The continuation of our phytochemical survey of Lobaria pulmonaria (L.) Hoffm. (Lobariaceae), has led to the isolation and identification of a new lichen depsidone (1) characterised by the normal analytical and spectroscopic techniques. Unlike previously isolated depsidone (2) from the same species, the compound 1 has not showed activity in the acetylcholinesterase inhibition test on Thin-layer chromatography plate

De Rosa, Salvatore

Iodice, Carmine

Pejin, Boris

Tešević, Vele

Tommonaro, Giuseppina

Vajs, Vlatka

Digest Journal of Nanomaterials and Biostructures

English

Central Repository of the Institute of Chemistry, Technology and Metallurgy (CER)

Digest Journal of Nanomaterials and Biostructures       Vol. 7, No. 4, October-December 2012, p. 1663-1666    A NEW LICHEN DEPSIDONE FROM L OBARIA PULMONARIA   BORIS PEJINa,b,c,*, GIUSEPPINA TOMMONAROa, CARMINE IODICEa, VELE TESEVICb, VLATKA VAJSd, SALVATORE DE ROSAa aNational Research Council of Italy Institute of Biomolecular Chemistry CNR-ICB,  Pozzuoli-Naples, Italy bDepartment of Organic Chemistry, Faculty of Chemistry, University of Belgrade, Belgrade, Serbia cDepartment of Life Sciences, Institute for Multidisciplinary Research, University of Belgrade, Belgrade, Serbia dCenter of Chemistry, Institute for Chemistry, Technology and Metallurgy, University of Belgrade, Belgrade, Serbia   The continuation of our phytochemical survey of Lobaria pulmonaria (L.) Hoffm. (Lobariaceae), has led to the isolation and identification of a new lichen depsidone (1) characterised by the normal analytical and spectroscopic techniques. Unlike previously isolated depsidone (2) from the same species, the compound 1 has not showed activity in the acetylcholinesterase inhibition test on Thin-layer chromatography plate.  (Received September 11, 2012; Accepted October 25, 2012)  Keywords: Lobariaceae, aromatic natural product, acetylcholinesterase inhibitory activity,        Alzheimer’s disease      1. Introduction  Lichens are by definition 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. The majority of organic compounds found in lichens are secondary metabolites of the fungal component, which are deposited on the surface of the hyphae rather than within the cells. These products are usually insoluble in water and can be only extracted with organic solvents. Of the acetyl-polymalonyl derived compounds, aromatic products are especially well represented, the most characteristic being depsides, depsidones, dibenzofurans, usnic acids and depsones. Other aromatic compounds of acetate-polymalonate origin, such as the chromanes, xanthones, and antraquinones are often identical or analogous to products of non-lichen forming fungi or higher plants [1].  Lobaria pulmonaria (L.) Hoffm. (Ascomycotina, Peltigerales, Lobariaceae) is a foliose lichen with broad lobes and a greenish, reticulate upper surface with deep hollows [2]. The genus Lobaria is represented by a variety species, in which L. pulmonaria has been extensively studied and ß-Orcinol depsidones were found to be main constituents [3].  As part of our ongoing project on natural products of lower marine and terrestrial organisms [4,5], a phytochemical investigation has been continued on the lichen L. pulmonaria.   2. Experimental   2.1. General  1H- and 13C-NMR spectra were recorded at the NMR Service of the Institute for Biomolecular Chemistry of National Council Research of Italy (CNR-ICB) on a Bruker Avance-																																																								*Corresponding author: borispejin@imsi.rs, brspjn@gmail.com 1664  400 spectrometer operating 400 and 100 MHz, respectively, using an inverse probe fitted with a gradient along the Z-axis, in CDCl3, using the solvent signal as an internal standard. Thin-layer chromatography was carried out on pre-coated silica gel 60 F254 (0.25 mm, Merck, Darmstadt, Germany). LRMS and HRMS were recorded on a JEOL JMS D-300 and an AEI MS-50, respectively.   2.2. Plant material  The lichen Lobaria pulmonaria (L.) Hoffm. (Lobariaceae) was collected from Fagus sylvatica on the mountain Zelengora (Bosnia and Herzegovina) in July 2009. Voucher specimen has been deposited in the Herbarium of the Institute of Botany, University of Belgrade, Serbia (BEOU 5997).  2.3. Extraction and isolation  Before extraction the lichen was carefully inspected for contaminants. Air-dried parts of L. pulmonaria (70 g) were ground and extracted three times with CHCl3, CHCl3-MeOH 1:1, MeOH, and MeOH-H2O 1:1, respectively, (500 mL each) at room temperature, for up to 1 day each, with the extractives pooled and then evaporated in vacuo. The dried CHCl3-MeOH (1:1) extract (5.81 g) was dissolved in H2O (50 mL) and partioned sequentially with CHCl3 (3 × 50 mL) and n-BuOH (3 × 50 mL). The crude insoluble colored residue (0.46 g), obtained after the partition, was classified as fraction rich in epsilons, by means of its spectroscopic data and typical chromatographic profile. In order to further characterise the residue, it was chromatographed on Sephadex LH-20 column (20 mg) and eluted with the system of methylene chloride/methanol 1:1 to yield the fraction with a new depsidone (5 mg, 0.0071% of dry weight).  New depsidone 1 (Figure 1): 1H NMR (DMSO-d6, 400 MHz) δ 10.44 (1H, s, H-9), 10.24 (1H, s, H-8’), 10.24  (1H, s, H-7’), 7.10 (1H, s, H-5), 3.92 (3H, s, 0CH3-4), 2.50 (3H, s, H-8), 2.19 (3H, s, H-9’); 13C NMR (DMSO-d6, 100 MHz) δ 186.7 (CHO, C-9), 170.6 (COOH, C-8’), 170.6 (COOH, C-7’), 163.2 (C, C-4), 163.2 (COO, C-7), 162.4 (C, C-2), 160.9 (C, C-2’), 147.9 (C, C-4’), 144.8 (C, C-6), 137.4 (C, C-6’), 136.2 (C, C-5’), 120.5 (C, C-3’), 114.4 (C, C-3), 114.4 (C, C-1’), 113.1 (C, C-1), 112.8 (CH, C-5), 56.8 (C, 0CH3-4), 21.6 (CH3, C-8), 9.6 (CH3, C-9’). ESIMS m/z 403 [M+H]+ (calcd for C19H15O10 403.3096).  2.4. Biological Assay  Acetylcholinesterase inhibition test was performed dissolving the sample in MeOH at a concentration of 1 mg/mL. From this main solution was performed a serial dilution in order to obtain lower concentration of the sample (0.1; 0.01; 0.001 mg/mL), and 10 µL of each solution was applied to TLC plates to test 10, 1, 0.1, and 0.01 µg of the sample to detect the minimum concentration that inhibited AChE. Galantamine was used as positive control. The assay was carried out as described Marston, Kissling, & Hostettmann [6]. It is a simple and rapid bioautographic enzyme assay. The test relies on the cleavage by acetylcholinesterase of                 1-naphthyl acetate to form 1-naphthol, which in turn reacts with Fast Blue B salt to give a purple-coloured diazonium dye. Briefly, a stock solution of acetylcholinesterase (1000 U in 150 mL of Tris-hydrochloric acid buffer pH 7.8) was obtained, which was stabilised adding bovine serum albumin (150 mg). A 10 µL aliquot of each solution of the sample was applied to the TLC plates, dried to remove the solvent, and then sprayed with enzyme stock solution. For incubation of the enzyme, the plate was kept at 37 C for 20 min in a humid atmosphere. For the detection of the enzyme, solutions of 1-naphthyl acetate (250 mg in 100 mL of EtOH) and of Fast Blue B salt (400 mg in 160 mL of distilled H2O) were mixed and sprayed onto the plate. Acetylcholinesterase inhibition activity was detected by a white spot on a purple background after 1-2 min.    1665  3. Results and discussion  High-resolution mass spectrometry established the molecular formula of a new lichen depsidone 1 (C19H14O10, Figure 1). The structure followed from 1-D and 2-D NMR spectra. The 1H-NMR spectrum showed three three-proton singlets between δ 2.0 and 4.0, one-proton singlet between   δ 7.0 and 10.0, carboxylic protons at δ 10.24 and an aldehyde proton at δ 10.44; C-carboxylic resonances were specific signals of the identified depsidone molecule. The 13C-NMR and DEPT spectra exhibited 19 carbon signals. A combination of 2-D NMR experiments (COSY, NOESY, HSQC and HMBC) allowed us to assign all signals in the 1H- and 13C- NMR spectra.     Fig. 1. A new lichen depsidone    The AChE inhibition test [6,7] showed no activity for the compound 1. In comparison, the alkaloid galantamine used clinically for the treatment of Alzheimer’s disease [8] inhibited the enzyme at 0.01 µg, while a new depsidone 2 isolated from the same lichen in the form of its diacetate derivative  (C21H18O9, Figure 2) was active at 1 µg [9]. According to our best knowledge, it is the only identified depsidone with acetylcholinesterase inhibitory activity till date. On the other hand, AChE inhibitors are still considered the best drugs currently available for the management of Alzheimer's disease [10]. Because most inhibitors of AChE are alkaloids that often possess several side effects, it is important to search for new AChE inhibitors not belonging to this structural class.     Fig. 2. Previously isolated depsidone in the form of its diacetate derivative  1666    4. Conclusion   Our results indicate the significance of covalent modification (acetylation) of depsidone structure in the AChE inhibition.    Acknowledgement  This work was supported by the Ministry of Science and Technological Development of the Republic of Serbia (Research grant Nos. 172053 and 173040). The assistance of Mr. V. Mirra of NMR Service CNR-ICB is gratefully acknowledged.   References    [1] T. H. Nash, Lichen Biology, University Press Cambridge, UK (2008).   [2] A. G. González, J. Bermejo Barrera, E. M. Rodríguez Pérez,  C. E. Hernández Padrón,          Biochem. Syst. Ecol. 22, 583 (1994).    [3] C. F. Culberson, Bryologist 72, 19 (1969).    [4] B. Pejin, C. Iodice, G. Tommonaro, S. De Rosa, J. Nat. Prod. 71, 1850 (2008).   [5] B. Pejin, M. Vujisic, M. Sabovljevic, V. Tesevic, V. Vajs, Cryptogamie Bryol.          32, 113 (2011).   [6] A. Marston, J. Kissling, K. Hostettmann, Phytochem. Anal. 13, 51 (2002).   [7] I. K. Rhee, M. van de Meent, K. Ingkaninan, R. Verpoorte,  J. Chromatogr. A          915, 217 (2001).    [8] L. J. Scott, K. L. Goa, Drugs 60, 1095 (2000).   [9] B. Pejin, G. Tommonaro, C. Iodice, V. Tesevic, V. Vajs, S. De Rosa, J.  Enzym. Inhib.        Med. Chem. in press, doi: 10.3109/14756366.2012.677839 (2012).  [10] L. P. Graham, An Introduction to Medicinal Chemistry, University Press Oxford,         UK (2005).                   

A New Lichen Depsidone from  L Obaria Pulmonaria

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A New Lichen Depsidone from L Obaria Pulmonaria

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