=14.0 Hz, H-10), 2.110 (1H, ddd, J=11.0, 5.5, 2.5 Hz, H-8), 2.812 (1H, d, J=5.5 Hz, H-17), 3.362 (3H, s, MPA-OCH3), 3.654 (1H, d, J=9.0 Hz, H-19), 3.779 (3H, s, COOCH3), 4.213 (1H, t, J=7.5 Hz, H-6), 4.630 (1H, s, MPA-CH), 5.074 (1H, s, H-24), 5.249 (1H, s, H24), 5.346 (1H, dd, J=7.5, 2.5 Hz, H-7), 5.393 (1H, br s, H-11), 5.400 (1H, br s, H-15), 5.594 (1H, dd, J=15.5, 7.5 Hz, H-5), 5.681 (1H, d, J=15.5 Hz, H-2), 5.977 (1H, dd, J=15.5, 11.0 Hz, H-4), 6.836 (1H, dd, J=15.5, 11.0 Hz, H-3), 7.325-7.345 (5H, m, MPA-Ph) C-NMR (CDCl3): δ 10.9 (C-22), 16.2 (C-23), 22.0 (C-25), 23.3 (C-26), 25.2 (C-21), 13 31.5 (C-10), 32.2 (C-9), 37.2 (C-20), 37.7 (C-13), 39.3 (C-14), 43.8 (C-8), 51.0 (C-17), -126- 51.5 (COOCH3), 57.3 (MPA-OCH3), 73.9 (C-6), 76.6 (C-7), 82.6 (C-19), 82.7 (MPACH), 117.1 (C-24), 121.2 (C-2), 121.9 (C-11), 127.2 (MPA-Ph), 127.9 (C-15), 127.9, 128.7 (MPA-Ph), 129.5 (C-4), 133.0 (C-16), 133.9 (C-12), 135.7 (MPA-Ph), 139.3 (C5), 144.1 (C-3), 145.4 (C-18), 167.3 (C-1), 169.2 (MPA-CO) NOESY: H-2/H-4, H-3/H-5, H-6/H-10eq, H-7/H-17, H-7/H-19, H-17/H-19 HMBC: COOCH3→C-1; H-2→C-1, 4; H-5→C-3; H-6→C-4, 5, 7,19; H-7→C-6, 8, 9; H-11→C-9, 13; H-15→C-14, 25; H-17→C-8, 9, 15, 16, 18; H-19→C-17, 18, 20, 21, 24; H2-21→C-20; H3-22→C-20, 21; H3-23→C-19, 20, 21; H2-24→C-17, 18, 19; H325→C-15, 16, 17; H3-26→C-11, 12, 13 HR-ESIMS m/z: 597.3191 (calcd for C36H46O6Na: 597.3194 [M+Na]+) New polyketide pyrenulic acid H (70) Colorless needles mp 146-147oC (CHCl3-MeOH) [α]D22 +86o (c=1.03, MeOH) UV λmax (EtOH) nm (log ε): 256 (4.39) IR (KBr) νmax cm-1: 3423, 2695, 1693, 1643, 1614, 1434, 1382, 1309, 1264 H-NMR: Table 13 13 C-NMR: Table 12 NOESY: H-2/H-4, H-3/H-5, H-5/H-7, H-6/H-10eq, H-7/H-19 HR-ESIMS m/z: 427.2503 (calcd for C26H35O5: 427.2486 [M-H]-) Chapter 4: Biological activity of isolated compounds The bio-assay was taken at Kobe-Gakuin University, Japan by Dr Y Mizushina 4.1 Enzymes DNA polymerase α was purified from thymus by immuno-affinity column chromatography as described previously.123) Recombinant rat DNA polymerase β was purified from E coli JMpβ5, as described by Date et al.124) A truncated form of polymerase κ (residues 1-560) with × His-tags attached at the C-terminus was overproduced in E coli and purified as described previously.125) -127- 4.2 DNA polymerase assays The standard reaction mixture for polymerase α (24 µl final volume) contained 50 mM Tris-HCl, pH 7.5, mM dithiothreitol, mM MgCl2, µM poly(dA)/oligo(dT)18 (= 2/1), 10 µM [3H]dTTP (100 cpm/pmol), 15% (v/v) glycerol and µl of an enzyme inhibitor solution The standard reaction mixture for polymerase β was the same, except that it also contained 150 mM KCl The reaction mixture for polymerase κ was the same as for polymerase α The compounds were dissolved in distilled DMSO at various concentrations and sonicated for 30 s Aliquots of µl sonicated samples were mixed with 16 µl of each enzyme (final amount 0.05 units) in 50 mM Tris-HCl (pH 7.5) containing mM dithiothreitol, 50% glycerol and 0.1 mM EDTA, and kept at 0oC for 10 These inhibitor-enzyme mixtures (8 µl) were added to 16 µl of each of the enzyme standard reaction mixtures and incubation was carried out at 37oC for 60 Activity without the inhibitor was considered 100% and the remaining activity at each concentration of the inhibitor was determined relative to this value One unit of polymerase activity was defined as the amount of enzyme that catalyzed the incorporation of nmol dTTP into synthetic DNA template-primers in 60 at 37oC under the normal reaction conditions for each enzyme 4.3 Cell culture and measurement of cell viability The cell human cancer cell line, HCT116 (colon carcinoma cells), was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) Human cancer cells were cultured in McCoy’s 5A medium supplemented with 10% fetal bovine serum, penicillin (100 units/ml) and streptomycin (100 µg/ml) HTC116 cells were cultured at 37oC in a humid atmosphere of 5% CO2/95% air For the cell growth assay, the cells were plated at × 104 cells into each well 96-well microplates with various concentrations of the isolated compounds These compounds were dissolved in DMSO at a concentration of 10 mM as a stock solution The stock solutions were diluted to the appropriate final concentrations with growth medium as 0.5% DMSO just before use Cell viability was determined by WST-1 assay.122) -128- References Nash T.H III, Lichen Biology, 2nd Edition, Cambridge University Press, New York (2008) Ahmadjian V., The lichen symbiosis, John Wiley and Sons, Inc., New York (1993) Huneck S., Naturwissenschaften, 86, 559-570 (1999) Brodo I.M., Sharnoff S.D., Sharnoff S., Lichens of North America, Yale University Press, New Haven, Connecticut (2001) Beecken H., Gottschalk E.-M., Gizycki U., Krämer H., Maassen D., Matthies H.G., Musso H., Rathjen C., Zdhorszky Ul., Biotech Histochem., 78, 289-302 (2003) Joulain D., Tabacchi R., Flavour Fragrance 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24 parmosidone B PM 25 parmosidone C PM 26 (+)-usnic acid RC, PM 27 skyrin RC, PM 28 5-chloroemodin 29 gyrophoric acid RC 30 salazinic acid RC 31 norstictic acid 32 stictic acid 33 psoromic acid 34 protocetraric acid 35 2-acetyl-3,5-dihydroxybenzoic acid GV 36 trans-5,7-dihydroxy-3-(1-hydroxyethyl)phtalide GV 37 cis-5,7-dihydroxy-3-(1-hydroxyethyl)phtalide GV 38 4,6-dihydroxy-3,9-dehydromellein GV 39 6,8-dihydroxy-3-(hydroxymethyl)isocoumarin GV 40 cis-4,6-dihydroxymellein GV 41 6,8-dihydroxyisocoumarin-3-carboxylic acid GV 42 14-membered macrolide GV 43 6-methoxy-8-hydroxyisocoumarin-3-carboxylic acid 44 mutolide 45 ergosterol peroxide ST 46 sporogen-AO ST 47 dihydrosporogen-AO ST 48 petasol ST 49 isopetasol ST 50 JBIR-27 ST 51 1β-hydroxypetasol ST 52 3-epi-petasol ST 53 dihydropetasol ST 54 sarcographol ST 55 petasin 56 S-petasin 57 petasinol 58 8-epi-dihydropetasol 59 cyclodebneyol 60 chrysophanol PS 61 emodin PS 62 1,5,8-trihydroxy-3-methylxanthone PS 63 pyrenulic acid A PS 64 pyrenulic acid B PS 65 pyrenulic acid C PS 66 pyrenulic acid D PS 67 pyrenulic acid E PS 68 pyrenulic acid F PS 69 pyrenulic acid G PS 70 pyrenulic acid H PS 71 cladobotric acid A 72 cladobotric acid C 73 lobaric acid RC: Rimelia clavulifera PM: Parmotrema mellissii GV: Graphis vestitoides ST: Sarcographa tricosa PS: Pyrenula sp ... phytochemical studies on Vietnamese lichens were undertaken The major aim of this thesis is to Investigate the lichen substances from the macrolichens collected in the Western Highlands of Vietnam (ca 1,500... respectively) These differences indicated that the methine proton H-5′′′ of 23 and 25 had coupling with only one of the vicinal methylene protons H2-4′′′ The coupling between H-5′′′ and the other proton of. .. from the cultured lichen mycobionts Lichen- forming fungi have been shown to retain in axenic the capacity to biosynthesize secondary products found in the lichenised state.16) In some cases, the