Turmeric Sesquiterpenoids: Expeditious Resolution, Comparative

Article pubs.acs.org/jnp

Turmeric Sesquiterpenoids: Expeditious Resolution, Comparative Bioactivity, and a New Bicyclic Turmeronoid Danilo Del Prete,† Estrella Millán,‡ Federica Pollastro,† Giuseppina Chianese,§ Paolo Luciano,§ Juan A. Collado,§ Eduardo Munoz,‡ Giovanni Appendino,*,† and Orazio Taglialatela-Scafati*,§ †

Dipartimento di Scienze del Farmaco, Università del Piemonte Orientale, Via Bovio 6, 28100, Novara, Italy Maimonides Biomedical Research Institute of Córdoba, Reina Sofía University Hospital, Department of Cell Biology, Physiology and Immunology, University of Córdoba, Avenida Menéndez Pidal s/n, 14004, Córdoba, Spain § Dipartimento di Farmacia, Università di Napoli Federico II, Via Montesano 49, 80131, Napoli, Italy ‡

S Supporting Information *

ABSTRACT: An expeditious strategy to resolve turmerone, the lipophilic anti-inflammatory principle of turmeric (Curcuma longa), into its individual bisabolane constituents (ar-, α-, and β-turmerones, 2−4, respectively) was developed. The comparative evaluation of these compounds against a series of anti-inflammatory targets (NF-κB, STAT3, Nrf2, HIF-1α) evidenced surprising differences, providing a possible explanation for the contrasting data on the activity of turmeric oil. Differences were also evidenced in the profile of more polar bisabolanes between the Indian and the Javanese samples used to obtain turmerone, and a novel hydroxylated bicyclobisabolane ketol (bicycloturmeronol, 8) was obtained from a Javanese sample of turmeric. Taken together, these data support the view that bisabolane sesquiterpenes represent an important taxonomic marker for turmeric and an interesting class of antiinflammatory agents, whose strict structure−activity relationships are worth a systematic evaluation.

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he anti-inflammatory activity of turmeric (Curcuma longa L.) is traditionally associated with the presence of curcumin, a mixture of three nonvolatile diarylheptanoids (1a−c),1 and of turmerone, a mixture of three volatile bisabolane sesquiterpene ketones (2−4).2 Curcumin and turmerone have been developed as dietary ingredients and food additives, both as stand-alone ingredients and in combination, and turmerone has also found use in cosmetics as an inducer of antioxidant response and as a whitening agent.3 There is growing evidence that the three constituents of curcumin show a distinct profile of anti-inflammatory activity,4 oral absorption,5 and stability at physiological pH values.6 Conversely, the three constituents of turmerone [ar-, α-, and βturmerone (curlone), 2−4, respectively] have not yet been individually investigated in terms of bioactivity. The clarification of this issue is of relevance to fill the gap between the dietary consumption of turmeric and the use of its purified constituents as healthfood ingredients. Thus, the ratio between the three diarylheptanoids of curcumin is markedly different between turmeric and commercial curcumin, since the concentration of the demethoxylated analogues 1b and 1c is significantly decreased during purification.7 On the other hand, steam distillation of turmeric to obtain turmerone partially degrades and/or aromatizes the dienic constituents α-and βturmerones (3 and 4, respectively), emphasizing the contents of ar-turmerone.8 © XXXX American Chemical Society and American Society of Pharmacognosy

The two symmetrical constituents of curcumin (1a and 1c) are readily available from the Pabon condensation of the boric complex of 2,4-pentandione and their corresponding benzaldehyde derivatives (vanillin for 1a, 4-hydroxybenzaldehyde for 1c).9 Conversely, the resolution of turmerone into its individual constituents is more complex and involves a series of tedious chromatographic separations, difficult to scale up and impractical to provide sufficient material for structure−activity studies.10 To fill this gap, we have capitalized on the different chemistry of the three constituents of turmerone to develop a Received: July 21, 2015

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DOI: 10.1021/acs.jnatprod.5b00637 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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scalable protocol for their separation and on the availability of single constituents to comparatively evaluate their activity against a series of anti-inflammatory targets. During the isolation of turmerone from commercial samples of turmeric of various geographical origin, differences were noticed in the profile of polar turmeronoids, and, as a corollary to the main aim of the study, we report the structure elucidation of bicycloturmeronol (8), a novel cyclobisabolane ketol obtained from a Javanese sample of the spice.

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RESULTS AND DISCUSSION Turmerone (2−4) was obtained by hexane extraction of commercial turmeric powder of Indian origin and gravity column chromatography on silica gel of the resulting extract. A 3.5:1:1.5 ratio between its three constituents (α-, β-, and arturmerones) was estimated by integration of diagnostic 1H NMR signals (the olefin protons of α- and β-turmerones and H-10, overlapped in all three compounds). Turmerone was partially resolved by gravity column chromatography on silica gel or by flash chromatography on RP18 silica gel into a less polar fraction containing ar-turmerone (2) and α-turmerone (3) and a more polar one corresponding to a mixture of α- and β-turmerones (3 and 4, respectively). HPLC could separate arturmerone (2) from α-turmerone, but the resolution of α- and β-turmerones was not satisfactory, in accordance with previous reports.10 Clearly, chromatography alone was not suitable to obtain pure turmerones in amounts sufficient to sustain structure−activity studies. Therefore, we decided to resolve turmerone by exploiting differences in reactivity, since arturmerone is a phenyl derivative, α-turmerone an s-cis diene, and β-turmerone an s-trans diene. Treatment of turmerone with an excess of MCPBA (metachloroperoxybenzoic acid) converted the dienic constituents (α- and β-turmerones, 3 and 4) into a complex mixture of polar (di)epoxides, leaving ar-turmerone (2) unscathed and easy to purify from the reaction mixture. Although plagued by the loss of the dienic constituents, this method is quick and can be easily scaled up, competing well with the published purification protocols to obtain ar-turmerone from turmerone.11 Notably, even with a large excess of MCPBA, the reaction was not plagued by Baeyer−Villiger oxidation of the methylene ketone moiety of ar-turmerone, at least when run at room temperature. The high reactivity of turmerone with peracids has been investigated as a model reaction for its antioxidant activity.12 However, the reaction is associated only with the dienic constituents of the mixture and is therefore of little general relevance, since the composition of turmerone is critically dependent on the nature of the starting plant biomass.8 The major issue in the chromatographic resolution of turmerone is the overlapping of α-turmerone (3) with arturmerone (2) and β-turmerone (4).4 Removal of 3 from the mixture is therefore expected to lead to a chromatographically more manageable mixture of 2 and 4. To this aim, turmerone was treated with the heterodienophile PTAD (N-phenyl-1,2,4triazolidinedione), which reversibly trapped the s-cis diene αturmerone (3) into the polar Diels−Alder adduct 5a, leaving unscathed the mixture of β- and ar-turmerones.13 These could be separated by gravity column chromatography, while αturmerone (3) was recovered by cycloreversion of its Diels− Alder adduct 5. For the large-scale purification of β-turmerone, PTAD could be replaced by the cheaper dienophile maleic anhydride, removing α-turmerone from the mixture by irreversible conversion into its maleic anhydride adduct 5b.

Scheme 1 summarizes the purification protocol for the three constituents of turmerone. While ar-turmerone and βScheme 1. Reactivity-Based Resolution Strategy of Turmeronea

a

GCC = gravity column chromatography.

turmerone are relatively stable compounds, α-turmerone is not and tends to slowly aromatize to ar-turmerone in the air. The anti-inflammatory activity of turmeric is traditionally associated with curcuminoids, but there is evidence that the plant also contains other bioactive constituents.7 In this context, there is a growing interest in turmeric oil and turmerone, but doubts in terms of efficacy and safety have been raised for the in vivo translation of the favorable cell experimental data on these compounds.14 Both turmeric oil and turmerone are mixtures, and to clarify this issue, it is therefore important to evaluate the profile of its individual constituents. To this purpose, the three purified constituents of turmerone (2, 3, and 4) were evaluated on well-defined targets involved in inflammation and in carcinogenesis. In the event, ar-turmerone (2) could inhibit both TNFα-induced NF-κB activation (Figure 1A) and γ-IFNinduced STAT3 activation (Figure 1B) with an IC50 of 22.7 ± 3.2 and 14.21 ± 4.7 μM, respectively, in accordance with previous observations.15 Conversely, its dienic analogues α- and β-turmerones (3 and 4, respectively) enhanced NF-κB activation and were ineffective in STAT3 activation. These observations explain, in part, the immunomodulatory activity of B



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Figure 1. Effects of turmerones on NF-κB and STAT3 activation. (A) NIF-3T3-KBF-Luc fibroblasts (15 × 104 cells/mL) preincubated with increasing concentrations of the compounds and next treated with TNFα (30 ng/mL) for 6 h. Luciferase activity was measured in the cell lysates, and results are represented as the percentage of inhibition considering 100% the value of TNFα-induced NF-κB activation. (B) HeLa-STAT3-Luc cells (15 × 104 cells/mL) preincubated with increasing concentrations of the extracts and next treated with IFNγ (50 U/ml) for 6 h. Luciferase activity was measured in the cell lysates, and results are represented as the percentage of inhibition considering 100% the value of IFNγ-induced STAT3 activation. (C) NIF-3T3-KBF-Luc cells were treated for 15 min with either ar-turmerone or the IKKβ inhibitor SC514 at the indicated concentrations and stimulated with TNFα (30 ng/mL) for 30 min. Cells were lysed and analyzed for the levels of endogenous protein expression by immunoblot.

Figure 2. Effects of turmerones on Nrf2 and HIF-1α activation. (A) HaCaT-ARE-Luc cells (15 × 104 cells/mL) treated with increasing concentrations of each compound for 6 h. Luciferase activity was measured in the cell lysates, and results are represented as the fold induction over basal levels. (B) NIH-3T3-EPO-Luc cells (15 × 104 cells/mL) preincubated with increasing concentrations of compounds and next treated with the hypoximimetic DFX for 6 h. Luciferase activity was measured in the cell lysates, and results are represented as the fold induction over basal levels.

turmeric oil16 as well as the contrasting data on its antiinflammatory activity,14 which should critically depend on the ratio between ar-turmerone and its dienic analogues. Next, we investigated the effect of ar-turmerone (2) on the canonical

pathway of NF-κB activation by analyzing the steady-state levels of phosphorylated IκBα and p65 (a subunit of the more common form of NF-κB heterodimers). Both IκBα and p65 proteins are phosphorylated by the IκB kinase β (IKKβ), which C



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Figure 3. (Left) COSY (bold) and HMBC (arrows) correlations for bicycloturmeronol. (Right) Dominant rotamer around the C-7/C-8 bond with the corresponding Murata parameters and the observed NOE correlations.

bonded hydrogen atom. One sp3 and three sp2 unprotonated carbon atoms, including a ketone carbonyl at δC 201.0, complete the carbon count implied by the molecular formula. This preliminary NMR investigation was indicative of the bicyclic sesquiterpenoid nature of 8, whose planar structure was then built up based on the combined inspection of 2D NMR COSY and HMBC spectra. The first experiment defined the three short spin systems, shown in bold in Figure 2, while the second was instrumental in merging the different substructures. Thus, the HMBC correlations of the allylic methyls Me-12 and Me-13 with C-11 and C-10, as well as those of H-10 with the ketone carbonyl C-9 and with the oxymethine C-8, indicated the linking of the dimethyl-α,β-unsaturated ketone at C-8. A bicyclo[3.1.0]hex-2-ene system was built on the basis of the network of HMBC correlations shown by H-6 and by the allylic Me-15 and H2-3 (see Figure 3). The presence of a cyclopropane ring was further supported by the high-field resonances of H2-5 (δH 0.88 and 0.20). Finally, the HMBC correlations H2-3/C-7 and Me-14/C-4 connected the side chain to the quaternary carbon C-4, eventually defining the planar structure of bicycloturmeronol (8). Bicycloturmeronol (8) is a new member of the class of sesquithujenes, first obtained as a volatile from ginger (Zingiber of f icinale) essential oil.22 A terpene synthase converting farnesyl pyrophosphate in sesquithujene has been recenty characterized in Zea mays.23 Configurational elucidation at the four adjacent stereogenic carbons of 8 (C-6, C-4, C-7, and C-8) was complicated by the limited amount of material available and by the occurrence of isomeric stereoparents within natural sesquiterpenoids. Along with sesquithujenes, epimers at C-7 (7-episesquithujenes) and at C-4 and C-6 (sesquisabinenes) have also been reported. In the event, the relative configuration at C-7/C-8 could be assigned using the NMR-based Murata method.24 Thus, the small value of the coupling constant 3 JH‑7/H‑8 (2.6 Hz) suggested the presence of a dominant rotamer with two synclinal protons around the C-7/C-8 bond. Next, the required heteronuclear coupling constants 2JC,H (H7/C-8) and 3JC,H (H-8/C-4; H-8/C-14; H-7/C-9) were qualitatively determined through the phase-sensitive PSHMBC spectrum. The resulting pattern of J data was unambiguously indicative of an erythro relationship between the two stereogenic carbons, further supported by the NOE interactions shown in Figure 1. Because of the lack of vicinal protons, the Murata method could not be used to establish the relative configuration around the C-4/C-7 bond. To this aim, we capitalized on an alternative computational approach based on a quantum-mechanical prediction of 13C NMR chemical shifts.25 The conformational behavior around the C-4/C-7 bond was explored in terms of the dihedral angle (θ) C-6/C-4/C-7/C-8 for the two possible

is activated by TNFα through the so-called canonical pathway.17 As depicted in Figure 1C, ar-turmerone (2) clearly inhibited the phosphorylation of both IκBα and p65 induced by TNFα in NIH-3T3-KBF-Luc cells. The inhibitory activity of arturmerone was not due to cytotoxic effects since none of the investigated compounds at the concentrations tested showed cytotoxicity in any of the cell lines used in this study (see the Supporting Information). Marked differences in the activity of turmeronoids were also observed in terms of activation of the antioxidant Nrf2 pathway, a major target of curcumin,9b,18 since, while 2 and 3 were inactive, β-turmerone potently activated Nrf2 (Figure 2A). To complete the evaluation of the anti-inflammatory profile, the turmeronoids 2−4 were assayed for the capacity to inhibit the activiation of HIF-1α, a member of the hypoxia-inducible transcription factors (HIFs) that plays a major role in the proinflammatory milieu in tissues.19 α-Turmerone (3) could inhibit the activation of the erythropoietin gene promoter induced by the hypoximimetic agent desferrioxamine (DFX), a surogate marker of HIF-1α activation (Figure 2B), but 2 and 4 were inactive in this assay. Taken together, these observations show that the three constituents of turmerone have a different, and sometimes contrasting, biological profile, making it difficult to predict its overall in vivo activity. In particular, differences should be marked between turmeric oil obtained from fresh rhizomes, rich in the immonomodulantory agent α-turmerone (3), and the one obtained from processed rhizomes, enriched in the anti-inflammatory agent ar-turmerone (2).8 We also obtained turmerone from a Javanese sample of fresh turmeric rhizomes. The differences in the composition of turmerone were those expected between fresh and processed turmeric rhizomes, namely, a higher content of the dienic constituents in turmerone from fresh rhizomes.8 Differences were, however, also observed in the profile of the more polar turmeronoids. Thus, the Indian sample contained the known hydroxylated bisabolane ketones 6 (turmeronol),20 7a (arturmeronol-A),21 and 7b (ar-turmeronol-B),21 while the Javanese sample lacked 7a and 7b and also contained the ketol 8, a novel compound that we have named bicycloturmeronol. The molecular formula of bicycloturmeronol (8) was established as C15H22O2 by HR-ESIMS, corresponding to five degrees of unsaturation. Its well-resolved 1H NMR spectrum (CDCl3) included three methyl singlets (δH 1.74, 1.93, and 2.19), one methyl doublet (δH 0.79), and the resonances of the remaining 10 protons, located between δH 0.20 and 6.02. 2D NMR HSQC experiment sorted out these protons into two sp2 methines, three sp3 methines, including one oxymethine (δC 79.8), and two sp3 methylenes. The doublet at δH 3.57, showing no HSQC correlations, could be attributed to an oxygenD



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Figure 4. (Left) Structure of the diastereomers 8a and 8b. (Middle) Dominant rotamer of 8b. (Right) Experimental ECD curve of bicycloturmeronol (black) and calculated EDC curves for 8b (red) and its enantiomer (blue).

suggests that even within the most common turmerone chemotype differences might exist or, at least, that the effect of processing on the composition of the polar bisabolane fraction should be systematically investigated. Taken together, our data show that turmerone, easily obtained from commercial and inexpensive turmeric powder, can be conveniently resolved into its constituents, providing functionalized bisabolanes for further studies.

diastereomers 8a and 8b (Figure 4) through a DFT (density functional theory) calculation (Gaussian03 program). This systematic search afforded 15 rotamers for each stereoisomer, which were geometrically optimized at the DFT level using a B3LYP functional and 6-31G(d) basis set. The relative energies of all conformations were calculated, and the equilibrium roomtemperature Boltzmann populations were obtained. Structure 8a was characterized by a dominant rotamer (86% of total population, θ = −160°), while three rotamers were significantly populated in 8b, with the one with θ = −75° (Figure 3) accounting for 65% of the population. 13 C NMR chemical shifts were next calculated for each reasonably populated conformer at the same level with the GIAO (gauge including atomic orbitals) option using the mPW1PW91/6-31G(d,p) DFT method (see the Supporting Information). Using the ab initio standard free energies as weighting factors, a Boltzmann average of 13C NMR chemical shifts for any given carbon atom was independently calculated for the pair of diastereomers. Since the computed chemical shifts for 8b matched the experimental values of 8 better than those of 8a (corrected mean absolute errors were 2.13 for 8b vs 3.47 for 8a), the relative configuration of 8b was assigned to bicycloturmeronol. To upgrade this relative configuration to the level of absolute configuration, the ECD spectrum of 8b was simulated. The excitation energies as well as the oscillator and rotatory strengths of the electronic excitation were calculated using the TDDFT methodology.26 The theoretical curve of 8b closely resembled the experimental one (Figure 4), allowing a confident assignment of absolute configuration to the natural product. Notably, the configuration at C-7 of bicycloturmeronol was found to be identical to that of the co-occurring turmerones. Bicycloturmeronol is one of the few bicyclobisabolanes whose relative and absolute configuration has been established in an unambiguous way. Both the spectroscopic data and the approach we have outlined will be of general relevance for the stereochemical assignment of this class of compounds, which encompasses three distinct stereoparents (sesquithujenes, 7episesquithujenes, and sesquisabinenes) difficult to distinguish on the basis of 1D NMR data alone. The taxonomic relevance of the isolation of 8 is unclear, since this compound was obtained from a fresh sample of turmeric purchased in loco (Jakarta, Indonesia), while the other polar analogues came from the processed (boiled and powdered) Indian turmeric usually available in Europe. Marked differences have been documented within the composition of the essential oil of turmeric, even within the plant of Indian origin, with five distinct chemotypes, all containing curcumin, having been characterized (turmerone, α-phellandrene, bisabolene, carvacrol, and curzerene types).27 Our observation

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations (CHCl3) were measured at 589 nm on a P2000 Jasco polarimeter. 1H (700 MHz) and 13C (175 MHz) NMR spectra were measured on a Varian INOVA spectrometer. Chemical shifts were referenced to the residual solvent signal (CDCl3: δH = 7.26, δC = 77.0). Homonuclear 1H connectivities were determined by the COSY experiment. One-bond heteronuclear 1H−13C connectivities were determined with the HSQC experiment. Through-space 1H connectivities were evidenced using a ROESY experiment with a mixing time of 250 ms. Two- and threebond 1H−13C connectivities were determined by gradient 2D HMBC experiments optimized for a 2,3J = 9 Hz. The PS-HMBC spectrum was recorded using 2K points in ω2, setting the delay for long-range coupling evolution (Δ) at 50 ms, with 32 scans/t1 (t1max 15.2 ms). Zero-filling (8 K × 1 K) was carried out in ω2 and ω1, respectively, to obtain a digital resolution of 0.9 Hz in ω2. Low- and high-resolution ESIMS spectra were obtained on a LTQ OrbitrapXL (Thermo Scientific) mass spectrometer. Silica gel 60 (70−230 mesh) used for gravity column chromatography (GCC) was purchased from Macherey-Nagel. Reactions were monitored by TLC on Merck 60 F254 (0.25 mm) plates, which were visualized by UV inspection and/ or staining with 5% H2SO4 in ethanol and heating. Organic phases were dried with Na2SO4 before evaporation. Chemical reagents and solvents were from Aldrich. For the sake of simplicity, the molecular weight of turmerone was calculated as that of ar-turmerone, its major constituent, in all stoichiometric calculations. Plant Material. Turmeric rhizome powder of Indian origin was purchased from A. Minardi & Figli (Bagnacavallo, RA, Italy, batch D140114130214, year of harvest: 2013). Fresh turmeric rhizomes were purchased in Jakarta at a local market in March 2014. Voucher specimens (number 01/15 for the powder and 02/15 for the frozen rhizomes) are kept at the laboratories of Novara. Isolation of Turmerone. (a) Indian sample: Powdered turmeric (800 g) was extracted with petroleum ether at room temperature (rt) (2 × 7.5 L). The pooled extracts were evaporated to afford 16.3 g (2.0%) of a pale yellow oil, which was purified by GCC on silica gel (400 g) with a petroleum ether−EtOAc gradient, taking fractions of 18 mL, and next combined according to their TLC profile (petroleum ether−EtOAc, 9:1, as eluant). Elution with petroleum ether−EtOAc, 98:2 (fractions 136−158), afforded 10.8 g of turmerone (1.3%) as a colorless oil (Rf 0.78, petroleum ether−EtOAc, 95:5). Elution with petroleum ether−EtOAc (9:1 and 8:2) afforded the more polar turmeronoid 6a (180 mg) and an almost equimolecular mixture of 7a and 7b (110 mg), which was resolved by HPLC on silica gel (petroleum ether−EtOAc, 9:1). (b) Javanese sample: Fresh roots (180 g) were cut and then mashed with a coffe grinder. The wet paste was E



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then extracted with acetone. Evaporation of the solvent left a residue that was partitioned between water and petroleum ether. Evaporation of the organic phase gave a residue (940 mg) that was purified by GCC as described for the Indian sample, affording 510 mg of turmerone and a ca. 4:1 mixture of 6 and 8 (85 mg), which was further partially resolved by GCC on silica gel (petroleum ether−ether, 9:1, as eluant). After final HPLC purification (petroleum ether−EtOAc, 98:2), 1.2 mg of 8 was eventually obtained. Bicycloturmeronol (8): colorless, amorphous solid; [α]25D −107 (c 0.1, CHCl3); UV (CH3CN) λmax 242 (ε 9650); ECD (CH3CN, c = 1.00 × 10−3) Δε (nm) −6.00 (200), + 2.4 (261); 1H NMR (CDCl3, 700 MHz) δH 6.02 (1H, bs, H-10), 4.95 (1H, bs, H-2), 4.30 (1H, dd, J = 5.5, 2.6 Hz, H-8), 3.57 (1H, d, J = 5.5 Hz, OH-8), 2.59 (1H, bd, J = 17.4 Hz, H-3a), 2.29 (1H, bd, J = 17.4 Hz, H-3b), 2.19 (3H, s, H-12), 1.93 (3H, s, H-13), 1.74 (3H, s, H-15), 1.51 (1H, overlapped, H-7), 1.47 (1H, overlapped, H-6), 0.88 (1H, dd, J = 7.5, 3.4 Hz, H-5a), 0.79 (3H, d, J = 7.0 Hz, H-14), 0.20 (1H, t, J = 3.4 Hz, H-5b); 13C NMR (CDCl3, 125 MHz) δC 201.0 (C-9), 159.4 (C-11), 144.0 (C-1), 122.1 (C-2), 119.4 (C-10), 79.8 (C-8), 42.0 (C-7), 36.7 (C-3), 33.9 (C-6), 32.7 (C-4), 28.0 (C-13), 23.1 (C-5), 21.2 (C-12), 16.1 (C-15), 10.8 (C-14); (+) ESIMS m/z 235 [M + H]+, 257 [M + Na]+; HR-ESIMS found m/z 257.1505; C15H22O2Na requires 257.1517. ar-Turmerone (2) from Turmerone. To a solution of turmerone (500 mg, 2.31 mmol) in CH2Cl2 (20 mL) was added an excess MCPBA (77%, 1.590 g, 9.2 mmol, 4 molar equiv), following the course of the reaction by TLC (petroleum ether−EtOAc, 95:5, Rf turmerone 0.78, Rf epoxides ca. 0.10). After stirring 1 h at rt, the reaction was worked up by dilution with brine and neutralization by dropwise addition of 2 N NaOH. After dilution with CH2Cl2 and washing with saturated Na2S2O3, the organic phase was dried, filtered, and evaporated. The residue was purified by GCC in silica gel (20 g, petroleum ether−EtOAc, 95:5, as eluant) to afford 200 mg of arturmerone (2) as a colorless oil. For the spectroscopic data, see the Supporting Information. α-Turmerone (3) and β-Turmerone (4) from Turmerone. To a solution of turmerone (500 mg, 2.3 mmol) in dry THF (10 mL) was added PTAD (405 mg, 2.3 mmol, 1 molar equiv) in small portions until the development of a persistent pinkish color of unreacted PTAD. After stirring 1 h at rt, the reaction was worked up by dilution with brine and extraction with EtOAc. The organic phase was dried, filtered, and evaporated, and the residue was purified by GCC on silica gel (5 g). Elution with petroleum ether−ether, 99:1, afforded 60 mg of β-turmerone, 160 mg of ar-turmerone, and 102 mg of the Diels−Alder adduct 5 as a colorless oil. The latter was dissolved in 2.1 M KOH in ethanol (4 mL) and refluxed for 3 h. The reaction was then worked up by dilution with brine and extraction with petroleum ether. The organic phase was dried, filtered, and evaporated, and the residue was purified by GCC on silica gel (6 g, petroleum ether−EtOAc, 9:1, as eluant) to afford 30 mg of α-turmerone as a colorless oil. For spectroscopic data on α- (3) and β-turmerone (4), see the Supporting Information. Reaction of Turmerone with Maleic Anhydride. A solution of turmerone (500 mg, mmol) and maleic anhydride (75 mg, 0.765 mmol) in dry THF (10 mL) was refluxed for 24 h, monitoring the course of the reaction by 1H NMR. After cooling, the solution was diluted with brine and extracted with EtOAc. After drying and filtration, the organic phase was evaporated, and the residue purified by GCC on silica gel (30 g, petroleum ether−EtOAc as eluant) to afford 82 mg of β-turmerone, 180 mg of ar-turmerone, and 50 mg of the Diels−Alder adduct 5b. Computational Calculations. DFT calculations were performed using the Gaussian03 package (Multiprocessor). A systematic conformational search for the diastereomers 8a and 8b around the C-4/C-7 bond was carried out at the B3LYP level using the 6-31G(d) basis set (range: −170 to +170; number of conformers = 15). All the conformers obtained were subsequently optimized at the B3LYP level using the 6-31G(d,p) basis set. GIAO 13C NMR calculations were performed using the mPW1PW91 functional and 6-31G(d,p) basis set, using as input the geometry previously optimized at the mPW1PW91/ 6-31G(d) level. TDDFT calculations were run using the functional

B3LYP and the basis sets TZVP including at least 30 excited states in all cases and using IEF-PCM for MeOH. Cell Lines. NIH-3T3-KBF-Luc, NIH-3T3-EPO-Luc, HaCaT-ARELuc, and HeLa-STAT-3-Luc cell lines were constructed in our lab and were grown at 37 °C and 5% CO2 in supplemented DMEM containing 10% heat-inactivated fetal bovine serum, 2 mM glutamine, penicillin (50 U/mL), and streptomycin (50 μg/mL). The characterization of the cell lines is shown in the Supporting Information. Luciferase Assays. For the anti-NF-κB activity NIH-3T3-KBFLuc cells were stimulated with TNFα (20 ng/mL) in the presence or the absence of the compounds for 6 h. For the activation of the antioxidant response element (ARE) that is activated by Nrf2 HaCaTARE-Luc cells were stimulated with the compounds for 6 h. For the anti-STAT3 activity HeLa-STAT-3-Luc cells were stimulated with γIFN (20 IU/mL) in the presence or the absence of the compounds for 6 h. For the inhibition of DFX-induced HIF-1α activation NIH3T3-EPO-Luc cells were preincubated with the compounds and then treated with DFX (150 μM) for 6 h. After the treatment the cells were washed twice in PBS and lysed in 25 mM Tris-phosphate pH 7.8, 8 mM MgCl2, 1 mM DTT, 1% Triton X-100, and 7% glycerol during 15 min at rt in a horizontal shaker. After centrifugation, luciferase activity in the supernatant was measured using an Autolumat LB 9510 (Berthold) following the instructions of the luciferase assay kit (Promega, Madison, WI, USA). Western Blots. NIH-3T3-KBF-Luc cells were stimulated as indicated and washed with PBS, and total cell extracts were extracted in 50 μL of lysis buffer (20 mM Hepes pH 8.0, 10 mM KCl, 0.15 mM EGTA, 0.15 mM EDTA, 0.5 mM Na3VO4, 5 mM NaF, 1 mM DTT, leupeptin 1 mg/mL, pepstatin 0.5 mg/mL, aprotinin 0.5 mg/mL, and 1 mM PMSF) containing 0.5% NP-40. Cells were incubated for 15 min at 4 °C, and cellular proteins were obtained by centrifugation at 10000g for 10 min. Protein concentrations were determined by the Bradford assay (Bio-Rad, Richmond, CA, USA), and 20 μg of proteins was boiled in Laemmli buffer and electrophoresed in 10% SDS polyacrylamide gels. Separated proteins were transferred to nitrocellulose membranes (0.5 Å at 100 V; 4 °C) for 1 h. Blots were blocked in TBS solution containing 0.1% Tween 20 and 5% nonfat dry milk overnight at 4 °C, and immunodetection of specific proteins was carried out with primary antibodies using an ECL system (GE Healthcare, NJ, USA). The antiphospho-p65 (3031S) and antiphospho-IκBα (14D4) antibodies were from New England Biolabs (Hitchin, UK). The mAb anti-tubulin was purchased from Sigma Co. (St. Louis, MO, USA), and the mAb anti-p65 (sc-8008) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Statistics. Statistical analyses and IC50 calculations were performed using GraphPad Prism (Graph Pad Software, San Diego, CA, USA). Sample population means were compared against control population means in an unpaired two-tailed Student’s t test. A p value < 0.05 was considered statistically significant.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00637. 1 H and 2D NMR spectra for bicycloturmeronol (8) and NMR data for turmerones (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +39-0321375744. Fax: +39-0321375744. E-mail: [email protected] (G. Appendino). *Tel: +39-081678509. Fax: +39-081678552. E-mail: scatagli@ unina.it (O. Taglialatela-Scafati). Notes

The authors declare no competing financial interest. F



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ACKNOWLEDGMENTS E.M., J.J.C., and E.M. are grateful to MINECO (Ministerio de Economiá y Competitividad, Spain) for a grant (SAF201453763-P). Work in the laboratories of Novara was supported by the Università del Piemonte Orientale, and that in Naples by the Università di Napoli Federico II. Mass and NMR spectra were recorded at “Centro di Servizio Interdipartimentale di Analisi Strumentale”, Università di Napoli Federico II. We are grateful to Dr. A. Leeman (Mensa Group, Jakarta, Indonesia) for providing fresh rhizomes of turmeric from Java cultivations.

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REFERENCES

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