Biologically Active Eremophilane-Type Sesquiterpenes from

Note pubs.acs.org/jnp

Biologically Active Eremophilane-Type Sesquiterpenes from Camarops sp., an Endophytic Fungus Isolated from Alibertia macrophylla Juliana R. Gubiani,† Maria L. Zeraik,† Camila M. Oliveira,‡ Valdecir F. Ximenes,§ Cláudio R. Nogueira,† Luiz M. Fonseca,⊥ Dulce H. S. Silva,† Vanderlan S. Bolzani,*,† and Angela R. Araujo*,† †

Departamento de Química Orgânica, NuBBE - Núcleo de Bioensaios, Biossíntese e Ecofisiologia de Produtos Naturais, Instituto de Química, Universidade Estadual Paulista, Rua Professor Francisco Degni, 55, 14800-900, Araraquara, São Paulo, Brazil ‡ Instituto de Ciências Exatas e Tecnologia, Universidade Federal do Amazonas, Rua Nossa Senhora do Rosário, 3863, Itacoatiara, Amazonas, Brazil § Departamento de Química, Faculdade de Ciências, UNESP, Universidade Estadual Paulista, Avenida Eng. Luiz Edmundo Carrijo Coube, 14-01, Bauru-SP, 17033-360, Brazil ⊥ Departamento de Análises Clinicas, Faculdade de Ciências Farmacêuticas de Araraquara, UNESP, Universidade Estadual Paulista, Rodovia Araraquara - Jaú Km 1, 14801-902, Araraquara-SP, Brazil S Supporting Information *

ABSTRACT: Two new eremophilane-type sesquiterpenes, xylarenones F (3) and G (4), have been isolated from solid substrate cultures of a Camarops sp. endophytic fungus isolated from Alibertia macrophylla, together with the known compounds xylarenones C (1) and D (2). The structures and relative configurations of 1−4 were elucidated by extensive NMR and HRESIMS spectroscopic analysis. Due to their effects on the respiratory burst of neutrophils, which included inhibition of the reactive oxygen species production, these sesquiterpenes exhibited potential anti-inflammatory and antioxidant properties.

E

Here, we describe the isolation, structure, and biological (antioxidant, anti-inflammatory, and cytotoxicity) activity of two new eremophilane sesquiterpenes, xylarenones F (3) and G (4), together with those of the known compounds xylarenones C (1) and D (2). An investigation of a CH3CN extract obtained from the cultures of Camarops sp. (a strain that had previously been isolated from healthy leaves of A. macrophylla)5 in corn led to the isolation of four eremophilane sesquiterpenes. The structures of the compounds were elucidated based on spectrometric and spectroscopic data (IR, UV, MS, and 1H and 13C NMR) and on a comparison of the data with previously reported NMR data.5 The compounds were identified as xylarenone C (1), xylarenone D (2), and two new compounds, namely, xylarenone F (3) and xylarenone G

ndophytes, fungi that grow in the inter- or intracellular spaces of higher plants and cause no apparent infections, are recognized as one of the most chemically promising groups of fungi in terms of diversity and pharmaceutical potential.1,2 Camarops sp. (Ascomycota: Boliniales, Boliniaceae) are typically wood-inhabiting endophytic fungi and are found in both temperate and tropical regions.3,4 A preliminary study on Camarops sp. fungus isolated from the leaves of Alibertia macrophylla (Rubiaceae) resulted in the isolation of three new eremophilane sesquiterpenes, which showed potent inhibitory activity against the protease pepsin.5 Eremophilane-type sesquiterpenes are well known to occur in both higher plants and fungi and belong to a small group of natural products with broad pharmaceutical applications.6 These compounds exhibit phytotoxic potential and mycotoxic, phytohormonal, and carcinostatic activities in addition to enzyme inhibition and antibacterial and cytotoxic activity.7,8 As part of ongoing research on new bioactive metabolites produced by endophytic fungi, two new eremophilane sesquiterpenes were isolated from an extract of Camarops sp. © 2014 American Chemical Society and American Society of Pharmacognosy

Special Issue: Special Issue in Honor of Otto Sticher Received: October 2, 2013 Published: March 4, 2014 668

dx.doi.org/10.1021/np400825s | J. Nat. Prod. 2014, 77, 668−672

Journal of Natural Products

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Table 1. 1H and 13C NMR Data for Xylarenones F and G (compounds 3 and 4, respectively), at 500 and 125 MHz, Respectively

(4) (Figure 1). Xylarenones C (1) and D (2) were identified by comparing the IR, MS, UV, 1H NMR, and 13C NMR data with data in the literature.5

xylarenone F (3) position 1 2 3 4 5 6 7 8 9 10 11 12

Figure 1. Structures of compounds 1−4 isolated from the endophyte Camarops sp.

Xylarenone F (3) was isolated as an optically active, colorless oil ([α]24D +1.5, c 0.1, CH3OH). HRESIMS analysis indicated the presence of an [M + H]+ ion at m/z 451.3054, corresponding to the formula C26H42O6. The IR spectrum of 3 showed a broad band at 3464 cm−1 (νO−H) as well as bands assigned to aliphatic ester (1734 cm−1) and α,β-unsaturated carbonyl (1618 cm−1) functionalities. The ultraviolet spectrum in CH3OH presented an absorption maximum at 240 nm, which indicated the presence of an α,β,α′,β′-unsaturated ketone. 13C NMR, gHSQC, and DEPT spectra revealed 26 signals attributable to six methyl carbons, seven methylene carbons, seven methine carbons, and six nonprotonated carbons, two of which were assigned to carbonyl groups at δ 186.7 (α,β-unsaturated ketone) and δ 174.7 (ester). The spectroscopic data obtained for this compound showed the same eremophilane-type skeleton of xylarenone D (2), which had previously been isolated from Camarops sp.5 A careful and detailed analysis and comparison of the 1H and 13 C NMR spectra (Table 1) for 3 and 2 demonstrated similarity between the two compounds; the main difference observed was the absence of an epoxide between C-6/C-7 and the absence of the terminal double bond at C-11/C-12 in 3. Additionally, we observed the presence of the methyl as a doublet at δ 1.07 and a hydrogen at δ 6.75 corresponding to an α,β,α′,β′-unsaturated ketone. Analysis of all spectroscopic data, including HMBC and NOESY correlations (Figures 2 and 3), and further MS interpretation (Supporting Information) allowed us to propose that compound 3 is a new eremophilane sesquiterpene compound, which we termed xylarenone F. Xylarenone G (4) was also isolated as an optically active, colorless oil ([α]25D +35.3, c 0.1, CH3OH). HRESIMS analysis indicated the presence of an [M + H]+ ion at m/z 451.3062, corresponding to the formula C26H42O6. Detailed analysis and comparison of the 1H and 13C NMR spectra (Table 1) of 4 with those of 3 demonstrated similarity between the two compounds; the main difference observed was the multiplicity observed for H-13. In compound 3, H-13 presented as a double doublet (dd), whereas in compound 4 H13 presented as a doublet. This difference in multiplicity suggests that these compounds differ in the stereogenic C-11, which would be related to isomers R and S. An explanation for H-13a and H-13b to be a dd, J = 10.6; 6.6 in compound 3 could be due to the hydrogen bond between the hydroxyl group and

δC 76.8, CH 31.4, 25.6, 35.3, 43.2, 151.8, 139.1, 186.7, 129.1, 158.8, 41.0, 16.1,

CH2 CH2 CH C CH C CO CH C CH CH3

δH (J in Hz) 5.55 tl (2.5) 2.05 m 1.50 m 2.96 m 6.75 s

6.28 s 1.51 m 1.07 d (7.0)

xylarenone G (4) δC 77.1, CH 31.7, 25.8, 35.6, 43.5, 152.2, 139.3, 186.9, 129.3, 159.1, 41.3, 16.3,

CH2 CH2 CH C CH C CO CH C CH CH3

13

67.2, CH2

14 15

19.8, CH3 15.6, CH3

1′ 2′ 3′

174.7, CO 78.9, C 41.7, CH2

4′ 5′

26.7, CH 45.8, CH2

6′ 7′ 8′

32.0, CH 28.6, CH2 11.1, CH3

1.40 1.40 1.56 0.83 0.86 2.05 1.23 0.76

9′

18.0, CH3

0.76 d (7.5)

18.2, CH3

10′

21.5, CH3

0.90 d (6.5)

21.8, CH3

11′

68.2, CH2

3.45 d (11.0)

68.5, CH2

3.58 dd (10.6, 6.6); 3.61 dd (10.6, 5.2) 1.19 s 1.06 d (7.5)

m m m m m m s t (7.0)

3.45 d (11.0)

67.3, CH2 20.1, CH3 15.9, CH3 175.0, CO 79.1, C 42.0, CH2 26.9, CH 46.0, CH2 32.4, CH 28.8, CH2 11.3, CH3

δH (J in Hz) 5.55 tl (2.7) 2.06 m 1.52 m 2.94 m 6.77 s

6.28 s 1.52 m 1.07 d (6.5) 3.53 d (6.0) 1.18 s 1.06 d (7.0)

1.62 m 1.58 m 1.54 m 1.38 m 1.38 m 2.06 m 1.23 s 0.76 t (7.0) 0.79 d (7.0) 0.90 d (6.0) 3.60 d (11.0) 3.60 d (11.0)

Figure 2. Key HMBC correlations (H−C) for compounds 3 and 4.

carbonyl group, which may cause an interruption of the free rotation of C-11. To confirm this assumption, HPLC analyses were performed, and compounds 3 and 4 exhibited distinct retention times (tR). The stereoisomer 3 showed tR = 35.4 min, and 4 presented tR = 29.8, which was confirmed by the co-injection of both 669



Note

result shows that the inhibitory effect produced by compounds 1−4 (Figure 4) was not mediated by cell death; that is, these effects were not the result of a cytotoxic effect on neutrophils. Compounds 1−4 were also tested as scavengers of superoxide anions (the first ROS produced via the NADPH oxidase complex by stimulated neutrophils), HOCl (the main strong oxidant produced by myeloperoxidase (MPO)), and MPO enzymatic activity, but the compounds were inactive and had IC50 values of >100 μM. Our findings show that the effect of 1−4 on the inhibition of respiratory burst was not due to direct ROS scavenging or the inhibition of MPO chlorinating activity but, rather, to NADPH oxidase inhibition. The effect of the xylarenones on NADPH oxidase inhibition in neutrophils affects the production of HOCl by these cells, which might be helpful in preventing oxidative damage because the deleterious effects of HOCl on biomolecules are correlated with tissue injury in several pathologies, including atherosclerosis, rheumatoid arthritis, and some inflammatory cancers.15−17

Figure 3. Selected NOEs observed in the NOESY-2D experiment of 3 and 4.

compounds. The higher tR for stereoisomer 3 corroborates our assumption, and these experimental features allowed us to propose that compound 4 is also a new eremophilane sesquiterpene, named xylarenone G. The further relative configurations of 3 and 4 were determined by analyzing 2D-NOESY spectra, which verified the presence of interactions between methyl H-15 and H-6 and H-14; these finding indicate that these hydrogens are in a cis ratio based on a comparison with data reported for compounds 1 and 2,5 whereas the relative configuration of C-1 was determined based on multiplicity and the coupling constant observed for H-1 (tl; 2.5) and the NOE between H-1 and H-9. Thus, the relative configuration of 3 was established as (1R*, 4S*, 5S*), and the relative configuration of 4 was established as (1R*, 4S*, 5S*), which is consistent with the relative configurations established for other eremophilane-type sesquiterpenes that have been obtained from endophytic fungi.9−11 Xylarenones 1−4 were identified as potent inhibitors of reactive oxygen species (ROS) produced by stimulated neutrophils, acting in a concentration-dependent manner (Figure 4). The inhibitory concentrations of 1 (IC50 = 6.13

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EXPERIMENTAL SECTION

General Experimental Procedures. 1H NMR (500 MHz), 13C NMR (126 MHz), HMBC, HMQC, and COSY experiments were recorded on a Varian DRX-500 spectrometer using the residual nondeuterated (CDCl3) signal as an internal standard. Mass spectra were measured using a Q-TOF Micromass spectrometer set to ESI mode and using MeOH−H2O (1:1) as the solvent (cone voltage 25 V). IR spectra were obtained using a Perkin-Elmer FTIR-1600 series spectrometer with the aid of KBr pellets. Optical rotations were measured using a Perkin-Elmer polarimeter equipped with a sodium lamp operating at 28 °C and a sample cell volume of 1 mL (MeOH). TLC analysis was performed using Merck silica gel 60 (230 mesh) and precoated silica gel 60 PF254. Spots on the TLC plates were visualized either under UV light or by spraying with anisaldehyde−H2SO4 reagent followed by heating at 120 °C. Preparative HPLC was performed on a Varian Prep-Star 400 system using a Phenomenex C18 (250 mm × 21.2 mm) preparative column. Analytical HPLC was performed on a Varian Pro Star 230 system using a Phenomenex C18 column (250 mm × 4.6 mm). Column chromatography (CC) was performed over reversed-phase silica gel, 230−400 mesh (Merck). Fluorescence bioassay data were collected using a SynergyTM HT Multidetection microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) equipped with 360 nm excitation and 460 nm emission filters and analyzed using KC4 software (BioTek Instruments) and Microsoft Windows XP. IC50 values were calculated using nonlinear regression fit analysis in GraphPad Prism 4 software. Zymosan, Histopaque, taurine, catalase (EC 1.11.1.6), luminol (5amino-2,3-dihydro-1,4-phthalazinedione), calcium chloride, magnesium chloride, glucose, and 5,5′-tetramethylbenzidine (TMB) were purchased from Sigma−Aldrich Chemical Co. (St. Louis, MO, USA). 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). MPO (EC 1.11.1.7) was purchased from Planta Natural Products (Vienna, Austria), and its concentration was determined based on its absorption at 430 nm (ε = 89 000 mol−1 cm−1 per heme).18 Hypochlorous acid was prepared by diluting a concentrated commercial bleach solution and calculating its concentration based on its absorption at 292 nm (ε = 350 mol−1 cm−1).18 All solutions were prepared with water that was purified using a Milli-Q system (Millipore, Bedford, MA, USA). Plant Material. Authenticated Alibertia macrophylla K. Schum. (Rubiaceae) was collected in Estaçaõ Ecológica Experimental de MogiGuaçu, Fazenda Campininha, Mogi-Guaçu, São Paulo, Brazil, in November 2003. The identification of A. macrophylla was performed by Dr. Inês Cordeiro (Institute of Botany, São Paulo, Brazil), and a

Figure 4. Effect of xylarenones 1−4 on the inhibition of total ROS produced by active neutrophils during a respiratory burst.

± 0.41 μM), 2 (IC50 = 5.73 ± 0.42 μM), 3 (IC50 = 5.90 ± 0.70 μM), and 4 (IC50 = 4.17 ± 0.81 μM) were similar to those of quercetin (IC50 = 4.86 ± 0.36 μM) and apocynin (IC50 = 3.90 ± 0.30 μM), an efficient inhibitor of the NADPH oxidase complex.12 This potent inhibition of the respiratory burst by xylarenones might be helpful in the prevention of oxidative damage.13,14 Cytotoxicity assays using the trypan blue exclusion assay showed that compounds 1−4 were not toxic to human neutrophils at concentrations of less than 100 μM, even after 60 min. At 10 μM, the concentration used for the respiratory burst studies, the viability of the cells was greater than 98%. This 670



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MPO Inhibitory Activity. The chlorination activity of MPO was determined based on the reaction of HOCl with taurine (TauNH2) to produce taurine chloramine (TauNHCl), which oxidizes TMB to a strongly absorbing blue product.23 This product was detected spectrophotometrically at 655 nm using a microplate reader (Synergy 2 Multi-Mode, BioTek, USA) as described previously.24,25 The compound 5-fluorotryptamine was used as positive control. Antioxidant Capacity. The scavenger capacities of xylarenones (1−4) were evaluated with respect to O2•− (produced during the respiratory burst by NADPH oxidase) and the potent oxidant HOCl (a compound generated by MPO). Superoxide anion radicals generated from the xanthine/xanthine oxidase system (X/XO) were measured based on reduction of the tetrazolium salt (WST-1) to produce a soluble formazan superoxide according to the modified method reported by Tan and Berridge.26 HOCl was assayed based on the reaction of HOCl with TauNH2 to produce TauNHCl, which oxidizes TMB.26 The oxidized product was measured spectrophotometrically at 655 nm using a microplate reader (Synergy 2 MultiMode, BioTek, USA) according to the procedure described by Ximenes et al.24 Quercetin was used as positive control. Trypan Blue Exclusion Assay. The cytotoxic effect of compounds 1−4 on human neutrophils was studied using the trypan blue exclusion assay according to Kitawaga et al.21

voucher specimen was deposited at the Herbarium of the Institute of Botany of São Paulo, Brazil (voucher no. SP 370915). Fungal Isolation and Identification. The endophytic fungus Camarops sp. was isolated from adult and healthy leaves of A. macrophylla, which were subjected to surface sterilization. The leaves were first washed with water and soap and then immersed in a 1% aqueous sodium hypochlorite solution for 5 min, followed by 70% aqueous EtOH for 1 min. After a second wash with H2O and soap, the leaves were immersed in sterile H2O for 10 min. The sterilized leaves were cut into 2 × 2 cm pieces and deposited on a Petri dish containing PDA (potato-dextrose agar) and anthramycin sulfate (50 μg/mL) with approximately 3 to 4 pieces per dish.19 The pure fungal strain was obtained after serial transfers onto PDA and was deposited as Camarops sp. at the NuBBE fungi collection in Araraquara, Brazil (stored in sterile water at 25 °C).20 The pure Camarops sp. culture was sent to the Divisão de Recursos Microbianos, Centro Pluridisciplinar ́ ́ de Pesquisas Quimicas Biológicas e Agricolas, State University of Campinas (CPQBA/UNICAMP), and was classified by Drs. Lara Durães Sette and André Rodrigues.5 Fungal Growth, Extraction, and Isolation. The endophytic fungus strain Camarops sp. was cultivated in 24 Erlenmeyer flasks, each containing 90 g of corn and 80 mL of H2O. The medium was autoclaved four times (on four consecutive days) at 121 °C for 40 min. After sterilization, the medium was inoculated with the endophyte and incubated while stationary at 25 °C for 21 days. At the end of the incubation period, the cultures were combined, ground, and extracted with CH3OH (7 × 350 mL). The solvent was evaporated, yielding a crude CH3OH extract (64.9 g). The CH3OH extract was dissolved in CH3CN and defatted with hexane by liquid partitioning. The CH3CN fraction was evaporated to yield 3.3 g of crude extract. The crude CH3CN extract was fractionated by C18 RPCC and eluted with an H2O−CH3OH gradient (70:30 → 0:100) and CH3OH−EtOAc (50:50 → 0:100), affording eight fractions (Fr1−Fr8). Fraction Fr3 (0.716 mg) was fractionated by silica gel CC and eluted with an nhexane−EtOAc gradient (95:05 → 0:100), affording 14 subfractions (Fr3-A−Fr3-N). Subfraction Fr3-H was identified as containing compound 2 (73.0 mg). Subfraction Fr3-I was subjected to preparative HPLC separation using H2O−CH3CN (35:65 → 0:100 v/v, 7.5 mL min, λmax = 254 nm) as an eluent. Compounds 2 (1.6 mg, tR = 14.5 min) and 3 (2.0 mg, tR = 12.8 min) were obtained. Subfraction Fr3-J was subjected to preparative HPLC separation using H2O−CH3CN (25:75 → 0:100 v/v, 8.0 mL min, λmax = 254 nm) as an eluent. Compounds 3 (2.8 mg, tR = 15.0 min) and 4 (3.2 mg, tR = 17.6 min) were obtained. Fraction Fr4 (1.017 mg) was purified using silica gel CC with a gradient of Hex−AcOEt (95:05 → 0:100) and yielded 10 subfractions (Fr4-A−Fr4-J). Subfraction Fr4-B (367.0 mg) was submitted to silica gel CC and eluted with a gradient of n-hexane− EtOAc (95:05 → 0:100) to yield 22 subfractions (Fr4-B1−Fr4-B22). Subfraction Fr4-B3 (32.3 mg) was identified as compound 1. Xylarenone F (3): colorless oil; [α]24D +1.5 (c 0.1, CH3OH); UV λmax (MeOH)/nm 235; IR (KBr) IR (KBr) νmax/cm−1 3464 (OH), 1734 (CO), 1618 (CO); 1H NMR and 13C NMR (see Table 1); HRESIMS m/z [M + H]+ 451.2276 (calculated for C26H42O6, 451.3054). Xylarenone G (4): colorless oil; [α]24D +35.3 (c 0.1, CH3OH); UV λmax (MeOH)/nm 235; IR (KBr) νmax/cm−1 3427 (OH), 1736 (CO), 1662 (CO); 1H NMR and 13C NMR (see Table 1); HRESIMS m/z [M + H]+ 451.3062 (calculated for C26H42O6, 451.3054). Inhibitory Activity with Respect to ROS Production by Activated Neutrophils. ROS production by stimulated neutrophils was measured using luminol-enhanced chemiluminescence assays.21 Human neutrophils were isolated from blood samples taken from healthy volunteers according to a protocol approved by the University of the State of São Paulo Ethics Committee (CEP/FCF-UNESP no. 29/2011). Neutrophil isolation and the LumCL assay were performed as previously reported.21,22 For control assays (blanks), the tested compounds were replaced by the corresponding volume of DMSO, under identical conditions (value considered to be 100%). The compounds quercetin and apocynin were used as positive controls.

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ASSOCIATED CONTENT

S Supporting Information *

Selected 1H and 13C NMR, 1H−1H COSY, HSQC, and HMBC spectra of compounds 3 and 4. Selected NOEs observed in the NOESY-2D experiments of compounds 3 and 4. Mass spectra of compounds 3 and 4. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +55-16-33019658. Fax: +55-16-33227932. E-mail: [email protected]; [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by grants from the Fundaçaõ de Amparo à Pesquisa do Estado de São Paulo (FAPESP) as part of BiotaFAPESP, the Biodiversity Virtual Institute Program (www. biota.org.br), grant no. 03/02176-7, awarded to V.S.B. J.R.G., C.M.O., and M.L.Z. acknowledge FAPESP and Coordenaçaõ ́ Superior (CAPES) for de Aperfeiçoamento de Pessoal de Nivel scholarships.

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DEDICATION Dedicated to Prof. Dr. Otto Sticher, of ETH-Zurich, Zurich, Switzerland, for his pioneering work in pharmacognosy and phytochemistry.

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REFERENCES

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Biologically Active Eremophilane-Type Sesquiterpenes from

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