Naphthoquinones of Sinningia reitzii and Anti-inflammatory

Article pubs.acs.org/jnp

Naphthoquinones of Sinningia reitzii and Anti-inflammatory/ Antinociceptive Activities of 8‑Hydroxydehydrodunnione Adson S. Soares,† Felipe L. Barbosa,‡ André L. Rüdiger,† David L. Hughes,§ Marcos J. Salvador,⊥ Aleksander R. Zampronio,‡ and Maria Élida A. Stefanello*,† †

Departamento de Química, Universidade Federal do Paraná, 81530-900, Curitiba, PR, Brazil Departamento de Farmacologia, Universidade Federal do Paraná, 81530-970, Curitiba, PR, Brazil § School of Chemistry, University of East Anglia, Norwich NR4 7TJ, England ⊥ Departamento de Biologia Vegetal, PPG-BTPB and PPG-BV, Universidade Estadual de Campinas, Instituto de Biologia, 13083-970, Campinas, SP, Brazil ‡

S Supporting Information *

ABSTRACT: Chemical investigation of the tubers of Sinningia reitzii led to the isolation of five new naphthoquinones, 8hydroxydehydrodunnione (1), 7-hydroxydehydrodunnione (2), 5-hydroxy-6,7-dimethoxy-α-dunnione (3), 5-hydroxy-6,7-dimethoxydunniol (4), and 8-hydroxy-7-methoxy-2-O-methylstreptocarpone (5). Three known naphthoquinones, 7-hydroxy-αdunnione, 8-hydroxydunnione, and 6,8-dihydroxy-7-methoxy-2O-methyldunniol, were also identified. When tested for antiinflammatory activity in a mouse model, compound 1 (50−500 pg/paw) reduced the edema induced by carrageenan in a dosedependent fashion. The highest dose showed a similar inhibition to that observed for the positive control dexamethasone. At lower doses (5−10 pg/paw), 1 also dose dependently reduced the mechanical hyperalgesia induced by carrageenan. Compound 1 (15 pg/paw) abolished the mechanical hyperalgesia induced by prostaglandin E2 and dopamine, but not that induced by dibutyryl cyclic AMP. Dipyrone (320 μg/paw) completely abolished the hyperalgesia induced by these algogens. Additionally, compound 1 did not alter heat-induced nociception. These results suggest that this new naphthoquinone exhibits important anti-inflammatory and antinociceptive activities, which is dissimilar to that of most known analgesics.

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tubers distributed in southern and southeastern Brazil, are reported. This plant has neither a vernacular name nor use in folk medicine. In addition to the isolation and identification of eight naphthoquinones, the anti-inflamatory and antinociceptive activities of compound 1 were evaluated. Successive chromatographic fractionation of the less polar extracts from S. reitzii tubers yielded five new (1−5) and three known naphthoquinones. All compounds were analyzed by 1D and 2D NMR spectroscopy, and the data were compared with literature data. UV, IR, and MS data of the new compounds were also acquired. Additionally, X-ray diffraction data for compound 1 were obtained. The anti-inflammatory and antinociceptive activities of 1 in standard animal models of inflammation and nociception were evaluated, and its mechanism of action was investigated.9,13−16

aphthoquinones are a large class of secondary metabolites found in fungi, bacteria, and higher plants. They are structurally diverse and display several biological activities, such as cytotoxic, antitumoral, antibacterial, fungicidal, antiparasitic, insecticidal, and anti-inflammatory.1−3 Naphthoquinones have been reported from the family Gesneriaceae in Chirita, Didymocarpus, Streptocarpus, and, more recently, Sinningia species.4−7 Sinningia comprises ca. 70 species of herbs, most of them native to Brazil. We are investigating this genus as part of a project of screening Brazilian Gesneriads, aiming at the isolation of new bioactive compounds. In previous studies we reported the isolation of cytotoxic and anti-inflammatory compounds from Sinningia allagophylla,8,9 as well as cytotoxic and antinociceptive compounds from S. aggregatta.10,11 Recently, a bioassay-guided fractionation led to the isolation of 7hydroxy-6-methoxy-α-dunnione, a naphthoquinone with antiinflammatory and antipyretic properties, from the less polar extracts of S. canescens tubers.12 These results support the continuation of phytochemical and biological studies on Sinningia species. Herein the results of the first study of S. reitzii (Hoehne) L. E. Skog, an evergreen subshrub with small © 2017 American Chemical Society and American Society of Pharmacognosy

Received: January 23, 2017 Published: June 9, 2017 1837

DOI: 10.1021/acs.jnatprod.6b01186 J. Nat. Prod. 2017, 80, 1837−1843

Journal of Natural Products

Article

no signal of a hydrogen-bonded hydroxy group. Analysis of HSQC and HMBC spectra confirmed the structure of 2 through the following cross-peaks: H-6 (δH 7.13) with C-10 (δC 119.0), C-8 (δC 117.6), and C-7 (δC 160.3); H-13 (δH 4.49, 4.92) with C-15 and C-14 (δC 27.7), C-12 (δC 171.3), and C-11 (δC 44.0); and H-14 (δH 1.54) with C-15 (δC 27.7), C-12, C11, and C-3 (δC 120.8) (Table 1). Therefore, compound 2 was identified as 7-hydroxydehydrodunnione. Compound 3 was isolated as an orange solid with the molecular formula C17H18O6, indicating nine indices of hydrogen deficiency. Its 1H NMR data (Table 2) showed Table 2. NMR Data (400 MHz, CDCl3) of Compound 3

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δC, type

position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 OMe-6 OMe-7 OH

RESULTS AND DISCUSSION Compound 1 was isolated as an orange solid with a molecular formula of C15H12O4 from HRESIMS and NMR data, which is consistent with 10 indices of hydrogen deficiency. The 1H NMR data of 1 (Table 1) showed signals of three protons in a spin system of a 1,2,3-trisubstituted benzene (δH 7.14−7.57). Two coupled olefinic methylene protons (δH 4.50 and 4.92), two equivalent methyl groups (δH 1.55), and one hydroxy group with an intramolecular H-bond (δH 11.93) were also observed. The 13C NMR data (Table 1) showed 15 carbons, including two carbonyl groups (δC 174.0 and 184.7), indicating a 1,2-naphthoquinone structural moiety.17 The structure 1 was deduced from the following HMBC correlations: H-5 (δH 7.27) with C-9 (δC 113.4), C-7 (δC 123.3), and C-4 (δC 164.6); H-14 (δH 1.55) with C-15 (δC 27.6), C-12 (δC 170.9), C-11 (δC 44.2), and C-3 (δC 123.1); and OH (δH 11.93) with C-9, C-8 (δC 164.8), and C-7 (Table 1). Therefore, compound 1 is a derivative of dehydrodunnione,17 named 8-hydroxydehydrodunnione. The structure of 1 was confirmed by X-ray diffraction analysis (Figure S1, Supporting Information). Compound 2 was isolated as a purple solid with the same molecular formula as 1. Its 1H NMR data (Table 1) were similar to those of compound 1, but the signals of the aromatic protons were in a spin system characteristic of a 1,2,4trisubstituted aromatic system (δH 7.13−7.61), and there was

177.4, 159.1, 130.3, 188.3, 155.7, 142.7, 156.5, 104.3, 127.0, 111.2, 45.1, 92.1, 14.2, 25.9, 20.7, 61.1, 56.5,

δH (J in Hz)

C C C C C C C CH C C C CH CH3 CH3 CH3 CH3 CH3

7.26 s

4.59 1.44 1.48 1.29 3.98 3.97 12.57

HMBC

1, 6, 9, 10

q (6.6) d (6.6) s s s s s

14, 15 11, 12 3, 11, 12, 15 3, 11, 12, 14 6 7 5, 6, 10

signals for one aromatic proton (δH 7.26), two methoxy groups (δH 3.98, 3.97), a hydroxy group with an intramolecular hydrogen bond (δH 12.57), and a 2,3-dihydro-2,3,3-trimethylfuran group (quartet at δH 4.59, two singlets at δH 1.29 and 1.48, and a doublet at δH 1.44). In the HMBC spectrum, the aromatic proton showed a cross-peak with a carbon at δC 177.4,

Table 1. NMR Data of Compounds 1 (400 MHz, CDCl3) and 2 (600 MHz, CDCl3) 1 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 OH

δC, type 184.7, 174.0, 123.1, 164.6, 117.5, 137.8, 123.3, 164.8, 113.4, 126.6, 44.2, 170.9, 87.0,

C C C C CH CH CH C C C C C CH2

27.6, CH3 27.6, CH3

δH (J in Hz)

7.27 dd (7.4, 0.8) 7.57 dd (8.6, 7.4) 7.14 dd (8.6, 0.8)

4.50 4.92 1.55 1.55 11.93

d (3.4) d (3.4) s s s

2 HMBC

4, 7, 9 8, 10 5, 8, 9

11, 12, 14, 15 3, 11, 12, 15 3, 11, 12, 14 7, 8, 9 1838

δC, type 181.1, 174.6, 120.8, 166.8, 126.9, 121.2, 160.3, 117.6, 132.9, 119.0, 44.0, 171.3, 87.0,

C C C C CH CH C CH C C C C CH2

27.7, CH3 27.7, CH3

δH (J in Hz)

HMBC

7.61 d (8.6) 7.13 dd (8.6, 2.1)

4, 7, 9, 10 7, 8, 10

7.59 d (2.1)

1, 6

4.49 4.92 1.54 1.54

11, 12, 14,15 3, 11, 12, 15 3, 11, 12, 14

d (3.4) d (3.4) s s



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Table 3. NMR Data (400 MHz, CDCl3) of Compounds 4 and 5 4 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 OMe-2 OMe-6 OMe-7 OH

δC, type 181.0, 156.5, 127.0, 190.9, 156.0, 143.9, 156.5, 103.5, 123.6, 111.5, 41.0, 148.2, 109.7,

C C C C C C C CH C C C CH CH2

28.6, CH3 28.6, CH3 61.1, CH3 56.5, CH3

5

δH (J in Hz)

HMBC

7.25 s

1, 6, 7, 10

6.27 4.96 4.99 1.56 1.56

11, 14, 15 11, 12

dd (17.5, 10.7) d (10.7) d (17.5) s s

4.00 s 3.97 s 12.74 s

3, 11, 12, 15 3, 11, 12, 14 6 7 5, 6, 10

which is characteristic of C-1 in the α-dunniones.17,18 Therefore, the hydroxy group was located at C-5 and the methoxy groups at C-6 and C-7. The remaining correlations in the HSQC and HMBC spectra (Table 2) led to the identification of 3 as 5-hydroxy-6,7-dimethoxy-α-dunnione. This compound was obtained both pure and as the major compound in a mixture with 4. Complete assignment of the 13C NMR data was achieved by analysis of the 13C NMR data of 3 + 4 in comparison with HSQC and HMBC data. Compound 3 is levorotatory, and its absolute configuration was thus assigned as S, considering that (+)-α-dunnione has the R absolute configuration.19 Compound 4, a yellow solid, had the same molecular formula as 3. Its 1H NMR data (Table 3) resembled those of 3, showing signals for one aromatic proton (δH 7.25), two methoxy groups (δH 3.97, 4.00), and one hydrogen-bonded hydroxy group (δH 12.74). The signals of a 1,1-dimethylallyl group (δH 1.56, 4.96, 4.99, and 6.27) also were observed. These data suggested a dunniol derivative.20,21 In the HMBC spectrum, a cross-peak was observed between the aromatic proton (δH 7.25) and a carbon at δC 181.0 that could be assigned to C-1 of the quinone group. Therefore, the aromatic hydrogen was located at C-8, and compound 4 was identified as 5-hydroxy-6,7-dimethoxydunniol, which was confirmed by the remaining correlations in the HSQC and HMBC spectra. The full assignment of the 13C NMR data was done via analyses of the 13C NMR spectrum obtained for the mixture of 3 + 4, where 4 was the minor component (Table 3). Compound 5 was isolated as an orange solid and also showed the same molecular formula as 3 and 4. The 1H NMR data (Table 3) showed signals for two ortho-coupled aromatic protons (δH 7.07, 7.57), two methoxy groups (δH 3.98, 4.05), three methyl groups, two of which were equivalent (δH 1.50) while the other had a chemical shift typical of an acetyl group (δH 2.17), and one hydrogen-bonded hydroxy group (δH 12.06). In the 13C NMR data, 16 peaks representing 17 carbons were observed, two of which could be assigned to the carbonyl groups of a 1,4-naphthoquinone (δC 183.9 and 187.0)

δC, type 187.0, 158.2, 141.5, 183.9, 120.8, 115.7, 153.7, 151.8, 114.3, 124.0, 51.0, 209.3, 25.5,

δH (J in Hz)

HMBC

C C C C CH CH C C C C C C CH3

7.57 d (8.4) 7.07 d (8.4)

4, 7, 9 8, 10

2.17 s

11, 12

24.9, CH3 24.9, CH3 61.5, CH3

1.50 s 1.50 s 4.05 s

3, 11, 12, 15 3, 11, 12, 15 2

56.4, CH3

3.98 s 12.06 s

7 7, 8, 9

while a third was attributed to a ketocarbonyl (δC 209.3). These data were compatible with those of a derivative of streptocarpone.17,21 Analyses of the HSQC and HMBC data led to structure 5, which was identified as 8-hydroxy-7methoxy-2-O-methylstreptocarpone. The most important correlations were the following: H-5 (δH 7.57) with C-9 (δC 114.3), C-7 (δC 153.7), and C-4 (δC 183.9); H-6 (δH 7.07) with C-10 (δC 124.0) and C-8 (δC 151.8); H-14 (δH 1.50) with C-15 (δC 24.9), C-12 (δC 209.3), C-11 (δC 51.0), and C-3 (δC 141.5); and OH (δH 12.06) with C-9, C-8, and C-7 (Table 3). The known compounds were identified as 8-hydroxydunnione,17 7-hydroxy-α-dunnione,18 and 6,8-dihydroxy-7-methoxy-2-O-methyldunniol.22 EtOH and EtOAc extracts were not studied, as our experience with other Sinningia species indicates that more important compounds are to be found in the less polar extracts. The edema observed during an inflammatory response depends on vasodilation and on the increase in small-vessel permeability caused by several inflammatory mediators to allow the flow of proteins and accumulation of fluids in the surrounding tissue.23 Local treatment with 1 (500 and 150 pg/paw) inhibited edema formation similarly to the positive control dexamethasone, a steroidal anti-inflammatory drug (Figure 1A). A lower dose of 1 did not change this response. Compound 1 was also effective against inflammatory nociception induced by carrageenan, specifically mechanical hyperalgesia (Figure 1B). However, the effective doses of this compound needed to obtain this effect were lower (10−50 pg/ paw) than those necessary to reduce edema formation (Figure 1). Together, these results suggest that 1 might be reducing the synthesis or action of some inflammatory mediators involved in both edema formation and the sensitization of the nociceptor. The antihyperalgesic doses of 1 were lower (from 0.04 to 0.2 pmol/paw) when compared to those of other compounds found in other Sinningia species, such as 8-methoxylapachenole from S. allagophyla (0.03−2.8 pmol/paw)9 or aggregatin D from S. aggregata (2.4−24 pmol/paw),11 suggesting that this compound is potent in attenuating nociception. In addition, 1 1839



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Figure 1. Effect of local treatment with 1 on carrageenan-induced edema and mechanical hyperalgesia. (A) Animals were treated with 1 (50−500 pg/paw) or dexamethasone (Dex, 30 ng/paw) or the same volume of vehicle (Veh, 0.1% Tween 20) by intraplantar route. After 15 min, the animals received an intraplantar injection of carrageenan (Cg 300 μg/paw) in the right paw, and paw thickness was measured at the indicated time points. (B) Basal mechanical threshold was measured before any injection. Animals were treated with 1 (15−50 pg/paw) or dipyrone (Dip, 320 ng/paw) or the same volume of Veh (20 μL, 0.1% Tween 20) by intraplantar route. After 15 min, the animals received an intraplantar injection of carrageenan (Cg, 300 μg/ paw), and the mechanical threshold was evaluated again 3 h after the injection of the nociceptive stimuli. Data show the mean ± SEM of 5− 8 animals. Symbols denote difference in comparison with the Veh/ Veh-treated group or basal (#p < 0.05) or with the Veh/Cg (*p < 0.05).

Figure 2. Effect of local treatment with 1 on mechanical hyperalgesia. Animals were treated with 1 (15 pg/paw) or dipyrone (Dip, 320 ng/ paw) or the same volume of vehicle (Veh, 20 μL of 0.1% Tween 20) by intraplantar route. After 15 min, the animals received an intraplantar injection of prostaglandin E2 (PGE2, 100 ng/paw, panel A), dopamine (DOPA 3 μg/paw, panel B), or dybutiryl cAMP (dbcAMP, 5 μg/paw, panel C) in the right paw. The mechanical threshold was evaluated again 3 h after the injection of the nociceptive stimuli. Bars represent the mean ± SEM of the mechanical threshold (n = 5−8). Symbols denote difference in comparison with the Veh/ stimulus group (*p < 0.05) or with the basal group (#p < 0.05).

was effective as an anti-inflammatory and antinociceptive agent when administered locally, suggesting that it acts directly at the inflammatory site and that no hepatic metabolic transformation is necessary for its activity. Carrageenan-induced mechanical hyperalgesia in mice depends on the release of mediators such as bradykinin, tumor necrosis factor-α, interleukin-1β, and keratinocyte chemoattractant, which subsequently induce the release of prostaglandins and sympathetic amines.15,16 We then attempted to establish whether the action of 1 occurred before or after the release of these final mediators of inflammatory hyperalgesia by injecting prostaglandin E2 (PGE2) and dopamine in the animal paw. Compound 1 (15 pg/paw) significantly reduced mechanical hyperalgesia induced by both PGE2 (Figure 2A) and dopamine (Figure 2B). PGE2 biosynthesis depends on the conversion of membrane arachidonic acid by the action of cyclooxygenase. Consequently, the blockade of PGE2-induced hyperalgesia by 1 suggests that this compound does not act by inhibiting cyclooxygenase-2 activity as do the nonsteroidal antiinflammatory drugs (NSAIDs). Additionally, NSAIDs usually do not reduce peripheral analgesia induced by sympathetic amines such as dopamine, which compound 1 was able to do. Once released, PGE2 interacts with EP3 and EP4 receptor

subtypes expressed in peripheral sensory neurons. The activation of these receptors results in an increase in the levels of cyclic AMP (cAMP), with subsequent activation of protein kinase A.24 Protein kinase A, in turn, has actions on various ion channels to sensitize nociceptors.25 Dopamine also induces hyperalgesia by an increase in the cAMP pathway.26 Compound 1 (15 pg/paw) did not reduce the hyperalgesia induced by dibutyryl cyclic AMP (dbcAMP) (Figure 2C). These results show that the site of action of 1 is between the activation of prostaglandin or dopamine receptors and the generation of cAMP. As expected, the positive control dipyrone reduced the mechanical hyperalgesia induced by PGE2, dopamine, and dbcAMP by activating the L-arg/NO/cGMP pathway.27,28 Therefore, compound 1 also possesses a mechanism of action distinct from dipyrone. The blockage of mechanical hyper1840



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in CDCl3 at 295 K on a Bruker AVANCE 400 or AVANCE III 600 NMR spectrometer, observing 1H at 400 or 600 MHz and 13C at 100 or 150 MHz, respectively. All 1H and 13C NMR chemical shifts are given in ppm (δ), using tetramethylsilane as internal reference, with coupling constants (J) in Hz. HRESIMS data were obtained on a Micromass ESI-Qq Tof or a Thermo Scientific LTQ-Orbitrap XL mass spectrometer, using the positive-ion mode. X-ray data were collected on a Bruker D8 Venture diffractometer, equipped with a Photon 100 CMOS detector, Cu Kα μS-microsource radiation, and Gobbel mirrors monochromator at 300(2) K. Accurate unit cell dimensions and orientation matrices were determined by least-squares refinement of the reflections obtained by θ−χ scans. The data were indexed and scaled with the ApexII Suite.32 Bruker SAINT and Bruker SADABS were used for integration and scaling of data, respectively. Scattering factors for neutral atoms were taken from ref 33. The structure was solved with WinGX, using the ShelXS structure solution program using direct methods and refined with ShelXL34−36 by a full-matrix least-squares technique on F2. The non-hydrogen atoms were refined with anisotropic thermal parameters. Hydrogen atoms were included in idealized positions, and their Uiso values were set to ride on the Ueq values of the parent carbon atoms. ORTEP-3 for Windows34 was also used for the drawing of molecular graphics and the preparation of figures for publication. Silica gel (Merck, 230−400 mesh) was used for column chromatographic separation, while silica gel 60 PF254 (Merck, Darmstadt, Germany) was used for analytical (0.25 mm) and preparative (1.0 mm) TLC. Compounds were visualized by exposure under UV254/366 light and spraying with 5% (v/v) H2SO4 in EtOH solution, followed by heating on a hot plate. Plant Material. Specimens of S. reitzii were collected from a natural population growing in Jundiai ́ do Sul, Paraná State, Brazil (23°26′45″ S, 50°15′33″ W), in April 2012. The plant was identified by Clarisse Bolfe Poliquesi, who deposited a voucher in the herbarium of Museu Botânico Municipal de Curitiba (MBM 253150). The plants were cultivated in the city of Curitiba, Paraná State, Brazil (25°25′44″ S, 49°16′03″ W). Tubers were collected in August 2012 (sample A) and April 2015 (sample B) from these cultivated plants. Extraction and Isolation. Dried and powdered tubers (A = 30.9 g; B = 46.6 g) were extracted at room temperature with hexanes (HEX, misture of isomers), CH2Cl2, EtOAc, and EtOH, successively (50 mL of each solvent for 10 g of material). After solvent removal, extracts in HEX (A = 0.30 g; B = 0.34 g), CH2Cl2 (A = 0.27; B = 0.28 g), EtOAc (A = 0.25 g; B = 0.26 g), and EtOH (A = 1.03 g; B = 1.22 g) were obtained. Extracts from sample A were kept frozen at −4 °C until fractionation. After TLC analyses, HEX and CH2Cl2 extracts from both samples were pooled, and an aliquot (0.14 g) was reserved for future assays. The remaining extract (1.05 g) was subjected to silica gel CC, using HEX, mixtures of HEX/EtOAc (95:5, 9:1, 8:2), EtOAc, and finally MeOH, to give 20 fractions (F1−F20) after TLC analyses. F3 (52.2 mg) yielded 1 (9.0 mg) after preparative TLC (PTLC) and recrystallization in CH2Cl2. Fraction F5 (55.8 mg) yielded 6,8dihydroxy-7-methoxy-2-O-methyldunniol (6.2 mg), 8-hydroxydunnione (13.2 mg), and 3 + 4 (3.3 mg) after PTLC in CH2Cl2. F6 (13.6 mg) yielded 3 (2.0 mg) and 4 (2.3 mg) after PTLC in CH2Cl2. F8 (48.7 mg) was subjected to further CC, eluted with CH2Cl2, a gradient of MeOH in CH2Cl2 (1−20%), and finally MeOH to give seven subfractions (F8.1−F8.7). Subfraction F8.1 (18.5 mg) yielded 7hydroxy-α-dunnione (2.2 mg) after repeated PTLC in CH2Cl2/MeOH (99:1). F10 (23.3 mg) yielded 2 + 5 (11.4 mg) after PTLC in HEX/ EtOAc (1:1). This mixture was subjected to further PTLC in CH2Cl2/ MeOH (99:1) to give 2 (3.3 mg) and 5 (7.6 mg). EtOAc and EtOH extracts were not studied. 8-Hydroxydehydrodunnione (1): orange solid; UV−vis (MeOH) λmax (log ε) 213 (4.51); 257 (4.19); 416 (3.64) nm; IR (KBr) νmax 3426, 3074, 2925, 1734, 1617, 1122, 891 cm−1; 1H and 13C NMR data see Table 1; HRESIMS m/z 257.08077 [M + H]+ (calcd for C15H13O4 257.08139). For crystallographic data see Supporting Information. The crystallographic data can be requested from the CSD with the deposit number CCDC 1502425. 7-Hydroxydehydrodunnione (2): purple solid; UV−vis (MeOH) λmax (log ε) 218 (3.66); 277 (3.58), 499 (2.44) nm; IR (KBr) νmax

algesia was not associated with a sedative effect of compound 1 since, at the time point at which the analgesic effect was observed, this compound did not affect motor performance, while diazepam significantly reduced the capacity of the animals to stay on the bar (Figure 3A).

Figure 3. Effect of local treatment with compound 1 on the motor performance and on the hot-plate test in mice. (A) Mice received 1 (15 pg/paw) or vehicle (Veh, 20 μL of 0.1% Tween 20) locally or diazepam (Diaz, 5.0 mg/kg, s.c.), and 3 h after 1 or 15 min after Diaz, they were tested for the Rotarod task for 180 s. (B) Mice basal latency in the hot-plate was assessed. Animals received the same doses of 1 or Veh localy or fentanyl (Fent, 100 μg/kg, s.c.), and 3 h after 1 or Veh or 15 min after Fent, the latency was assessed again. The maximal possible effect percentage (%MPE) was calculated. Bars represent the mean ± SEM (n = 6). Symbols denote difference in comparison with the Veh-treated group (p < 0.001).

Naphthoquinones are known for their ability to inhibit the activation of nuclear factor-κB (NF-κB) in several cell types,29−31 which may result in an anti-inflammatory/ antinociceptive action by the inhibition of cytokine and cyclooxygenase-2 expression. Although we did not evaluate cyclooxygenase-2 synthesis, the effectiveness of 1 on PGE2induced hyperalgesia suggests that it does not act by inhibiting NF-κB activity and consequently cyclooxygenase-2 expression. Compound 1 also does not appear to act similarly to opiods, another class of commonly used analgesic drugs, because opioids effectively block physiological heat-induced nociception, while 1 was ineffective in this (Figure 3B). In conclusion, compound 1 had an anti-inflammatory and antinociceptive activity at low doses and significantly reduced inflammatory pain by primarily preventing the sensitizing actions of the major mediators of inflammatory pain, such as PGE2 and sympathetic amines. Its antinociceptive effect appeared to be peripheral and different from classic NSAIDs, dipyrone, and opioids.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation was measured in MeOH on a Rudolph Research polarimeter. The UV spectra were obtained in MeOH on a Shimadzu UV-2401PC spectrophotometer. 1D and 2D NMR experiments were carried out 1841



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3131, 2922, 1734, 1639, 1175, 900, 833 cm−1; 1H and 13C NMR data see Table 1; HRESIMS m/z 257.08023 [M + H]+ (calcd for C15H13O4 257.08139). 5-Hydroxy-6,7-dimethoxy-α-dunnione (3): orange solid; [α]20D −13 (c 0.03, MeOH); UV−vis (MeOH) λmax (log ε) 212 (3.67), 263 (3.29), 330 (2.80) nm; IR (KBr) νmax 3436, 2922, 1734, 1630, 1135 cm−1; 1H and 13C NMR data see Table 2; HRESIMS m/z 319.11705 [M + H]+ (calcd for C17H19O6 319.11817). 5-Hydroxy-6,7-dimethoxydunniol (4): yellow solid; UV−vis (MeOH) λmax (log ε) 219 (3.71); 263 (3.54) nm; IR (KBr) νmax 3414, 2918, 1734, 1630, 1122 cm−1; 1H and 13C NMR data see Table 3; HRESIMS m/z 319.11705 [M + H]+ (calcd for C17H19O6 319.11817). 8-Hydroxy-7-methoxy-2-O-methylstreptocarpone (5): orange solid; UV−vis (MeOH) λmax (log ε) 217 (3.70); 268 (3.36) nm; IR (KBr) νmax 3423, 3138, 2918, 1725, 1630, 1265 cm −1; 1H and 13C NMR data see Table 3; HRESIMS m/z 319.11704 [M + H]+ (calcd for C17H19O6 319.11817). Animals. The experiments were conducted using male Swiss mice (25−35 g) from the Biological Sciences Section standard breeding unit of the Federal University of Paraná. Animals were housed five per cage under a 12 h light/dark cycle, with controlled humidity (60−80%) and temperature (22 ± 1 °C), and food and water were freely available. Animals were acclimatized to the experimental room at least 2 h before testing and were used only once throughout the experiments. The studies were performed in accordance with the current Brazilian and International guidelines for the care of laboratory animals. The animal procedures were approved by the Institutional Animal Care and Use Committee (CEUA/BIO-UFPR, authorization no. 745). The number of animals used was the minimum number necessary to show consistent effects, and all efforts were made to minimize animal suffering. Edema Tests. Edema was measured as previously described.9 Briefly, the mice received an intraplantar injection of 1 (50−500 pg/ paw), vehicle (0.1% Tween 20), or dexamethasone (1 mg/kg, positive control). After 1 h, they received a 50 μL subcutaneous injection of carrageenan (300 μg into the right hind paw) suspended in sterile 0.9% saline. The contralateral paw received only saline and was used as a control. The paw thickness was measured using a digital micrometer 1 h before and 1, 2, and 4 h after the carrageenan injection. Results were expressed as the difference between the paw thicknesses (in μm) before and after the carrageenan injection. Mechanical Hyperalgesia Tests. The mechanical threshold was measured by using von Frey filaments in the up-and-down paradigm as described previously.13,14 The mice were acclimatized (1 h) in individual clear Plexiglas boxes (9 × 7 × 11 cm) on an elevated wire mesh platform to allow access to the plantar surface of the hindpaws. The test consisted of touching the paw with a series of eight von Frey filaments with logarithmic increments of force (0.008, 0.02, 0.07, 0.16, 0.4, 1.0, 2.0, and 4.0 g). The test always started with the filament of 0.4 g, and the absence of paw lifting after this 2−4 s led to the use of the next filament with increased force. Paw lifting indicated a positive response and led to the use of the next weaker filament. This paradigm continued until six measurements were collected. The von Frey filaments were applied perpendicularly to the plantar surface with sufficient force to cause slight buckling against the paw. The 50% mechanical paw withdrawal threshold was then calculated from these scores as described before.14 The animals were then treated with 1 (5− 50 pg into the right paw) or the respective vehicle (0.1% Tween 20 in sterile saline, 20 μL applied locally). Fifteen minutes after this treatment, the animals received a 20 μL injection of carrageenan (300 μg/paw) into the right hindpaw. The withdrawal threshold was measured again 3 h after the carrageenan injection. Animals were also treated with 1 (15 pg/paw) or the positive control dipyrone (320 μg/ paw) or vehicle (0.1% Tween 20). After 15 min they received local injections of different nociceptive stimuli: PGE2 (100 ng/paw), dopamine (3 μg/paw) or dbcAMP (5 μg/paw). Mechanical hyperalgesia was measured after 3 h. Motor Performance Tests. Mice were previously selected based on their ability to remain on a Rotarod that rotated at 22 rotations per

minute for at least 60 s (one of three trials). Animals were treated with vehicle or 1 (15 pg directly in the paw). Positive control group received diazepam (5 mg/kg, subcutaneously). Three hours after local treatment or 30 min after diazepam treatment, the animals were subjected to the Rotarod test as previously described.9 Hot-Plate Tests. Animals were exposed to a metal platform at 55 °C in an acrylic box. When the animal withdrew or licked its paws, the time (in seconds) of exposure to the hot-plate was recorded and the exposure interrupted. Only animals that presented a basal latency of 7−15 s were used. The mice were treated with the vehicle or 1 (15 pg) in the paw or the μ-opioid receptor agonist, fentanyl (100 μg/kg, subcutaneously). Three hours after local treatment or 15 min after subcutaneous injection of fentanyl, the mice were re-exposed to the hot-plate. The cutoff time was 30 s. Hot-plate latencies were converted to a percentage of maximal possible effect (%MPE): %MPE = (postdrug latency − basal latency)/(cutoff time − basal latency) × 100. Statistical Analysis. The data are expressed as mean ± standard error of the mean (SEM). The data were analyzed using one- or twoway analysis of variance (ANOVA) followed by Bonferroni’s test. Values of p < 0.05 were 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.6b01186. Crystallographic data of 1; UV, IR, 1H, and 13C NMR spectra of compounds 1−5 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +55 41 3361-3177. E-mail: [email protected] (M. E. A. Stefanello). ORCID

Maria Élida A. Stefanello: 0000-0001-5821-6339 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to C. B. Poliquesi, at Museu Botânico Municipal de Curitiba, for collection and identification of the plant, and to CNPq and CAPES for scholarships.

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REFERENCES

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Naphthoquinones of Sinningia reitzii and Anti-inflammatory

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