Chemistry of two new Leprarioid lichens from Sri Lanka

The hexane and dichloromethane extracts of the lichen Leproloma sipmantanum, collected from Beragala, contained the compounds, atranorin 1, /Lsitosterol 2, (+)-usnic acid 3, zeorin 4 and tritetracontylpentanoate 5 while the methanol extract furnished the tr i terpenoid 3P-acetoxyfern-9(11)-ene 6 . In addit ion t o compounds 1-5, the lichen powder yielded glyceryl trilinolate 7 and 3, 6-dimethyl-2-hydroxy-4-methoxybenzoic acid 8. Chromatography of the lichen powder of Lepraria atrotomentosa led to the isolation of five compounds, including compounds 1-4 along with methyl-~orcinolcarboxylate 9. (+)-Usnic acid exhibited potent antitermite activity against a common pest of tea at low elevations, Glyptotermes dikztatus. 3,6-Dimethyl-2-hydroxy-4-methoxybenzoic acid 8 showed moderate larvicidal activity against the second instar larvae of Aedesaegypti.


IIVTRODUCTION
Lichens are biologically distinct entities composed of an algal or cyanobacterial partner (photobiont) and a fungal partner (mycobiont) living in a symbiotic state. ' The mycobiont plays an important role in absorbing mineral nutrients and protects the lichen from exposure to intense sunlight and desiccation. The mycobiont, in turn, obtains sugars and in some cases organic nitrogen from the photosynthetic ~a r t n e r .~ Lichens synthesize a wide variety of metabolites with different structures and potential biological activities. These activities range from being poisonous (to insects, snails and nematodes) to antibiotic, antitumour, antiherbivore and growth and enzyme inhibitory.? Lichens play a prominent role in air pollution studies because of their sensitivity to different gaseous pollutants, trace metals and radioactive elements.?

METHODS AND MATERIALS
General methods: Melting points were determined by using a Kofler hot stage apparatus and are uncorrected. UV absorptions were measured in a Shimadzu 1601 UV spectrophotometer. IR spectra were recorded on a Shimadzu 160 spectrophotometer on KBr pellets. Optical rotations were determined in a Bellingham-Stanley ADP 220 polarimeter. 'H NMR, "C NMR/ DEPT, HETCOR and HMBC spectra were recorded on a VARIAN ('H 300 and I3C 75.45 MHz) in CdC1, with TMS (tetramethylsilane) as the internal standard. Low and high resolution electron impact mass spectra were recorded on a Kratos/AEI MS-902 spectrometer detector. FAB (Fast Atom Bombardment) mass spectra were measured on a Varian Mat C H 4-B spectrometer using 1-thioglycerol as the matrix. Silica gel used was ~e r c k Kieselgel (230 -400 mesh ASTM).
C o l h k , txiractkand isobtwn: L. sipmdnldnum was collected from Beragala (Uva Province). Specimens were cleaned, airdried and ground to a fine powder (100 g), which then was sequentially extracted into hexane, dichloromethane and methanol using a bottle shaker at room temperature. The methanol extract (10 g) was subjected to Medium Pressure Liquid Chromatography (MPLC) (eluent: CH,Cl, to MeOH) to yield 12 fractions. The first two fractions upon further MPLC {eluent: CH2C12: hexane (1: 4) CH,Cl,) gave 3Bacetoxyfern-9(1l)-ene 6 (68.7 mg) which was crystallized from CH,Cl, while fractions 3 and 4 of the CH2Cl, to MeOH MPLC yielded atranorin 1 (Figure 1).   Hydrolysis ofritetrdcontyIpeniizmate 5 Tritetracontylpentanoate 5 (10 mg) was dissolved in T H F (tetrahydrofuran), mixed with 10 % NaOH (3 ml) and stirred for two h. The reaction mixture was monitored b y T L C (Thin Layer Chromatography) for the disappearance of the starting material. After the completion of the reaction, the mixture was acidified with dilute HC1 until the p H of the solution was acidic. The solid obtained was filtered and the filtrate was extracted with CH2C12 (10 ml x 3). The CH2C1, was evaporated in a rotavapor and the resultant crude solid was used to obtain the 'H NMR spectrum. Hydrolysis of 3~-acetoxyfern-9(11)-ene 6: 3~-acetoxyfern-9(11)-ene 6 (8 mg) was refluxed with 6 O/ O methanolic K O H (5 ml) for 4.5 h. The product was diluted with water (10 ml), extracted into ethyl acetate and dried with MgSO,. The solvent was removed and the cnlde product was crystallized from CH2C1, to obtain the alcohol 6' (7 mg) as colourless scales. Journal of the National Sctence Foundation of Sri Ldnka 34 (2) Chemistry ofLeprarioid lichens from Sri Lanka Mosquito larvicidal assay13: A laboratory culture of Aedes aegypti was used for the assay. Test solutions were prepared by dissolving crude extracts (100 mg) or pure compounds (I mg) in acetone (1 ml) and diluting with water up to 200 ml (500 ppm for extracts; 10 ppm for pure compounds). Polyethylene glycol (120 p1) was added to homogenize the test solutions. These test solutions were used as treatments to screen against the second instar larvae of A. aegypti. Four replicates (five larvae in each beaker) were used for each treatment and the control contained all the ingredients except the lichen extract or the pure compound. The number of larvae moribund or dead was recorded after 24 and 48 h. To the resultant mixture distilled water (10 ml) was added and heated (40-45OC) for 30 min to produce the slurry. An agar solution (1.0 g in 15 ml of distilled water) was heated (50-S°C) and mixed well wirh the cellulose/ compound slurry. T h e resultant mixture was compressed using a pellet block t o prepare approximately five pellets (thickness: 1 cm; diameter: 2.5 cm). Pellets for the control experiment were prepared in the same manner without the test compound. Each pellet (five with the test compound and two without) was transferred to apetri dish and Glyptotermes dilatatus (10 each), which had been starved for 24 h, were introduced. Mortality counts were recorded after 5 d from the start of the experiment and were continued for 30 d. Kampferol was used as the positive control, which had 100 O/ O mortality in 23 d under the above conditions.

RESULTS AND DISCUSSION
L. sipmanianum described in this study was collected from an exposed and well-lit, siliceous bed-rock in Beragala (elevation 1077 m above sea-level), Central Province, Sri Lanka. It is a Leprose (loose powdery surface without a cortex) lichen. The thallus contains short distinct lobes with narrow upturned margins and deep cream in colour. L. sipmanianum has previously been reported from South Africa, Columbia and Brazil15and the report of it from Beragala is a new record to the entire Asian region. 16 Previously, we had reported on the presence of L. sipmanianum lichen compounds namely, atranorin I, psitosterol 2, (+)-usnic acid 3, zeorin 4 and tritetracontylpentanoate 5 in the butterfly Talicada nyseus which is found in the vicinity of the lichen in Beragala. 17 The butterfly, whose natural food source is Bryophyllum calycinum (Sinhala: akkapana; Tamil: malai-kalli), found in Beragala was very likely using the lichen as an alternative food source. Although moths of the family Arctiidae are known to be lichen feeders, that there are no records of a Lycaenid butterfly feeding on lichens, makes the association between L. sipmanianum and 7: nyseus unique. In this study, we report on the isolation and structure elucidation of compounds 1-5 and 3pacetoyfern-9(1 1)-ene 6 from the methanol extract of L. sipmanianum, and glyceryl trilinolate 7 and 3, 6-dimethyl-2-hydroxy-4methoxybenzoic acid 8 directly from the lichen powder.
L. atrotomentosa was collected from partly shaded, overhung rock surfaces and on soil sheltered from rain in Ramboda (elevation 1700 m above sea-level), Central Province.I7 It is a Crustose (crust like, thallus stretching over and firmly fixed to the substratum by the whole lower surface and generally lacking of rhizines) type lichen. The thallus is pale blue to grey in colour. There are no previous reports on the chemistry of this species. Chromatography of the lichen powder, in addition to compounds 1-4, yielded methyl-b-orcinolcarboxylate 9.
The HREIMS of the compound 5 gave a peak at m / z 704.7365, suggesting that the molecular formula was Base hydrolysis of t h e compound gave tritetracontanol whose 'H NMR spectrum showed a peak at d 4.05 (CH,-0), which was shifted to downfield compared to the same peak in the starting ester (d 4.25) confirming that the C,, chain belongs to the alcoholic part of the ester. The 'H NMR, COSY and the mass spectral data taken together to confirm this compound 5 as tritetracontylpentanoate. To the best of our knowledge this is a new natural product.
HREIMS of the compound 6 gave a molecular ion at 468.3967 suggesting an elemental composition of C3,H,,0,which was confirmed by "C NMR (32 peaks) and DEPT (9 x CH,, 9 x CH,, 7 x C H and 5 quarternary carbons) analysis.
The 'H Nh4R spectrum of compound 6 indicated that it was a triterpenoid with oxygenation in ring A at C-3: it showed six methyl singlets at d 0.82 (Me-28), 0.86 (Me-

June 2006
Jotlmal of the National Science Eotlndation of Sri Lanka 34 (2) Chemistry of Ltprarioid lichens from Sri Lanka the position of the gemdimethyl group at CX with an acetoxy moiety at C-3 and the C-25 methyl is attached to an allylic quaternary carbon (G10) adjacent to the double bond between C-9 and C-11. Furthermore, the methyls at C26, C27 and C-28 with respect to the isopropyl group at C-21 was discerned from the above data.
3,6-Dimethyl-2-hydroxy-4-methoxybenzoic acid 8 at 10 ppm showed moderate mosquito larvicidal activity (90 O/ O moribund after 24 hours) against the second instar larvae of Aedes aegypti, which is a major vector of dengue fever. The well-known antimicrobial compound known as (+)-usnic acid 4 showed significant antitermite activity (80 O/ O mortality at 10 mg) against Glyptotermes dilatatus, which is a major live-wood attacking tea-termite at low elevations.