BIODIVERSITY OF THE CHEMICAL CONSTITUENTS IN THE EPIPHYTIC

AIM
The identification and evaluation of lichen metabolite production by the epiphytic lichenized ascomycete Ramalina lacera collected from different substrates: Crataegus sinaicus, Pinus halepensis, and Quercus calliprinos.


METHODS
Chemical constituents were characterized by GC MS, HPLC, HR TLC, and other chemical methods.


RESULTS
The most abundant fatty acids were alpha-linolenic acid, oleic acid, and palmitic acid but a considerable variability of the ester composition from one to another was found. A comparison of neutral lipids, glycolipids, polar lipids and fatty acid composition of the tree growing lichen Ramalina lacera was done. Diacylglyceryl N,N,N trimethylhomoserine, diaclyglycerylhydroxymethyl N,N,N trimethyl beta alanine, phosphatidylcholine, and phosphatidylinositol were found as major components among polar lipids. Diffractaic, lecanoric, norstictic, protocetric, and usnic acids were isolated as major aromatic compounds in all samples of R. lacera.


CONCLUSIONS
We evaluated a diversity of fatty acids, lipids, and aromatic compounds produced by the samples of Ramalina lacera growing on different tree substrates, Crataegus sinaicus, Pinus halepensis and Quercus calliprinos.


INTRODUCTION
Lichens are symbiotic associations between certain types of fungi (usually ascomycetes) and various green algae or cyanobacteria 1,2 .Lichens have been used for a number of years as natural bioindicators for various heavy metals and as sources of information for environmental monitoring 3,4 .These organisms have both algal and fungal properties and produce n-alkanes 5 , unusual betaine ether glycerols 6 , glyco-and phospholipids 7,8 , and saturated, unsaturated, branched 8,9 , and halogenated fatty acids 10 .Many diff erent bioactive secondary metabolites have also been isolated from lichen species [11][12][13] which have been used in pharmaceutical and biotechnological sciences and industry [14][15][16] .
Some Ramalina species are usually used as food in some Central and South Eastern Asian countries.Thus, lichens are used as traditional food by Rai and Limbu communities of East Nepal.Lichens Ramalina farinacea, R. conduplicans, R. sinensis, and R. subfarinacea are cooked mixed with various foods 17 .Ramalina farinacea and R. conduplicans are usually used as traditional food by Rai and Limbu communities of East Nepal.Lichens are cooked mixed with various foods 17 .Since the beginning of the 20th century hair powder of Ramalina calicaris, and Ramalina spp.Have been used in cosmetics in Europe 18 , and India 19 respectively.Extracts from the lichen Ramalina farinacea were evaluated against fi fteen clinical isolates of Staphylococcus aureus 20 .The aqueous extract of the Ramalina farinacea has folkloric reputation for the treatment of mental disorders in Africa; and tinctures have also been used for treatment of ringworm tinea in Nigeria 21 .In southwestern province Yunnan (China), the Yi, Dai, and Han ethnic peoples cook these two species of Ramalina (R. conduplicans and R.sinensis) to prepare a traditional cold dish served at marriage banquets 22 .
The lichen substances are unique as they are unknown in other plant sources.Lichens contain many characteristic aromatic compounds with known antiviral, antimicrobial, antiproliferative, antimitotic, antioxidant activities 23 .
Lichens may be a good potential source of bioactive phytochemicals 7,13,24 .
In this study, we evaluated which fatty acids, lipids, and aromatic compounds are produced by the samples of Ramalina lacera growing on diff erent tree-substrates, Crataegus sinaicus, Pinus halepensis and Quercus calliprinos.
Fraction 1 was concentrated in vacuum at 35 °C, water layer was lyophilized, and then dissolved in 2 ml of ethanol.Fraction 2 and 3 were separately concentrated to dryness in vacuum at 5 °C under reduced pressure, and then dissolved in 2 ml of a cold mixture ethanol-dichloromethane (1:1, v/v), which was used for separation by HPLC, TLC, and followed chemical analysis.

Gas chromatographic-mass spectrometric analysis
A Hewlett Packard 6890 (series II) gas chromatograph modifi ed for glass-capillary work and a HP-GC mass selective detector (5973B MSD) were used.Fatty acid methyl esters were prepared and analyzed by GC fi tted with serially coupled capillary columns: the RTX 1 column (30 m, ID 0.32 mm, fi lm thick-ness 0.25 μm; Restek, USA) was coupled with a second capillary column (RTX 1701, 30 m, 0.32 mm, 0.25 μm fi lm; Restek, USA).The instrumental Biodiversity of the chemical constituents in the epiphytic lichenized ascomycete Ramalina lacera grown on diff erence substrates Crataegus sinaicus, Pinus halepensis, and Quercus calliprinos

High-Performance Liquid Chromatographic Analysis
The dried samples from fractions were reconstituted in 200 μL of methanol and analyzed using a Hewlett Packard 1100 HPLC system (Hewlett-Packard 1100 HPLC System w/ UV/VIS detector, includes: G1311A Quaternary Pump, G1314A UV/Vis Detector, G1313A Autosampler, G1322A Vacuum Degasser, Solvent Module, HP Chemstation with Computer System) with a photo diode array detec-tor set at a range of 200-450 nm; all peaks were analyzed at 254 nm.An analytical reverse phase C 18 column (A Spherisorb 5 ODS 2 column 250 × 4.6 mm, 5 μm; (Kontron) was used as the stationary phase.Mobile phase A contained 10 % methanol and 90 % water brought to a pH of 2.0 with phosphoric acid, and mobile phase B was 100% methanol.A linear gradient was applied over 30 min starting with 100% of mobile phase A at the start to 100% mobile phase B at the end.Chromatograms were analyzed by Hewlett Packard software; retention time and absorbance spectra were used to identify compounds, and also pure compounds were used for spectral analysis.Orsellinic and usnic acids were obtained from Sigma-IL, and used also as standard compounds.Isolated metabolites were identifi ed with 13 C-NMR, IR, UV, and chemical methods as described previously 11 .

RESULT AND DISCUSSION
Ramalina lacera is a moderately xeric epiphytic fruticose lichen that grows in the Mediterranean areas on diff erent shrubs and trees.These epiphytic species belonging to the family Ramalinaceae were chosen for a comparative examination of their fatty acid, polar lipids, and aromatic compounds.The GC-MS analysis of fatty acids in R. lacera, which grows on Crataegus sinaicus, Pinus halepensis, and Quercus calliprinos, revealed a high polyenoic content in species which grows on the Palestine oak Q. calliprinos (54.28 %, Table l), but much lower amounts of such acids in two other ones, viz.49.16 % and 47.84 %, respectively.The amounts of trienoic acids, however, were higher in the samples from tree-growing species, 42.75 % (sample 1), 37.95 % (sample 2) and 38.62 % (sample 3).A total of 11 saturated fatty acids were identifi ed, with n-16:0 and n-18:0 as major ones (8.47-12.94% and 3.29-5.13%, respectively).Total saturation in various species varied from 18.36 (sample 3) to 26 % (sample 1).All three species studied had almost identical monoenoic and dienoic acid contents.Among other interesting acids, 16:4(n-3), the presence of which is characteristic for green marine algae 27 , and 18:4(n-3), characteristic for brown marine algae 28 , were detected.It seems likely that the lichen photobiont synthesize these acids 5 .In sample 3, the total level of isomers of 20:4(n-6) and 20:4(n-3) was considerably higher than that from the rest of the examined species, reaching 5.91 %.These amounts of arachidonic acid isomers are the highest known in the lichen literature with regard to the total lipid extract, although still higher levels have been found in individual lipid classes, for example, Peltigera aphthosa 8 .
Total lipid content was studied in all collected lichen species, having common climatic peculiarities.Table 2 shows total lipid compositions in lichens collected during July; total lipid content in such lichens show variations from 36.9-51.3mg/g dry wt.Neutral lipids make up the highest percentage among of total lipids (Table 2) and vary from 22.1-32.1 mg/g dry wt.Examination of neutral lipids using HR-TLC revealed the domination of TAG and diacylglycerols over the rest of neutral lipids thereby representing more than 50% in the majority of lichen species.Free fatty acids, free sterols and its esters were also detected.The amount of glycolipid is comparatively lower than that of neutral lipids and varies between 8-11 %.
Examination of the lichen polar lipids, including betaine lipids (DGTA and DGTS) and phospholipids showed phosphatidylcholine (PC) to be the major phospholipids with concentrations varying from 34.9 to 44.2 % of total polar lipids (Table 2).The PC content in various fungal species is known to vary from 20 to 55%, whereas PC contained in red algal species varies from 61.6 to 77.8 % (ref. 23).Phosphatidylethanolamine (PE) was detected in all lichen species studied; its level was low in the fi rst two species (ca 6.8 %), but reached 14.3 % in sample 3. Phospatidylinositol was also found in all species studied; its level was highest in sample 3 (16.7 %).Both betaine lipids were detected in all samples; their level varies from 8.9 to 14.2 % for DGTA, and 18.6-25.2% for DGTS.
DGTS, one of the three known betaine lipids, has been the object of many studies 24 .Betaine lipids occur in bacteria 30 , fungi 31,32 , moss species 33 , and in a number of brown, green and red algae 26,29,34 as well as in lichens 7,35,36 .As for higher plants, betaine lipids have been found in bryophytes 31,37 , in ferns 38 , and other plants 39 .DGTA was detected in fungi 24 , marine brown algae 27 , and microalgal species 34 .
HPLC has become more widely used as an eff ective analytical tool for the separation and identifi cation of lichen substances 40 .Feige and co-workers 41 used HPLC with reversed-phase columns and gradient elution for separation of 331 lichen compounds.We used HPLC for separation aromatic compounds from three lichen samples.Seven aromaric acids were identifi ed (Table 3).Total aromatic compounds varies from 145.2 (sample 1) to 179.4 μg/100 g dry wt (sample 3).Examination of the lichen aromatics showed lecanoric, protocetric, diff ractaic, usnic, and norstictic acids to be the major metabolites (Table 3).
All samples of R. lacera produced the same aromatic acids but in diff erent amounts.Some biological activities of isolated aromatic compounds from R .lacerahave also been reported.Thus, we recently reported that orsellic, lecanoric, diff ractaic, protocetraric, usnic and norstictic acids from R. lacera possess antibacterial and antifungal activities 45 .Diff ractaic acid exhibited antifungal activity against the phytopathogenic fungus Cladosporium sphaerospermum 52 .Potent antiproliferative agents, usnic and diff ractaic acids showed inhibitory activities against the human keratinocyte cell line HaCaT, with IC 50 values of 2.1 and 2.6 μM, respectively 53 .Diff ractaic acid also showed strong inhibitory activity against tumor promoter-induced Epstein-Barr virus 54 .Orsellic acid revealed antibacterial activity against Escherichia coli, Ralstonia solanacearum, Staphylococcus aureus, and Xanthomonas campestris vesicatoria 55 .Protocetraric acid showed activity against yeasts Candida albicans and C. glabrata 56 , and norstictic acid was active against Aeromonas hydrophila, Bacillus subtilis, Listeria monocytogenes, Proteus vulgaris, Staphylococcus aureus, Streptococcus faecalis, Candida albicans, and C. glabrata 56 .(+)-Usnic acid and (-)-usnic acid isolated from the lichen Ramalina farinacea showed cytotoxic and genotoxic activities against V-79 (Chinese hamster lung fi broblast-like) and A549 (human lung carcinoma epithelial-like) cell lines 57 .Usnic acid exhibited antiviral, antiprotozoal, antiproliferative, anti-infl ammatory and analgesic activities as reported in recent review article 58 .

Table 1 .
Fatty acid composition of the Ramalina lacera growing on diff erent substrates

Table 3 .
Identifi ed aromatic compounds of the Ramalina lacera growing on diff erent substrates 300 °C, fi nal time, 20 min; injection port, 180 °C; carrier gas, He: fl ow rate, 25.0 mL/min.The MS detector operated at 194 °C; ionization energy, 70 eV.The scan range, 30 to 700 m/z at 0.9 scan per sec.Solvent delay, 9 min.Fatty acid methyl esters were identifi ed using mass spectral libraries search (Wiley 7 th , and NIST-98).