Fitoterapia 83 (2012) 1693–1698 Contents lists available at SciVerse ScienceDirect Fitoterapia journal homepage: www.elsevier.com/locate/fitote Lobarin from the Sumatran lichen, Stereocaulon halei☆ Friardi Ismed a, Françoise Lohézic-Le Dévéhat a,⁎, Olivier Delalande b, Sourisak Sinbandhit c, Amri Bakhtiar d, Joël Boustie a a b c d UMR CNRS 6226 ISCR, Equipe PNSCM, Faculté des Sciences Pharmaceutiques et Biologiques, Univ. Rennes 1, 35043 Rennes Cedex, France UMR CNRS 6290 IGDR, Equipe SIM, Faculté des Sciences Pharmaceutiques et Biologiques, Univ. Rennes 1, 35043 Rennes Cedex, France Centre Régional de Mesures Physiques de l'Ouest, Univ. Rennes 1, 35042 Rennes Cedex, France Faculty of Pharmacy, Andalas University, 26163 Padang, Indonesia a r t i c l e i n f o Article history: Received 5 June 2012 Accepted in revised form 21 September 2012 Available online 4 October 2012 Keywords: Lichen Stereocaulon halei Photoprotection Diphenyl ether Pseudodepsidone Antioxidant a b s t r a c t The diphenyl ether, lobarin (1) (syn. lobariol carboxylic acid) related to lobaric acid was isolated for the first time as a natural product along with five known compounds from Stereocaulon halei, a fruticose lichen collected in Indonesia. The structure of lobarin was elucidated by spectroscopic data analysis and its most stable conformers were determined by molecular mechanic dynamic calculations. A marked superoxide anion scavenging was found for compound 1 while no cytotoxicity on the B16 murine melanoma and HaCaT human keratinocyte cell lines was observed. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Stereocaulon is a widely distributed worldwide fruticose lichen genus which counts about 130 species [1]. These lichens grow mostly in upland regions on siliceous rock, particularly on recent volcanic rock, on metal-rich spoil heaps and on acidic soil among mosses. Some of them are used in traditional medicine e.g., S. paschale and S. vulcani used as antihemorrhagic, treating high blood pressure, diabetes symptoms, wounds and ulcers, and also syphilis [2–4]. About Abbreviations: SPF, Sun Protection Factor; UVA-PF, UVA-Protection Factor; MD, molecular dynamic; RMSD, root mean square deviation. ☆ Dedicated to the memory of Dr. Habil. Siegfried Huneck, Halle/Saale, Germany, for his outstanding contribution to lichen chemistry. ⁎ Corresponding author at: Department of Pharmacognosy-Mycology, Fac. Pharmacy, 2 av. Pr Leon Bernard, 35043, Rennes Cedex, France. Tel.: +33 223234817; fax: +33 223234704. E-mail address: francoise.le-devehat@univ-rennes1.fr (F. Lohézic-Le Dévéhat). 0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2012.09.025 40 lichen compounds are described from three dozens of Stereocaulon species. Although some lichen metabolites are frequently encountered, the number of lichen metabolites isolated was over 800 in the reference work published by Huneck and Yoshimura [5] and is now estimated to be over 1050 [6], but poorly investigated for their biological properties [7]. In our search to look for new photoprotective compounds, we investigated a tripartite lichen, Stereocaulon halei Lamb belonging to the subgenus Holostelidium. This species was exposed to sun on basalt rocks of Mount Singgalang (altitude 2877 m) in West Sumatra (Indonesia) and not yet phytochemically investigated. Herein, we describe the isolation and structural elucidation of a diphenyl ether, lobarin (1), along with the common metabolite atranorin (2) as well as four known compounds (3–6). Compounds 1–3 and 6 were tested for antioxidant activities using superoxide anion scavenging and DPPH assays and as UV-blockers by calculation of their Sun Protection Factor (SPF) and UVA-PF. Their cytotoxic activities against the B16 murine melanoma and HaCaT human keratinocyte cell lines were also evaluated. 1694 F. Ismed et al. / Fitoterapia 83 (2012) 1693–1698 2. Experimental 2.1. General Melting points were measured on a Kofler hot-stage apparatus. Optical rotation was determined with a PerkinElmer model 341 polarimeter. UV spectra were performed on a Uvikon 931 spectrophotometer. FT-IR spectra were run on a Perkin-Elmer 16 PC IR spectrometer. 1H and 13C NMR spectra were recorded at 500 and 125 MHz on a Bruker DMX 500 WB NMR spectrometer, at 400 and 100 MHz on a Bruker Avance III 400 or at 300 and 75 MHz, on a Bruker 300 NMR spectrometer respectively, using CDCl3, DMSO-d6, acetone-d6 and methanol-d4 as solvents. HRMS measurements for exact mass determination were performed on a Varian MAT 311 mass spectrometer for electrospray and a Micromass ZABSpecTOF mass spectrometer for chemical ionization at the Centre Régional de Mesures Physiques de l'Ouest. Chromatographic separations were performed using vacuum liquid chromatography on silica gel (Merck 35–70 μm) and Sephadex LH-20 (BioChemika Fluka). Medium-pressure chromatography was conducted on a SPOT Liquid Chromatography Flash® (Armen Instrument) using silica or C18 pre-packed columns (Super Vario Flash D26 cartridge SI60 40–63 μm, 30 g Merck, normal phase; SVF D26-RP18 25–40 μm, 31 g, Merck, reversed phase) or manually-packed silica columns (40–63 μm, Kieselgel 60, Merck, 7667). The circular preparative chromatography was a Chromatotron® (Harrison Research model 8924) operating with a peristaltic pump and a circular glass plate (diameter 20 cm) coated with silica (Silica GF254, 35–70 μm, Merck 7730) to a thickness of 3 mm. TLC plates (Merck silica gel 60F254) were eluted using standard solvent systems [5] and toluene– EtOAc–formic acid (139:83:8) (G). Visualization of plates was carried out under UV light (254 and 365 nm) and using anisaldehyde–H2SO4 reagent then heating. 2.2. Lichen material S. halei Lamb was collected in November 2009 on rocks from Mount Singgalang (2877 m), West Sumatra, Indonesia. After identification by Harrie Sipman (Berlin Museum), the voucher specimens were deposited at the Herbarium of the Department of Pharmacognosy and Mycology, Rennes and Biota Sumatran Laboratory, Andalas University, West Sumatra (Indonesia) with the reference numbers JB/09/117 and GSb 1 respectively. 2.3. Extraction and isolation The air-dried whole thalli of S. halei (1 kg) were successively macerated with n-hexane, ethyl acetate and acetone (3 times × 2.5 l). Each extract was concentrated in vacuo and the precipitates formed after solvent evaporation at room temperature in the n-hexane and the ethyl acetate mother liquors afforded compound 2 (25 g). The n-hexane mother liquor (2 g) was chromatographed on a vacuum liquid chromatography silica gel column (150 g, 4 × 30 cm) eluted with a solvent gradient consisting of n-hexane–EtOAc (100:0→0:100) as the mobile phase. Four sub-fractions (AP1– AP4) were obtained. Sub-fraction AP3 (407 mg) was selected for further chromatography using flash chromatography and was eluted employing toluene–CH2Cl2 (70:30). From this, six smaller fractions were obtained and compound 3 (50 mg) was recrystallized in n-hexane. Sub-fraction AP2 (1.2 g) was further purified by vacuum liquid chromatography over a Silica gel 60 (50 g, 4×30 cm) using n-hexane–CH2Cl2 (80:20) and followed by radial chromatography (Chromatotron®) with n-hexane– EtOAc (98:2) as the mobile phase. Compound 4 (20 mg) and compound 5 (5 mg) were obtained as crystalline residues. Silica gel vacuum liquid chromatography of the ethyl acetate filtrate (6 g) with an increasing gradient solvent system of n-hexane– diethylether–CH2Cl2–MeOH (100:0→0:100), 200 ml of each solvent, yielded 4 sub-fractions (E1–E4). The purification of E1 (2.2 g) by flash chromatography on C18 with solvent system H2O–TFA 0.1%–MeOH (50:50) yielded a mixture of 1 and 6 further purified by silica gel column with diethylether–CHCl3 (60:40). Compound 6 (300 mg) and compound 1 (30 mg) were finally separated on Sephadex LH-20 with CHCl3–Acetone (40:60). 2.3.1. Lobarin (1) Brown amorphous solid; [α] 25D 0.0 (c 0.6, CHCl3); UV (MeOH) λmax (log ɛ): 213 (3.74), 252 (3.41) and 295 (3.11) nm; IR (KBr) vmax 3350, 2955, 2928, 1736, 1614, 1484, 1439, 1247 cm −1; 1H (CDCl3, 400,1 MHz) and 13C NMR (CDCl3, 125.75 MHz) spectroscopic data see Table 1; HR-ESI-MS (negative) m/z 473.1817 [M − H] − (calcd. for C25H29O9, 473.1817), m/z 495.1638 [M − 2H + Na] −; Rf 0.44 (G). 2.3.2. Lobaric acid (6) Colorless needles (CHCl3); m.p. 196–197 °C; 1H and 13C NMR (acetone-d6, 500 MHz) data comparable to published values [8]; HR-ESI-MS (negative) m/z 455.1715 [M − H] − (calcd. for C25H27O8, 455.1715); Rf 0.56 (G). Table 1 1 H and 13C NMR spectroscopic data for compound 1 (CDCl3, 400 MHz for 1H NMR and 125 MHz for 13C NMR). Position δC δH (mult., J in Hz) 1 2 3 4 5 6 7 8 9 10 11 12 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ OCH3-4 106.63 157.48 101.95 167.67 100.74 153.77 167.57 107.23 38.10 25.16 22.44 13.80 103.45 163.74 103.65 155.58 132.73 140.87 174.16 28.35 30.30 32.10 21.97 13.85 56.11 – – 6.12, d (1.6) – 6.67, d (1.6) – – – 2.21, m −2.06, m 1.41, m −1.16 m 1.35, m 0.90, t (7.1) – 11.50, s(OH) 6.55, s – – – – 2.86, m −2.65, m 1.48, m −1.35,m 1.18, m 1.17, m 0.76, t (7.1) 3.81, s F. Ismed et al. / Fitoterapia 83 (2012) 1693–1698 1695 2.4. Molecular models and dynamic simulations 3. Results and discussion Lobarin initial structures were built using the Yasara program and parameterized for the Yamber3 force field following the automated AutoSMILE procedure [9]. Only R configuration has been considered for the chiral atom C-8. Four different conformers are discriminated from value combinations of the two torsion angles [C4′–C5′–O–C2] and [C5′–O–C2–C1], defined as torsions θ1 and θ2. To enhance the conformational space exploration, starting structures used as initial point for the two independent MD simulations were set arbitrary to θ1/θ2 combinations of −90°/−160° and −55°/−35°, respectively. Each conformer was placed in an explicit methanol solvent box and simulated under periodic boundary conditions at a constant temperature of 298 K. Structures were relaxed during a 1 nanosecond (ns) MD simulation to reach an equilibrated state (stable root mean square deviation, rmsd). Production trajectories of 32 ns were collected at 2 ps intervals for each of the two structural models. MD trajectory analyses (rmsd and clustering) were performed using Gromacs tools [10]. The known compounds atranorin (2, 25 g), methyl-βorcinol carboxylate (MOC) (3, 50 mg), and methyl- and ethylhaematommate (4, 20 mg and 5, 5 mg) and lobaric acid (6, 300 mg) (Fig. 1) were identified by direct comparison of their physical and spectral data in the literature [5,14]. Along with these common lichen metabolites, the unusual compound 1 (Fig. 1) was obtained as a brown solid. Its molecular formula was assigned as C25H30O9 on the basis of HRESIMS m/z 473.1817 [M− H]− (calcd. for C25H29O9, 473.1817). The UV spectrum of compound 1 with absorption maxima at 213, 252 and 295 nm suggested that 1 has a depside- or a depsidonetype structure [5]. Nevertheless, considering that the degrees of unsaturation were 11, 1 was postulated to be more related to a diphenyl ether such as sakisacaulon [15] than to a depsidone such as lobaric acid (6). IR bands suggested the presence of hydrogen-bonded hydroxyl groups (3350 and 2955 cm−1) and two carbonyl groups corresponding to a lactone ring (1736 cm−1) and a carboxylic group (1614 cm−1). The 1H NMR spectrum (Table 1) indicated the presence of signals for three aromatic protons (δ 6.12, 6.55 and 6.67), one methoxy group (δ 3.81) and two methyl groups (δ 0.76 and 0.90). Multiplet signals at δ 1.17–2.86 corresponding to a 14 H methylene proton group suggested the presence of aliphatic chains. The COSY 1H–1H NMR experiment confirmed the presence of n-butyl (C-9 to C-12) and n-pentyl (C-8′ to C-12′) side chains and the connectivity between two protons H-3 and H-5. The complete structure was established using HMBC (Fig. 2) with the following connectivities: aromatic H-3 (δ 6.12) with C-2 (δ 157.48), C-4 (δ 167.67), C-1 (δ 106.63) and C-5 (δ 100.74), aromatic H-5 (δ 6.67) with C-3 (δ 101.95), C-1 (δ 106.63), C-2 (δ 157.48) and C-4 (δ 167.67) and aromatic H-3′ (δ 6.55) with C-1′ (δ 103.45), C-2′ (δ 163.74), C-5′ (δ 132.73), C-6′ (δ 140.87), C-4′ (δ 155.58), C-7′ (δ 174.16). Using HSQC TOCSY experiments, the butyl side chain showed methyl H-12 (δ 0.90) to be correlated with C-11 (δ 22.44), C-10 (δ 25.16) and C-9 (δ 38.10) and the pentyl chain showed methyl H-12′ 2.5. Antioxidant testing Scavenging activity of compounds 1–3 and 6 on the 1,1′diphenyl-2-picrylhydrazyl free radical (DPPH) was measured as previously described [11]. For same compounds, measurement of superoxide anion scavenging activity in 96-well microplates was based on the non-enzymatic method described previously with some modifications. The reaction mixture in the sample wells consisted of NADH (78 μM), NBT (50 μM), PMS (10 μM), and lichen compounds (350, 116, 39, 13 μM). The reagents were dissolved in 16 mM trishydrochloride buffer, at pH= 8 except for all the lichen compounds which were dissolved in DMSO. After 5 min of incubation at room temperature, the spectrophotometric measurement was performed at 560 nm against a blank sample without PMS. Ascorbic acid was used as positive control. The percentage inhibition at steady state for each dilution was used to calculate the IC50 values. This gave the amount of antioxidant required (measured as the concentration of the stock solution added to the reaction mixture) to scavenge 50% of O2−•, with lower values indicating more effective scavenging of O2−•. All tests were done in triplicate and the results averaged. 2.6. Cytotoxicity testing Cytotoxic activities of compounds 1–3 and 6 were evaluated against B16-F10 (melanoma; ATCC CRL-6475) and HaCaT cells (HaCaT, ATCC) by using the MTT assay performed according to the method previously described [12]. 2.7. In vitro Sun Protection Factors (SPF) calculation SPF, UVA PF, critical wavelength (λc) calculation was assessed by an in vitro screening method [13] and Tinosorb M was used as positive control. Fig. 1. Structures of compounds 1–6. 1696 F. Ismed et al. / Fitoterapia 83 (2012) 1693–1698 Fig. 2. Selected 2D NMR correlations for compound 1. (δ 0.76) to be correlated with C-11′ (δ 21.97), C-10′ (δ 32.10), C-9′ (δ 30.30), and C-8′ (δ 28.35). The C-8 substitution by the butyl side chain was revealed by an HMBC correlation between H-9 and C-8 and C-6 but also with a NOESY correlation between H-5 and H2-9/H2-10. The connection between C-2 and C-5′ through an ether linkage was confirmed by a NOESY correlation between H-3 and H2-8′, H-9′ and H-10′. In CDCl3 solution at 296 K, compound 1 was found to be a mixture of two conformers as shown in NMR spectra by the broadening of both H-5 and H-3 signals. 1H NMR spectra were recorded at lower temperatures (203 K) in methanol-d4 and two sets of peaks in a 65/35 ratio were observable, suggesting the co-existence of two rotamers related to the ether bond. Two lobarin molecular models were extracted from molecular dynamics [16] trajectories using a cluster analysis (Fig. 3). These structures are representing respectively 40% and 30% of both first and second trajectories and correspond to the most stable MD conformers (Yamber internal energy). The geometry of these two conformers (lobarin 1 and lobarin 2) diverged in their θ1/θ2 torsion angles (− 90.78°/− 162.57° and 99.02°/166.81°). It is worthy to notice that a spontaneous conformational change occurred in the second MD simulation from the initial conformation (form 3 in Fig. 3) to a most favorable state (lobarin 2). We did not observed the fourth putative conformer corresponding to approximately −θ1/−θ2 torsion angles of the less stable form 3. Based on molecular dynamics calculations, lobarin in MeOH was predicted to present a conformational equilibrium between forms 1 and 2 which was in accordance to NMR records. Moreover, by comparison of inter-proton distance recording upon MD simulation and NOESY spectrum analysis, it appeared that the main conformer present in solution should be the lobarin form 1 (Supplementary Table A). Therefore, compound 1 was determined to be a diphenyl ether, identified as lobarin and has two stable conformers (forms 1 and 2). Diphenyl ethers are not so common in lichens compared to depsides and depsidones and most of them have the ether bond joined at the meta-position of the B-ring. Consequently, they are often related to depsidones [17,18]. Two pseudodepsidones have been reported from Stereocaulon alpinum collected in polar regions, one being distinguished from compound 1 by the 8-OCH3 substitution [19]. The co-occurrence of a depsidone having a carbonyl group in α-position of the side chain linked to ring A and its related diphenyl ether with a lactol group has been also reported for loxodin and norlobaridone [20,21]. Some diphenyl ethers have been formed during extraction [22,23] or by treatment of depsidones using hot alkali as described for lobariol carboxylic acid or lobarin from lobaric acid [24,25]. In order to check that 1 was not an artifact of isolation, the extraction process using n-hexane then EtOAc, was conducted Fig. 3. RMSD matrix measured for both MD simulations. Conformational space is chronologically represented along the square's diagonal (from bottom left to top right) and RMSD values increase towards the darkest points. The two most stable molecular models built from MD cluster analysis are shown in blue and red. Both main representative models observed all along trajectories are superimposed (with the six-membered cycle as reference) in the middle caption. 1697 F. Ismed et al. / Fitoterapia 83 (2012) 1693–1698 again on a new lichen sample and at the same time with lobaric acid, at room temperature and under hot temperature conditions (50 °C). Extracts and products obtained were then analyzed by TLC. Extracts were found to have the same profile at both temperature conditions with a spot unambiguously corresponding to 1 (Rf =0.44, (G)), while lobaric acid remained unchanged under these conditions. It is therefore concluded that lobarin is a natural metabolite of this lichen. Based on pathways already described [20,26], the diphenyl ether lobarin (1) is thought to result from the ring opening of the depsidone linkage of lobaric acid through hydrolysis, followed by a nucleophilic attack and a prototropic arrangement (Supplementary Fig. A) but without decarboxylation or O-methylation as described for the biosynthesis of loxodin and norlobaridone, respectively [26]. The compounds 1–3 and 6 were tested for radicalscavenging activity against superoxide anion (O2−•) and 2,2diphenyl-1-picrylhydrazyl (DPPH) radicals along with their cytotoxic activity against B16 murine melanoma and HaCaT human keratinocyte cell lines (Table 2). No compounds were active on DPPH assay and this lack of activity may be explained by hydrogen bonds between aldehyde and hydroxyl groups but also by steric hindrance [27,28]. Compound 1, was an active superoxide anion scavenger three times more efficient than the control (ascorbic acid) while atranorin was within the range of the control. This activity was all the more remarkable in that no cytotoxicity was observed on the two tested cell lines. Compound 1 obtained from lobaric acid was recently patented with regard to its remarkable effect on protein tyrosine phosphatase-1b (PTP-1b) suggesting a possible use as a drug to treat diabetes or obesity [23]. Whatever some valuable SPF values, atranorin 2 is found to have the better profile from the tested compounds but do not reach the requirements to be developed as a UV blocker. Acknowledgments We gratefully acknowledge the French Ministry of Research and Education (Bio-Asia Program, DREIC [International Relations and Cooperation Department]) and the French Embassy in Indonesia for a Ph.D. grant to Friardi Ismed. Thanks also to Prof. Philippe Uriac and Prof. Sophie Tomasi for fruitful discussion of lichen biogenesis. We also thank Isabelle Rouaud and Solenn Ferron for the biological assays and Nova Syafni and the Sumatran Biota Laboratory team for their help in collecting the lichens used in this study. We are grateful to P. Jéhan, F. Lambert and N. Le Yondre, CRMPO, Rennes, France, for the mass spectrometer measurements. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2012.09.025. References [1] Kirk PM, Cannon PF, David JC, Stalpers JA. Ainsworth and Bisby's dictionary of the fungi. 10th ed.Wallingford: CAB International; 2011. [2] Lavergne R. Plantes medicinales indigenes tisanerie et tisaneurs de la Reunion. Sciences biologiques. Montpellier: Université des Sciences et Techniques du Languedoc; 1989. p. 519-21. [3] Hanssen H-P, Schadler M. Pflanzen in der traditionellen chinesischen medizin. Dtsch Apoth Ztg 1985;24:1239-43. [4] Fraser M-H, Cuerrier A, Haddad PS, Arnason JT, Owen PL, Johns T. 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