TNF-α Inhibition Elicited by Mansoins A and B, Heterotrimeric

J. Nat. Prod. , 2014, 77 (4), pp 824–830. DOI: 10.1021/np400929g. Publication Date (Web): February 27, 2014. Copyright © 2014 The American Chemical...
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TNF‑α Inhibition Elicited by Mansoins A and B, Heterotrimeric Flavonoids Isolated from Mansoa hirsuta Priscilla R. V. Campana,†,‡ Christina M. Coleman,§ Mauro M. Teixeira,⊥ Daneel Ferreira,§ and Fernaõ C. Braga*,† †

Faculty of Pharmacy, Universidade Federal de Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil Division of Pharmaceutical Sciences, Fundaçaõ Ezequiel Dias, Belo Horizonte, MG, 30.510-010, Brazil § Department of Pharmacognosy and Research Institute of Pharmaceutical Sciences, University of Mississippi, University, Mississippi 38677, United States ⊥ Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, MG, 31.270-901, Brazil ‡

S Supporting Information *

ABSTRACT: Mansoins A (1) and B (2) isolated from the fruits of Mansoa hirsuta represent new glucosylated heterotrimeric flavonoids with a flavanone core linked to two 1,3diarylpropane C6−C3−C6 units. Their structures and absolute configurations were established by analysis of their NMR and electronic circular dichroism spectroscopic data. Compounds 1 and 2 inhibited TNF-α release by LPS-stimulated THP-1 cells with different potencies, with mansoin B (2) being active at lower concentrations than mansoin A (1) (IC50 values 20.0 ± 1.4 and 48.1 ± 1.8 μM, respectively). These results indicate potential anti-inflammatory properties for this structural type of oligoflavonoids, especially for mansoin B (2).

B

and inflammatory bowel disease.9 Presently, there is considerable interest in finding new TNF-α inhibitors that may be utilized as templates for new anti-inflammatory drugs, as current biologically derived agents in clinical use require intraarticular administration, have high costs, and are associated with severe side effects.10 As part of a search for new THF-α inhibitors from plants, reported herein are the structures and activities of two new glucosylated heterotrimeric flavonoids (1 and 2) with a flavanone core linked to two 1,3-diarylpropane C6−C3−C6 units from M. hirsuta.

ignoniaceae species are distributed primarily in neotropical regions, with Brazil being regarded as the diversity center for this large botanical family.1 The genus Mansoa of this family comprises 11 species that occur in the dry and wet forests of Brazil and from Argentina to southeast Mexico. Mansoa species found in the Amazonian forest are popularly named “cipó-dealho” (garlic vine), in view of the pungent garlic-like smell of the leaves when crushed. Such species are used in folk medicine to treat colds, fever, pain, and inflammation related to arthritis and rheumatism.2 The genus is considered to be a source of organosulfur compounds such as alliin, which may account for the alleged biological properties of Mansoa spp. and similarities with Allium sativum.2 Mansoa hirsuta DC. is a liana found in the Brazilian Atlantic forest, traditionally used to treat sore throats and diabetes.3 There is limited data on the chemical composition of M. hirsuta; the leaves contain terpenoids and sterols4 as well as mono- and dihydric aliphatic alcohols, with the latter compound classes being identified as the antifungal constituents of the low-polarity fractions.5 The EtOH extract of M. hirsuta leaves inhibited angiotensin-converting enzyme (ACE) in vitro6 and caused endothelium-dependent vasorelaxation in rat thoracic aorta.7 A MeOH fraction derived from this extract inhibited COX-1 enzyme activity in vitro,8 while the EtOAc fraction inhibited NO production and lymphoproliferation.4 TNF-α is a key mediator of inflammatory responses, and the inhibition of this cytokine is established as a therapeutic approach to treat human diseases such as rheumatoid arthritis © 2014 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION In a preliminary screening of Brazilian medicinal plants traditionally used to treat inflammatory conditions, the crude EtOH extract from M. hirsuta fruits was shown to significantly reduce the release of TNF-α by THP-1 cell cultures upon stimulation with LPS.11 Mansoin A (1), the major peak observed in the HPLC fingerprint of the fruits of M. hirsuta (data not shown), and mansoin B (2) were isolated using preparative RP-HPLC. Both compounds were obtained as amorphous, yellow powders. The HRESIMS of 1 showed an ion at m/z 1281.4064 [M + Na]+, while the spectrum of 2 showed an ion at m/z 1119.3508 [M + Na]+. This, in conjunction with NMR data, provided chemical formulas of C63H70O27 and C57H60O22 for compounds 1 and 2, respectively. Received: November 6, 2013 Published: February 27, 2014 824

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The UV spectrum for both compounds revealed absorption bands at 225, 284, and 325 nm, consistent with a flavanone core structure.12 Analysis of the 13C NMR and DEPT-135 spectra of 1 indicated resonances of 36 aromatic carbons, comprising 17 methine and 19 quaternary carbons (including 11 oxygenated carbons), consistent with the presence of six aromatic rings. Altogether, 17 aromatic protons were evident in the 1H NMR spectra, with splitting patterns characteristic of two paradisubstituted aromatic rings (δH 6.54, 2H, d, J = 8.2 Hz; 6.83, 2H, d, J = 8.2 Hz; 6.52, 2H, d, J = 7.9 Hz; 6.73, 2H, d, J = 7.9 Hz) and three 1,2,4-trisubstituted aromatic rings (δH 6.39, 1H, dd, J = 8.4, 2.2 Hz; 6.64, 1H, d, J = 2.2 Hz; 7.14, 1H, d, J = 8.4 Hz; 6.47, 1H, dd, J = 8.4, 2.2 Hz; 6.66, 1H, d, J = 2.2 Hz; 7.45, 1H, d, J = 8.4 Hz; 6.88, 1H, d, J = 8.2 Hz; 6.99, 1H, dd, J = 8.2, 1.4 Hz; 7.31, 1H, d, J = 1.4 Hz). On the basis of these data, the presence of one fully substituted aromatic ring was also inferred. The proton resonances centered at δH 5.27 (1H, dd, J = 13.3, 2.6 Hz), 3.18 (1H, dd, J = 16.9, 13.3), and 2.64 (1H, dd, J = 16.9, 2.6 Hz), along with the corresponding carbon resonances at δC 80.7 and 44.3 are typical of the methine and methylene groups of a flavanone moiety.13 The flavanone unit of 1 was identified as (2S)-eriodictyol using chemical shift values, proton coupling patterns, electronic circular dichroism (ECD), and chemical degradation data (vide infra). In addition, two C6−C3−C6 moieties, designated side chains 1 and 2 for these units at C-6 and C-8, respectively, were identified as constituents by the resonances of two methine (δC 35.0 and 35.2, δH 4.69 and 4.78, m) and four methylene groups (δC 34.8−36.7, δH 2.07−2.45, m). Such a structural pattern was confirmed by HMBC correlations between the C-1 methine protons (δH 4.78 and 4.69) and the aromatic carbons C-1′, C2′, and C-6′ of the side chains, as well as by correlations between the C-3 methylene protons and the aromatic carbons C-1″, C-2″, and C-6″ of the side chains. Owing to the lack of appropriate 3J HMBC correlations especially from H-2 to C-9 and from HO-5 to C-6 and C-10 of the flavanone moiety, the chemical shift values of the C-5, -6, -7, -8, and -9 resonances could not be assigned unequivocally. However, such an inability to differentiate the chemical shifts of C-6 and C-8 was not crucial to proper structural assignment because of the identity of the C-6 and C-8 substituents. Thus, the connectivity between the C6−C3−C6 moiety of side chain 1 and the flavanone unit was established by HMBC correlation (Figure 1) between the C-1 methine proton at δH 4.78 and the resonances ascribed to C-5 and C-7. Likewise, the linkage site of side chain 2 was revealed by HMBC cross-peaks between the C-1 methine proton at δH 4.69 and C-7 and C-9 of the flavanone moiey. These data permitted identification of compound 1 as a heterotrimeric flavonoid. The 1H NMR spectrum of 1 showed extensive overlapping of resonances in the 2−4 ppm region. The presence of three sugar units was indicated by three anomeric protons (δH 4.92, 1H, d,

Figure 1. Important 1H−13C HMBC (via 3J) correlations for mansoin A (1).

J = 7.2; 4.88, 1H, d, J = 7.4; 4.84, 1H, d, J = 7.3 Hz) and their corresponding carbons (δC 103.5, 104.0, and 103.7). The 13C NMR and DEPT-135 spectra showed 15 carbon resonances in the 60−80 ppm region that were assigned to the 12 oxymethine and three oxymethylene carbons of three hexose residues (Table 1). Acid-catalyzed hydrolysis of the crude extract of M. hirsuta and 1 followed by chiral derivatization14 of the sugar fraction permitted the identification of all three sugar units as D(+)-glucose. The hydrolysis reaction also afforded the flavanone (2S)-eriodictyol, but led to decomposition of the acyclic C6− C3−C6 moieties under the prevailing acidic conditions. When subjected to depolymerization using acid-catalyzed phloroglucinolysis,15,16 compound 1 afforded the diglucosylated heterodimers 3 and 4, formed by cleavage of the side chain bonds to C-8 and C-6 of the flavanone moiety, respectively (Table 2).

The connectivity of the glucosyl moieties to the aglycone was established by HMBC correlations between the anomeric protons and C-3′ of the eriodictyol unit and C-2′ of the 1,3diarylpropane moieties, respectively (Figure 1). Compound 2 showed 1D- and 2D-NMR spectra similar to those obtained for mansoin A (1). The major differences were in the 2−4 ppm region and the chemical shifts of the resonances belonging to the protons and carbons of the flavanone B-ring. The HRESIMS showed a difference of 162 mass units between compounds 1 and 2, which, in conjunction with the NMR data, indicated that compound 2 is a diglucosylated flavonoid heterotrimer that differed from 1 by the absence of the glucose residue at C-3′ of the flavanone moiety. The absolute configurations of compounds 1 and 2 were established by ECD. Both mansoins A and B possess a 2S absolute configuration as indicated by the weak positive Cotton effect for the n → π* transition at ca. 355 nm and the negative Cotton effect for the π → π* transition at ca. 280 nm (Figure 825

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Table 1. 1H and 13C NMR Data in Methanol-d4 for Mansoins A (1) and B (2) mansoin A δH (J in Hz)

position 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 1 2 3 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 1 2 3 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 1 1 1

Flavanone Moiety 5.27 dd (13.3, 2.6) 3.18 dd (16.9, 13.3); 2.64 dd (16.9, 2.6)

2, 1′, 3′, 4′, 6′

6.95 bs

1, 3′, 4′, 6′ 1′, 2′, 4′, 5′ 5,b 1′, 2 6,b 1′, 1, 3, 1″ 1, 2, 1″, 2″, 6″

6.80 6.80 Side 4.69 2.66 2.39

1′, 2′, 4′, 5′

6.63 d (1.7)

1′, 3′, 4′, 6′ 1, 1′, 2′, 4′, 5′

6.41 dd (8.4, 1.7) 7.22 d (8.4)

3, 1′, 3′/5′, 4′ 1′, 2′/6′, 4′

6.82 d (7.7) 6.54 d (7.7)

1′, 2′/6′, 4′ 3, 1′, 3′/5′, 4′ 7,b 9,b 1′, 2 8,b 1′, 1, 3, 1″ 1, 2, 1″, 2″, 6″

6.54 6.82 Side 4.63 2.15 2.39

1′, 2′, 4′, 5′

6.66 d (1.6)

1′, 3′, 4′, 6′ 1, 1′, 2′, 4′, 5′

6.46 dd (8.5, 1.6) 7.46 d (8.5)

3, 1′, 3′/5′, 4′ 1′, 2′/6′ 4′

6.71 d (7.7) 6.52 d (7.7)

1′, 2′/6′, 4′, 3, 1′, 3′/5′, 4′,

6.52 d (7.7) 6.71 d (7.7) glc-1

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

(8.0) (8.0)

35.2, 36.2, 34.8, 126.7, 157.2, 104.8, 157.1, 110.6, 131.3, 135.2, 130.3, 115.9, 155.8, 115.9, 130.3,

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

(7.3)

103.6, CH

3′b

(7.4)

104.0, CH

2′c

(7.2)

103.5, CH

2′d

6.46 dd (8.3, 1.4) 7.47 d (8.3) 6.84 d (8.1) 6.55 d (8.1) d (8.1) d (8.1) Chain 2 m m; 2.07 m m

6.62 d (1.4) 6.39 dd (8.4, 1.4) 7.13 d (8.4) 6.71 d (8.0) 6.52 d (8.0) 6.52 d 6.71 d glc-1 4.84 d glc-2 4.88 d glc-3 4.92 d

Flavanone Moiety 5.20 dd (13.5, 1.9) 3.12 dd (16.8, 13.5); 2.64 m

35.0, 36.6, 35.2, 127.0, 157.2, 104.2, 156.8, 110.5, 131.3, 135.1, 130.3, 115.9, 155.7, 115.9, 130.3,

6.64 d (1.4)

6.55 6.84 Side 4.69 2.37 2.37

3, 1′, 2′, 6′ 2, 4, 1′

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

d (8.2) dd (8.2, 1.4) Chain 1 m m; 2.15 m m

δH (J in Hz)

HMBCa

80.7, 44.3, 198.2, 162.4, 112.6, 164.0, 111.8, 160.9, 104, 132.1, 116.4, 146.6, 148.6, 117.0, 123.6,

7.31 d (1.4)

6.99 6.99 Side 4.78 2.65 2.45

mansoin B δC

δC

m m Chain 1 m; 2.39 m m

d (7.7) d (7.7) Chain 2 m; 2.07 m m

glc-2 4.86 d (7.4) glc-3 4.89 d (7.3)

80.9, 44.6, 198.9, 161.1, 112.3, 164.0, 111.5, 162.3, 104.9, 131.9, 115.0, 146.4, 146.8, 116.3, 119.7,

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

35.0, 36.1, 35.1, 126.8, 156.9, 104.9, 157.3, 110.8, 131.4, 135.0, 130.3, 115.9, 155.7, 115.9, 130.3,

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

35.2, 36.3, 34.7, 126.5, 156.8, 104.5, 157.3, 110.7, 131.2, 134.9, 130.3, 115.9, 155.7, 115.9, 130.3,

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

103.7 CH 103.4 CH

HMBC correlations are from proton(s) to the indicated carbon(s). Carbons of the flavanone moiety. C-2′ from side chain 1. C-2′ from side chain 2. a

b

c

d

consistent with those of (2S)-flavanones.17 This configurational assignment is consistent with P-helicity of the heterocycle

2). Similar Cotton effects were observed for eriodictyol (data not shown), obtained by hydrolysis of 1 (vide supra), and are 826

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Table 2. 1H NMR Data (400 MHz, Methanol-d4) for Compounds 3 and 4 position 2 3 6 8 2′ 5′ 6′ 1 2 3 3′ 5′ 6′ 2″/6″ 3″/5″

3

4

δH (J in Hz)

δH (J in Hz)

Flavanone Moiety 5.25 dd (13.1, 3.2) 3.09 dd (16.9, 13.1); 2.63 dd (16.9, 3.2) 5.87 7.34 6.94 7.04 Side 4.78 2.56 2.43 6.56 6.37 7.49 6.86 6.65

s d (1.6) d (8.4) dd (8.4, 1.6) Chain 1 m m; 2.11 m m d (2.4) dd (8.4, 2.4) d (8.4) d (8.4) d (8.4)

active than mansoin B (2), it also elicited TNF-α inhibition (IC50 48.1 ± 1.8 μM). Their results indicate that the loss of one glucopyranosyl moiety, as observed in 2, contributes to improved biological activity probably by favoring intracellular transport. Flavonoids are secondary metabolites involved in several aspects of plant development and defense. Antioxidant, antihypertensive, anti-inflammatory, and antiproliferative activities have been described for these compounds.20,21 The antiinflammatory activities of flavonoids have been extensively investigated, and several mechanisms are believed to be responsible for this activity. Compounds of this class have been shown to modulate pro-inflammatory gene expression through pathways involving mostly NF-κB and MAPK signaling.22 Oligomeric flavonoids are complex C6−C3−C6 compounds found in some plant species, usually formed by oxidative coupling of flavones, flavonols, flavanones, aurones, chalcones, or dihydrochalcones. The proanthocyanidins are thought to originate by ionic coupling of appropriate nucleophilic and electrophilic flavanyl moieties and are typically considered as flavan-3-ol oligomers.23,24 This is the first report of naturally occurring polyphenolic heterotrimers bearing a flavanone moiety attached to two glucosylated 1,3-diarylpropane moieties. “Unusual flavonoids” have been previously isolated from the resins of a Dracaena species, popularly known as Chinese dragon’s blood.25,26 These compounds were identified as oligomeric flavonoids composed of a dihydrochalcone unit condensed with one or more chalcone, flavan, or homoisoflavan moieties, but their absolute configurations were not defined. Flavonoids are considered one of the most diverse classes of natural products, with thousands of compounds already identified. They have been extensively studied, and some have applications in the pharmaceutical, chemical, cosmetic, and food industries. Therefore, the new structural type reported for compounds 1 and 2, combined with their observed biological activity, may have potential for pharmaceutical development.

Flavanone Moiety 5.24 dd (12.5, 2.5) 3.03 dd (16.9, 12.5); 2.60 dd (16.9, 2.5) 5.86 s 7.22 7.08 6.94 Side 4.91 2.66 2.39 6.63 6.41 7.22 6.82 6.54

d (1.4) d (8.3) dd (8.3, 1.4) Chain 2 m m; 2.39 m m d (1.7) dd (8.4, 1.7) d (8.4) d (7.7) d (7.7)

resulting from the thermodynamically favored conformation with an equatorial C-2 aryl substituent. The ECD spectrum is dominated by a positive couplet near 220 nm, permitting definition of the S absolute configuration for C-1 of both 1,3diarylpropane moieties. This assignment is based on comparison with the ECD data of 1,3-diarylpropanes generated via the photolysis of catechin in the presence of phloroglucinol.18 On the basis of these data, the structures of compounds 1 and 2 (mansoins A and B) were defined unambiguously as (2S)-6,8-di[(1S)-(2′-O-β-D-glucopyranosyl-4′-hydroxyphenyl)3-(4″-hydroxyphenyl)propyl]-3′-O-β-D-glucopyranosyleryodyctiol and (2S)-6,8-di[(1S)-(2′-O-β-D-glucopyranosyl-4′-hydroxyphenyl)-3-(4″-hydroxyphenyl)propyl]eryodyctiol, respectively. Compounds 1 and 2 presumably originate via acid-mediated coupling of a dihydrochalcone and the (2S)-flavanone, eriodictyol, followed by dehydration and reduction of the olefinic bond (Scheme 1). The potential anti-inflammatory activity of compounds 1 and 2 was investigated by measuring TNF-α release by LPSstimulated THP-1 cells employing an immunoassay, as previously described.19 Mansoin B (2) induced reduction of the release of TNF-α (IC50 20.0 ± 1.4 μM) in comparison with untreated cells (Figure 3). Although mansoin A (1) was less

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EXPERIMENTAL SECTION

General Experimental Procedures. UV and ECD absorbance spectra were obtained on a JASCO J-815 spectrometer using highpurity MeOH as the solvent and a 1 cm path length quartz cuvette. Multiple concentrations were examined for each compound to determine the concentration that gave an optimum ECD spectrum. A binomial smoothing algorithm with 10 passes, as provided with the JASCO Spectra Manager Ver. 1.54 software, was applied to the raw data to produce the spectra shown. 1H and 13C NMR spectra were recorded in deuterated solvents on a Bruker AVIII 400 Ultra Shield

Figure 2. ECD spectra for compounds 1 (A) and 2 (B). 827

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Scheme 1. Proposed Biosynthesis Route to 1

Figure 3. Curves of inhibition of TNF-α release (%) in LPS-stimulated cells elicited by mansoins A (1) and B (2). Each point represents the mean ± SEM of three replicates. NMR spectrometer (Bruker Biospin AG, Switzerland) with TMS as the internal standard for both nuclei. Mass spectra were recorded on a Bruker maXis UHR-TOF mass spectrometer (Bruker Daltonik GmbH, Germany). Analytical and preparative HPLC were performed on a Waters 600 HPLC system equipped with a Delta 600 quaternary solvent pump, an in-line degasser, a 2996 photodiode array (PDA) detector, and Empower 2 software (Waters, MA, USA). The separations and purifications were carried out on C18 Luna columns (21.2 × 250 mm, 5 μm and 10 × 250 mm, 5 μm) (Phenomenex, CA, USA). Thin-layer chromatography was performed on silica gel 60 F254 plates (0.20 mm thickness, Merck, Germany). The optical density (O.D.) for the TNF-α assay was read with a Tecan microplate reader (model Infinite 200 Pro, Tecan, Switzerland) using iControl software (Tecan). The ELISA kit for TNF-α was purchased from R&D Systems (DY210 TNF-a duo set, R&D Systems, Minneapolis, MN, USA). Plant Material. The fruits of M. hirsuta were collected in Caratinga, MG, Brazil, in August 1996 and identified by Dr. Julio A. Lombardi (UFMG). A voucher specimen was deposited at the herbarium of the Instituto de Ciências Biológicas UFMG under the number BHCB 23862. Extraction and Isolation. The dried fruits of M. hirsuta (25 g) were powdered and extracted by percolation with EtOH at room temperature. The extract was concentrated on a rotatory evaporator and kept in a desiccator for complete removal of the solvent to give a

light brown residue (4.6 g). A portion of this residue (1.0 g) was fractionated by preparative HPLC on an ODS column (Phenomenex Luna, 250 × 21.2 mm, 5 μm) using H2O (A) and MeCN (B) as follows: 0−8 min, 15−26% B; 8−22 min, 26−29% B; 22−52 min, 29− 44% B; 52−55 min, 44−50% B; 55−60 min, 50−95% B; and 60−70 min, 95% B. The flow rate was 5.0 mL/min, and the detection was set at λ 280 nm. Altogether, eight fractions were collected, and fractions 4 (18−19 min) and 7 (21−22 min) were further purified over a Phenomenex ODS column (10 × 250 mm, 5 μm), using similar chromatographic conditions at a flow rate of 3 mL/min, to afford mansoins A (1) (130 mg, tR 18.3 min) and B (2) (26 mg, tR 21.6 min). Mansoin A (1): pale yellow powder; UV (MeOH) λmax 225, 283, and 303 nm; HRESITOFMS [M + Na]+ m/z 1281.4064 (calcd for C63H70O27 [M + Na]+ 1281.4104); 1H and 13C NMR data, Table 1. Mansoin B (2): pale yellow powder; UV (MeOH) λmax 224, 283, and 303 nm; HRESITOFMS [M + Na]+ m/z 1119.3508 (calcd for C63H70O27 [M + Na]+ 1119.3576); 1H and 13C NMR data, Table 1. Degradation Reactions of Mansoin A (1). Acid Hydrolysis. A solution of mansoin A (1) (5.0 mg) in 1 N HCl (5 mL) was kept under reflux for 1 h, and the mixture was cooled and extracted with EtOAc (5 × 15 mL). The organic phase showed one major compound that was further purified by RP-HPLC and identified as the flavanone, (2S)-eriodictyol, by NMR and ECD data. The aqueous phase was subjected to sugar analysis using a published method.14 Briefly, the aqueous phase was neutralized with AgCO3, filtered through a 0.20 μm 828

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Journal of Natural Products nylon membrane, and dried. L-Cysteine methyl ester (1 mg) and pyridine (100 μL) were added, and the mixture was heated at 60 °C under reflux for 1 h. A solution of phenyl isothiocyanate in pyridine (10 mg/mL; 100 μL) was added, and the mixture refluxed at 60 °C for another hour. The reaction mixture was immediately analyzed by RPHPLC (Gemini C18 column; Phenomenex, 250 × 4.6 mm; 5 μm) using a gradient of MeCN and H2O, 10% to 90% MeCN over 70 min, flow rate of 1.0 mL/min, and UV detection at 250 nm. D-(+)-Glucose was used as a reference standard. A blank reaction was performed but without the addition of the sample or reference sugar. The hydrolysis and derivatization steps were carried out using 10 mg of the crude extract by the same procedure to investigate the presence of other sugar units in the crude extract. Phloroglucinolysis Reaction.15,16 Compound 1 (5 mg) and phloroglucinol hydrate (10 mg) were added to a round-bottom flask, and the mixture was stirred at 60 °C in EtOAc/0.1 N HCl (1:4) for 4 h. The mixture was extracted with EtOAc (6 × 15 mL), the organic phase dried over Na2SO4, and the solvent removed under vacuum at 40 °C. The products were purified by RP-HPLC over a Phenomenex ODS column (10.0 × 250 mm i.d., 5.0 μm), using a gradient of MeCN and H2O, with 10% to 65% MeCN over 40 min, a flow rate of 1.0 mL/min, and UV detection at 280 nm. Compounds 3 and 4 were identified by NMR data as the dimers formed by the cleavage of side chains 2 and 1 in mansoin A (1), respectively. TNF-α Production Assay. THP-1 cells (ATCC TIB-202) were cultivated in RPMI 1640 (Sigma-Aldrich, St. Louis, MO, USA) medium supplemented with 0.05 mM 2-mercaptoethanol, 10% FBS, 100 U/mL penicillin, and 100 μg/mL gentamicin at 37 °C in an atmosphere containing 5% CO2. The medium was renewed twice a week when the cell concentration reached 1.0 × 106 cells/mL. The cells were transferred to a 96-well microplate at a concentration of 100 000 cells per well and incubated for 18 h with RPMI supplemented with 1% SFB to initiate serum starvation, which was kept throughout the experiment. The cells were pretreated with mansoins A (1) and B (2) at concentrations from 3.9 to 250 μmol/L for 3 h. LPS (SigmaAldrich), added at a concentration of 200 ng/mL, was employed as the inflammatory stimulus. The plate was incubated at 37 °C overnight. After this period, the plate was centrifuged (1.800g, 5 min, 16 °C), the supernatant collected, and TNF-α release measured using the cytokine-specific sandwich quantitative ELISA according to the manufacturer’s instructions (TNF-α duo set, DY210, R&D Systems, Minneapolis, MN, USA). The cell viability was evaluated for the test samples employing the MTT method27 using untreated cells as the reference for viability. Samples were considered nontoxic for the THP1 cell line when cell viability was higher than 90%. The percentage of TNF-α inhibition was calculated from the ratio between the observed TNF-α amount secreted by treated cells (pg/mL) and the baseline secretion of TNF-α (pg/mL) observed for the solvent control (0.1% DMSO). The statistical significance of differences was calculated employing the software GraphPad Prism, version 5.0 (GraphPad Software Inc., San Diego, CA, USA), using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. Results were considered different when p < 0.05. IC50 values were determined by nonlinear regression using GraphPad Prism, version 5.0. All the experiments were performed in triplicate.

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ACKNOWLEDGMENTS

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REFERENCES

This work was funded by CNPq, Brazil (SWE grant for P.R.V.C., 200972/2011-01), in part, by the United States Department of Agriculture, ARS, Specific Cooperative Agreement number 58-6408-2-009, and from the European Community’s Seventh Framework Programme [FP7-20072013] under grant agreement number HEALTH-F4-2011281608. CNPq also provided a research fellowship for F.C.B. (307649/2009-1). We thank Dr. D. Rosado, Department of Chemistry and Biochemistry at Mississippi College, Clinton, MS, for the use of the JASCO J-815 spectrometer.

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S Supporting Information *

1D- and 2D-NMR spectra and HRMS of compounds 1 and 2 are available free of charge via the Internet at http://pubs.acs. org.

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