Fitoterapia 83 (2012) 1693–1698

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Fitoterapia

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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 UMR CNRS 6226 ISCR, Equipe PNSCM, Faculté des Sciences Pharmaceutiques et Biologiques, Univ. Rennes 1, 35043 Rennes Cedex, Franceb UMR CNRS 6290 IGDR, Equipe SIM, Faculté des Sciences Pharmaceutiques et Biologiques, Univ. Rennes 1, 35043 Rennes Cedex, Francec Centre Régional de Mesures Physiques de l'Ouest, Univ. Rennes 1, 35042 Rennes Cedex, Franced Faculty of Pharmacy, Andalas University, 26163 Padang, Indonesia

a r t i c l e i n f o

Abbreviations: SPF, Sun Protection Factor; UVAFactor; MD, molecular dynamic; RMSD, root mean squ☆ Dedicated to the memory of Dr. Habil. SiegfriedGermany, for his outstanding contribution to lichen⁎ Corresponding author at: Department of Pharmaco

Pharmacy, 2 av. Pr Leon Bernard, 35043, Rennes Ced223234817; fax: +33 223234704.

E-mail address: francoise.le-devehat@univ-rennes(F. Lohézic-Le Dévéhat).

0367-326X/$ – see front matter © 2012 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.fitote.2012.09.025

a b s t r a c t

Article history:Received 5 June 2012Accepted in revised form 21 September 2012Available online 4 October 2012

The diphenyl ether, lobarin (1) (syn. lobariol carboxylic acid) related to lobaric acid wasisolated for the first time as a natural product along with five known compounds fromStereocaulon halei, a fruticose lichen collected in Indonesia. The structure of lobarin waselucidated by spectroscopic data analysis and its most stable conformers were determined bymolecular mechanic dynamic calculations. A marked superoxide anion scavenging was foundfor compound 1 while no cytotoxicity on the B16 murine melanoma and HaCaT humankeratinocyte cell lines was observed.

© 2012 Elsevier B.V. All rights reserved.

Keywords:LichenStereocaulon haleiPhotoprotectionDiphenyl etherPseudodepsidoneAntioxidant

1. Introduction

Stereocaulon is a widely distributed worldwide fruticoselichen genus which counts about 130 species [1]. Theselichens grow mostly in upland regions on siliceous rock,particularly on recent volcanic rock, on metal-rich spoil heapsand on acidic soil among mosses. Some of them are used intraditional medicine e.g., S. paschale and S. vulcani used asantihemorrhagic, treating high blood pressure, diabetes symp-toms, wounds and ulcers, and also syphilis [2–4]. About

-PF, UVA-Protectionare deviation.Huneck, Halle/Saale,chemistry.gnosy-Mycology, Fac.ex, France. Tel.: +33

1.fr

ll rights reserved.

40 lichen compounds are described from three dozens ofStereocaulon species. Although some lichen metabolites arefrequently encountered, the number of lichen metabolitesisolated was over 800 in the reference work published byHuneck and Yoshimura [5] and is now estimated to be over1050 [6], but poorly investigated for their biological proper-ties [7]. In our search to look for new photoprotectivecompounds, we investigated a tripartite lichen, Stereocaulonhalei Lamb belonging to the subgenus Holostelidium. Thisspecies was exposed to sun on basalt rocks of MountSinggalang (altitude 2877 m) in West Sumatra (Indonesia)and not yet phytochemically investigated. Herein, we describethe isolation and structural elucidation of a diphenyl ether,lobarin (1), alongwith the commonmetabolite atranorin (2) aswell as four known compounds (3–6). Compounds 1–3 and 6were tested for antioxidant activities using superoxide anionscavenging and DPPH assays and as UV-blockers by calculationof their Sun Protection Factor (SPF) and UVA-PF. Theircytotoxic activities against the B16 murine melanoma andHaCaT human keratinocyte cell lines were also evaluated.

Table 11H and 13C NMR spectroscopic data for compound 1 (CDCl3, 400 MHz for 1HNMR and 125 MHz for 13C NMR).

Position δC δH (mult., J in Hz)

1 106.63 –

2 157.48 –

3 101.95 6.12, d (1.6)4 167.67 –

5 100.74 6.67, d (1.6)6 153.77 –

7 167.57 –

8 107.23 –

9 38.10 2.21, m −2.06, m10 25.16 1.41, m −1.16 m11 22.44 1.35, m12 13.80 0.90, t (7.1)1′ 103.45 –

2′ 163.74 11.50, s(OH)3′ 103.65 6.55, s4′ 155.58 –

5′ 132.73 –

6′ 140.87 –

7′ 174.16 –

8′ 28.35 2.86, m −2.65, m9′ 30.30 1.48, m −1.35,m10′ 32.10 1.18, m11′ 21.97 1.17, m12′ 13.85 0.76, t (7.1)OCH3-4 56.11 3.81, s

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2. Experimental

2.1. General

Melting points were measured on a Kofler hot-stageapparatus. Optical rotation was determined with a Perkin-Elmermodel 341 polarimeter. UV spectra were performed on aUvikon 931 spectrophotometer. FT-IR spectra were run on aPerkin-Elmer 16 PC IR spectrometer. 1H and 13C NMR spectrawere recorded at 500 and 125 MHz on a Bruker DMX 500 WBNMR spectrometer, at 400 and 100 MHz on a Bruker Avance III400 or at 300 and 75 MHz, on a Bruker 300 NMR spectro-meter respectively, using CDCl3, DMSO-d6, acetone-d6 andmethanol-d4 as solvents. HRMS measurements for exact massdetermination were performed on a Varian MAT 311 massspectrometer for electrospray and a Micromass ZABSpecTOFmass spectrometer for chemical ionization at the CentreRégional de Mesures Physiques de l'Ouest. Chromatographicseparations were performed using vacuum liquid chromatog-raphy on silica gel (Merck 35–70 μm) and Sephadex LH-20(BioChemika Fluka). Medium-pressure chromatography wasconducted on a SPOT Liquid Chromatography Flash® (ArmenInstrument) using silica or C18 pre-packed columns (SuperVario Flash D26 cartridge SI60 40–63 μm, 30 g Merck, normalphase; 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 aChromatotron® (Harrison Research model 8924) operatingwith a peristaltic pump and a circular glass plate (diameter20 cm) coatedwith 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 wascarried out under UV light (254 and 365 nm) and usinganisaldehyde–H2SO4 reagent then heating.

2.2. Lichen material

S. halei Lamb was collected in November 2009 on rocksfrom Mount Singgalang (2877 m), West Sumatra, Indonesia.After identification by Harrie Sipman (Berlin Museum), thevoucher specimens were deposited at the Herbarium of theDepartment of Pharmacognosy and Mycology, Rennes andBiota Sumatran Laboratory, Andalas University, West Sumatra(Indonesia) with the reference numbers JB/09/117 and GSb 1respectively.

2.3. Extraction and isolation

The air-dried whole thalli of S. halei (1 kg) were succes-sively macerated with n-hexane, ethyl acetate and acetone(3 times×2.5 l). Each extract was concentrated in vacuo andthe precipitates formed after solvent evaporation at roomtemperature in the n-hexane and the ethyl acetate motherliquors afforded compound 2 (25 g). The n-hexane motherliquor (2 g) was chromatographed on a vacuum liquidchromatography silica gel column (150 g, 4×30 cm) elutedwith 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 selectedfor further chromatography using flash chromatography and

was eluted employing toluene–CH2Cl2 (70:30). From this, sixsmaller fractions were obtained and compound 3 (50 mg) wasrecrystallized in n-hexane. Sub-fraction AP2 (1.2 g) was furtherpurified by vacuum liquid chromatography over a Silica gel 60(50 g, 4×30 cm) using n-hexane–CH2Cl2 (80:20) and followedby radial chromatography (Chromatotron®) with n-hexane–EtOAc (98:2) as the mobile phase. Compound 4 (20 mg) andcompound 5 (5 mg)were obtained as crystalline residues. Silicagel 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 eachsolvent, yielded 4 sub-fractions (E1–E4). The purification of E1(2.2 g) by flash chromatography on C18 with solvent systemH2O–TFA 0.1%–MeOH (50:50) yielded a mixture of 1 and 6further purified by silica gel column with diethylether–CHCl3(60:40). Compound 6 (300 mg) and compound 1 (30 mg)werefinally 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 publishedvalues [8]; HR-ESI-MS (negative) m/z 455.1715 [M−H]−

(calcd. for C25H27O8, 455.1715); Rf 0.56 (G).

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2.4. Molecular models and dynamic simulations

Lobarin initial structures were built using the Yasara pro-gram and parameterized for the Yamber3 force field followingthe automated AutoSMILE procedure [9]. Only R configurationhas been considered for the chiral atom C-8. Four differentconformers are discriminated from value combinations of thetwo torsion angles [C4′–C5′–O–C2] and [C5′–O–C2–C1], de-fined as torsions θ1 and θ2. To enhance the conformationalspace exploration, starting structures used as initial pointfor the two independent MD simulations were set arbitrary toθ1/θ2 combinations of −90°/−160° and −55°/−35°, respec-tively. Each conformer was placed in an explicit methanolsolvent box and simulated under periodic boundary conditionsat a constant temperature of 298 K. Structures were relaxedduring a 1 nanosecond (ns) MD simulation to reach anequilibrated state (stable root mean square deviation, rmsd).Production trajectories of 32 nswere collected at 2 ps intervalsfor each of the two structural models. MD trajectory analyses(rmsd and clustering) were performed using Gromacs tools[10].

2.5. Antioxidant testing

Scavenging activity of compounds 1–3 and 6 on the 1,1′-diphenyl-2-picrylhydrazyl free radical (DPPH) was measuredas previously described [11]. For same compounds, measure-ment of superoxide anion scavenging activity in 96-wellmicroplates was based on the non-enzymatic method de-scribed previously with some modifications. The reactionmixture 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 tris-hydrochloride buffer, at pH=8 except for all the lichencompounds which were dissolved in DMSO. After 5 min ofincubation at room temperature, the spectrophotometricmeasurement was performed at 560 nm against a blanksample without PMS. Ascorbic acid was used as positivecontrol. The percentage inhibition at steady state for eachdilution was used to calculate the IC50 values. This gave theamount of antioxidant required (measured as the concentra-tion of the stock solution added to the reaction mixture) toscavenge 50% of O2

−•, with lower values indicating moreeffective scavenging of O2

−•. All tests were done in triplicateand the results averaged.

2.6. Cytotoxicity testing

Cytotoxic activities of compounds 1–3 and 6were evaluatedagainst B16-F10 (melanoma; ATCC CRL-6475) and HaCaT cells(HaCaT, ATCC) by using the MTT assay performed according tothe method previously described [12].

Fig. 1. Structures of compounds 1–6.

2.7. In vitro Sun Protection Factors (SPF) calculation

SPF, UVA PF, critical wavelength (λc) calculation wasassessed by an in vitro screening method [13] and TinosorbM was used as positive control.

3. Results and discussion

The known compounds atranorin (2, 25 g), methyl-β-orcinol carboxylate (MOC) (3, 50 mg), and methyl- and ethyl-haematommate (4, 20 mg and 5, 5 mg) and lobaric acid (6,300 mg) (Fig. 1) were identified by direct comparison of theirphysical and spectral data in the literature [5,14]. Along withthese common lichen metabolites, the unusual compound 1(Fig. 1) was obtained as a brown solid. Its molecular formulawas assigned as C25H30O9 on the basis of HRESIMS m/z473.1817 [M−H]− (calcd. for C25H29O9, 473.1817). The UVspectrum of compound 1 with absorption maxima at 213, 252and 295 nm suggested that 1 has a depside- or a depsidone-type structure [5]. Nevertheless, considering that the degrees ofunsaturation were 11, 1was postulated to be more related to adiphenyl ether such as sakisacaulon [15] than to a depsidonesuch as lobaric acid (6). IR bands suggested the presence ofhydrogen-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 1HNMR spectrum (Table 1) indicated the presence of signals forthree aromatic protons (δ 6.12, 6.55 and 6.67), one methoxygroup (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 Hmethylene proton group suggested the presence of aliphaticchains. The COSY 1H–1H NMR experiment confirmed thepresence 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 andH-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 HSQCTOCSY experiments, the butyl side chain showed methyl H-12(δ 0.90) to be correlatedwith C-11 (δ 22.44), C-10 (δ 25.16) andC-9 (δ 38.10) and the pentyl chain showed methyl H-12′

Fig. 2. Selected 2D NMR correlations for compound 1.

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(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 thebutyl side chain was revealed by anHMBC correlation betweenH-9 and C-8 andC-6 but alsowith aNOESY correlation betweenH-5 and H2-9/H2-10. The connection between C-2 and C-5′through anether linkagewas confirmed by aNOESY correlationbetween H-3 and H2-8′, H-9′ and H-10′.

In CDCl3 solution at 296 K, compound 1 was found to be amixture of two conformers as shown in NMR spectra by thebroadening of both H-5 and H-3 signals. 1H NMR spectrawere recorded at lower temperatures (203 K) in methanol-d4

Fig. 3. RMSD matrix measured for both MD simulations. Conformational space is chrright) and RMSD values increase towards the darkest points. The two most stableBoth main representative models observed all along trajectories are superimposed

and two sets of peaks in a 65/35 ratio were observable,suggesting the co-existence of two rotamers related to theether bond. Two lobarin molecular models were extractedfrom molecular dynamics [16] trajectories using a clusteranalysis (Fig. 3). These structures are representing respec-tively 40% and 30% of both first and second trajectories andcorrespond to the most stable MD conformers (Yamberinternal energy). The geometry of these two conformers(lobarin 1 and lobarin 2) diverged in their θ1/θ2 torsionangles (−90.78°/−162.57° and 99.02°/166.81°). It is worthyto notice that a spontaneous conformational change occurredin the second MD simulation from the initial conformation(form 3 in Fig. 3) to a most favorable state (lobarin 2). We didnot observed the fourth putative conformer corresponding toapproximately−θ1/−θ2 torsion angles of the less stable form3. Based on molecular dynamics calculations, lobarin in MeOHwas predicted to present a conformational equilibrium be-tween forms 1 and 2 whichwas in accordance to NMR records.Moreover, by comparison of inter-proton distance recordinguponMD simulation andNOESY spectrumanalysis, it appearedthat the main conformer present in solution should be thelobarin form 1 (Supplementary Table A). Therefore, compound1 was determined to be a diphenyl ether, identified as lobarinand has two stable conformers (forms 1 and 2).

Diphenyl ethers are not so common in lichens compared todepsides anddepsidones andmost of themhave the ether bondjoined at themeta-position of the B-ring. Consequently, they areoften related to depsidones [17,18]. Two pseudodepsidoneshave been reported from Stereocaulon alpinum collected inpolar regions, one being distinguished from compound 1 by the8-OCH3 substitution [19]. The co-occurrence of a depsidonehaving a carbonyl group inα-position of the side chain linked toring A and its related diphenyl ether with a lactol group hasbeen also reported for loxodin and norlobaridone [20,21]. Somediphenyl ethers have been formed during extraction [22,23] orby treatment of depsidones using hot alkali as described forlobariol carboxylic acid or lobarin from lobaric acid [24,25]. Inorder to check that 1 was not an artifact of isolation, theextraction process using n-hexane then EtOAc, was conducted

onologically represented along the square's diagonal (from bottom left to topmolecular models built from MD cluster analysis are shown in blue and red(with the six-membered cycle as reference) in the middle caption.

.

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again on a new lichen sample and at the same timewith lobaricacid, at room temperature and under hot temperature condi-tions (50 °C). Extracts and products obtained were thenanalyzed by TLC. Extracts were found to have the same profileat both temperature conditions with a spot unambiguouslycorresponding to 1 (Rf=0.44, (G)),while lobaric acid remainedunchanged under these conditions. It is therefore concludedthat lobarin is a natural metabolite of this lichen. Based onpathways already described [20,26], the diphenyl ether lobarin(1) is thought to result from the ring opening of the depsidonelinkage of lobaric acid through hydrolysis, followed by anucleophilic attack and a prototropic arrangement (Supple-mentary Fig. A) but without decarboxylation or O-methylationas described for the biosynthesis of loxodin and norlobaridone,respectively [26].

The compounds 1–3 and 6 were tested for radical-scavenging activity against superoxide anion (O2

−•) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals along with theircytotoxic activity against B16 murine melanoma and HaCaThuman keratinocyte cell lines (Table 2). No compounds wereactive on DPPH assay and this lack of activitymay be explainedby hydrogen bonds between aldehyde and hydroxyl groupsbut also by steric hindrance [27,28]. Compound 1, was an activesuperoxide anion scavenger three timesmore efficient than thecontrol (ascorbic acid) while atranorin was within the range ofthe control. This activity was all the more remarkable in thatno cytotoxicity was observed on the two tested cell lines.Compound 1 obtained from lobaric acid was recently patentedwith regard to its remarkable effect on protein tyrosinephosphatase-1b (PTP-1b) suggesting a possible use as a drugto treat diabetes or obesity [23]. Whatever some valuableSPF values, atranorin 2 is found to have the better profilefrom the tested compounds but do not reach the require-ments to be developed as a UV blocker.

Acknowledgments

Wegratefully acknowledge the FrenchMinistry of Researchand Education (Bio-Asia Program, DREIC [International Re-lations and Cooperation Department]) and the French Embassyin Indonesia for a Ph.D. grant to Friardi Ismed. Thanks alsoto Prof. Philippe Uriac and Prof. Sophie Tomasi for fruitfuldiscussion of lichen biogenesis. We also thank Isabelle Rouaudand Solenn Ferron for the biological assays andNova Syafni andthe Sumatran Biota Laboratory team for their help in collecting

Table 2Antioxidant, cytotoxic activities and UV blocker properties of 1–3 and 6.

Compounds Antioxidant activities Cytotoxic

DPPH assay% activity at 3000 μM

NBT assayIC50±SD (μM)

B16IC50±SD (

1 17±3 9±1 >1002 0 38±8 >1003 0 0 63±36 0 78±3 38±0.1Ascorbic acida 71±3 35±1 –

Doxorubicinb – – 0.1±0.05Tinosorb Mc – – –

a Antioxidant positive control.b Cytotoxic positive control.c UV filter positive control.

the lichens used in this study. We are grateful to P. Jéhan, F.Lambert andN. Le Yondre, CRMPO, Rennes, France, for themassspectrometer measurements.

Appendix A. Supplementary data

Supplementary data to this article can be found online athttp://dx.doi.org/10.1016/j.fitote.2012.09.025.

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