Mansoins CâF, Oligomeric Flavonoid Glucosides Isolated from

Article pubs.acs.org/jnp

Mansoins C−F, Oligomeric Flavonoid Glucosides Isolated from Mansoa hirsuta Fruits with Potential Anti-inflammatory Activity Priscilla R. V. Campana,†,‡ Christina M. Coleman,§ Lirlândia P. Sousa,† Mauro M. Teixeira,⊥ Daneel Ferreira,§ and Fernaõ C. Braga*,† †

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

ABSTRACT: Continued investigation of the polyphenolic pool of the fruits of Mansoa hirsuta afforded four additional members of the new class of glucosylated oligomeric flavonoids comprising a flavanone core linked to 1,3-diarylpropane C6−C3−C6 units. The structures and absolute configurations of mansoins C−F (3−6) were established by analysis of NMR and electronic circular dichroism data. Mansoin C (3) was identified as a diglucosylated heterodimer, whereas mansoins D (4), E (5), and F (6) were identified as triglucosylated heterotrimers, isomeric with mansoin A (1). Mansoin F (6) inhibited TNF-α release by lipopolysaccharide-stimulated THP-1 cells (IC50 of 19.3 ± 1.3 μM) and, as with mansoin A (1), reduced the phosphorylation levels of p-65-NF-κB, when assayed at 50 μM. These results indicate that the potential anti-inflammatory properties of mansoin F (6) are probably due to inhibition of the NF-κB pathway and inhibition of TNF-α release. linked to one or more chalcone, flavan, or homoisoflavan moieties, but their absolute configurations were not defined. In a continuation of the phytochemical investigation of M. hirsuta fruits, this report describes the structures of mansoins C−F (3−6), four new glucosylated oligomeric flavonoids related to mansoins A (1) and B (2), and the in vitro antiinflammatory activity of mansoin F (6).

T

he liana Mansoa hirsuta DC. (Bignoniaceae) occurs in the Brazilian Atlantic forest and is used traditionally to treat sore throats and diabetes.1 The known constituents of M. hirsuta include terpenoids and sterols2 isolated from the leaves and mono- and dihydric aliphatic alcohols that are regarded as the antifungal constituents of less polar fractions.3 An EtOH extract of the leaves was reported to inhibit angiotensinconverting enzyme (ACE) in vitro4 and to induce endotheliumdependent vasorelaxation in the rat thoracic aorta.5 A MeOH fraction, obtained by chromatographic fractionation of the ethanolic extract from M. hirsuta leaves on a silica gel column, was described to inhibit COX-1 enzyme activity in vitro,6 while an EtOAc fraction was shown to inhibit NO production and lymphoproliferation.2 In a previous study, the isolation was reported of two new triglucosylated flavonoid heterotrimers from the fruits of M. hirsuta, named mansoins A (1) and B (2), which inhibited the release of TNF-α by lipopolysaccharide (LPS)-stimulated THP-1 cells. Mansoins A (1) and B (2) represent a group of glucosylated oligomeric flavonoids featuring a flavanone core linked to two 1,3-diarylpropane moieties.7 Other flavonoids with related structures have been isolated from the resin of a Dracaena species (Dracaenaceae), popularly known as Chinese dragon’s blood.8,9 These compounds were identified as oligomeric flavonoids composed of a dihydrochalcone unit © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The isolation and structure elucidation were previously reported of two new heterotrimeric flavonoid glucosides, mansoins A (1) and B (2), from the EtOH extract of M. hirsuta fruits.7 Compounds 1 and 2 were obtained as the major constituents of the EtOH extract as determined by its HPLC profile. Further investigation of the polyphenolic pool of the fruits has afforded four minor constituents of the oligomeric flavonoid fraction, and all four were found to be additional members of the mansoin class of oligomeric flavonoid glucosides, namely, mansoins C (3, 2.5 mg), D (4, 4.7 mg), E (5, 6.5 mg), and F (6, 23.3 mg). All four new mansoins were obtained as amorphous, yellowish powders. In general, their Received: May 1, 2016

A

DOI: 10.1021/acs.jnatprod.6b00390 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

UV spectra revealed absorption bands at 225, 284, and 325 nm, similar to those reported for mansoins A (1) and B (2).7 Comparison of the 1H and 13C NMR spectroscopic data of mansoins C−F (3−6) with those reported for 1 indicated substantial similarities, suggesting that all five of these compounds are structurally related. Therefore, mansoin A (1) served as a model compound to elucidate the structures of mansoins C−F (3−6). The HRESIMS data of mansoin C (3) showed a sodium adduct ion at m/z 877.2547 [M + Na]+, which, in conjunction with its 13C NMR data, indicated a molecular formula of C42H46O19. Analysis of the 13C NMR and DEPT-135 spectra of 3 disclosed signals of 24 aromatic carbons, comprising 11 methine, five quaternary, and eight oxygenated tertiary carbons, compatible with the presence of four aromatic rings. Altogether, 11 aromatic protons were evident in the 1H NMR spectrum, with splitting patterns characteristic of a p-disubstituted aromatic ring (δH 6.64, 2H, d, J = 8.2 Hz; 6.86, 2H, d, J = 8.2 Hz) and two 1,2,4-trisubstituted aromatic rings (δH 6.27, 1H, dd, J = 8.4, 1.9 Hz; 6.53, 1H, d, J = 1.9 Hz; 7.05, 1H, d, J = 8.4 Hz; 6.87, 1H, d, J = 8.0 Hz; 6.93, 1H, d, J = 8.0 Hz; 7.22, 1H, br s). The presence of a pentasubstituted aromatic ring was inferred by the presence of a one-proton singlet at δH 5.98. The proton signals centered at δH 5.30 (1H, dd, J = 12.7, 2.7 Hz), 3.08 (1H, dd, J = 16.9, 12.7 Hz), and 2.68 (1H, dd, J = 16.9, 2.7 Hz), and the corresponding carbon signals at δC 80.5 and 43.8 are, respectively, characteristic of the oxygenated methine and methylene groups of a flavanone unit.10 The flavanone constituent unit of 3 was identified as eriodictyol by comparison with the NMR data reported for mansoin A (1). Comparison between the MS and NMR data of mansoins A (1) and C (3) indicated that the latter differs from 1 by the absence of one glucosylated C6−C3−C6 moiety. The presence of only one C6−C3−C6 moiety in mansoin C (3) was confirmed by the resonances of a methine (δC 34.1, δH 4.76, m) and two methylene groups (δC 34.8 and 36.1, δH 2.10−2.31, m). The connectivity between the C6−C3−C6 moiety and the flavanone core was established via the HMBC correlations between the C-1 methine proton at δH 4.77 and the carbon resonances at δC 162.9 and 167.7. The unambiguous assignment of the C-5, C-7, and C-9 resonances was problematic due to the absence of long-range correlations, e.g., from H-2 to C-9. In the HMBC spectra of mansoins A (1) and B (2) it was observed that the C-1 methine proton of the 1,3-diarylpropyl moiety at C-8 gave two strong correlations indicative of coupling with C-7 and C-9. In contrast, a single correlation between C-5 and the same proton of the C6−C3− C6 unit located at C-6 was observed. This finding is explicable in terms of a ca. 90° dihedral torsion angle between the C-1 methine proton and C-7 and, hence, a coupling constant approximating zero according to the Karplus equation.11 Since two strong correlations, reminiscent of heteronuclear JH,C coupling for the C-1 methine proton, were found in the HMBC spectrum of 3; the structure of mansoin C (3) therefore was assigned as a heterodimeric flavonoid with the C6−C3−C6 moiety attached to C-8 of the flavanone core. The presence of two sugar units in mansoin C (3) was indicated by two anomeric protons resonating at δH 4.84 (1H, d, J = 7.4 Hz) and 4.79 (1H, d, J = 7.3 Hz) and their corresponding carbons resonating at δC 103.4 and 102.0. The 13 C NMR and DEPT-135 spectra showed 10 carbon resonances in the 60−80 ppm region that were assigned to eight oxymethine and two oxymethylene carbons of the two

hexosyl residues (Table 1). Acid-catalyzed hydrolysis of the ethanol extract from M. hirsuta fruits and mansoin A, followed by chiral derivatization of the sugar fraction, previously permitted the identification of D-(+)-glucose as the only B



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Table 1. 1H NMR Spectroscopic Data in Methanol-d4 of Mansoins A (1), C (3), D (4), E (5), and F (6)a position 2 3

a

mansoin A (1)7

mansoin C (3)

mansoin D (4)

mansoin E (5)

mansoin F (6)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

4.79, m 2.57 dd (17.0, 13.3); 3.13 dd (17.0, 2.6)

5.34 d (13.0) 3.12 dd (16.9, 13.0); 2.68 dd (16.9, 1.9)

7.29 br s 6.95 m 6.95 m

7.33 br s 7.01 d (8.2) 6.87 d (8.2)

4.72 2.47 2.47 6.70 6.42 6.88 6.94 6.67

m m; 2.21 m m d (2.4) dd (8.5, 2.4) d (8.5) m d (843)

4.65 2.51 2.21 6.64 6.46 7.39 6.95 6.66

m m m m dd (8.4, 1.8) d (8.4) d (8.3) d (8.3)

4.35 2.52 2.47 6.53 6.07 7.33 6.94 6.71

m m; 2.21 m m d (2.4) dd (8.4, 2.4) d (8.4) m d (8.3)

4.40 2.68 2.25 6.64 6.39 7.11 6.78 6.60

m m m m dd (8.4, 2.1) d (8.4) d (8.2) d (8.2)

5.75 dd (13.3, 2.6) 3.18 dd (16.9, 13.3); 2.64 dd (16.9, 2.6)

6 2′ 5′ 6′

7.31 d (1.4) 6.99 d (8.2) 6.99 dd (8.2, 1.4)

1 2 3 3′ 5′ 6′ 2″/6″ 3″/5″

4.69 2.37 2.37 6.62 6.39 7.13 6.71 6.52

m m; 2.07 m m d (1.4) dd (8.4, 1.4) d (8.4) d (8.0) d (8.0)

1 2 3 3′ 5′ 6′ 2″/6″ 3″/5″

4.78 2.65 2.45 6.64 6.46 7.47 6.84 6.55

m m; 2.15 m m d (1.4) dd (8.3, 1.4) d (8.3) d (8.1) d (8.1)

1

4.92 d (7.2)

1

4.88 d (7.4)

1

4.84 d (7.3)

Flavanone Moiety 5.30 dd (12.7, 2.7) 4.83 dd (13.4, 2.1) 3.08 dd (16.9, 12.7); 2.68 dd 3.01 dd (17.2, 13.4); (16.9, 2.7) 2.60 m 5.97 s 7.22 br s 7.27 d (1.4) 6.87 d (8.0) 6.93 m 6.93 br.d (8.0) 6.99 dd (8.3, 1.4) Side Chain 1 4.51 m 2.50 m; 2.21 m 2.50 m 6.63 d (1.8) 6.13 dd (8.3, 1.8) 6.94 m 6.94 m 6.59 d (8.2) Side Chain 2 4.76 m 4.66 m 2.31 m; 2.10 m 2.69 m; 2.29 m 2.31 m 2.59 m 6.53 d (1.9) 6.76 d (2.0) 6.27 dd (8.4, 1.9) 6.54 dd (8.4, 2.0) 7.05 d (8.4) 7.47 d (8.4) 6.86 d (8.2) 6.94 m 6.64 d (8.2) 6.66 d (8.1) glc-1 4.84 os 4.78 d (7.6) glc-2 4.69 os glc-3 4.79 os 4.71 os

4.80 d (7.8)

4.76 d (7.4)

4.58 d (7.5)

4.65 d (7.5)

5.24 d (7.8)

5.16 os

Data acquired on a Bruker AVIII NMR spectrometer, at 400 MHz (br s, broad singlet; os, overlapped signal).

constituent sugar unit.7 The connectivities of the β-Dglucopyranosyl moieties in 3 were established by HMBC correlations between the anomeric protons and C-3′ and C-2′ of the eriodictyol and 1,3-diarylpropane moieties, respectively. The 1H NMR spectrum of compound 3 was identical to the spectrum of the heterodimer previously produced by the phloroglucinolysis of mansoin A (1).7 The electronic circular dichroism (ECD) spectrum of 3 (Figure 1) contained a negative Cotton effect (CE) at ∼290 nm, indicating a 2S configuration for the flavanone moiety. The ECD spectrum also contained sequential positive and negative CEs at 238 and 211 nm, respectively. The UV absorption maximum at 225 nm indicated that this split CE was due to exciton coupling between the aromatic moieties of the C-1 biphenylmethine chromophore of the 1,3-diarylpropyl moiety. Thus, similar to the ECD spectrum of mansoin A (1), the P-helicity of this chromophore reflected a 1S absolute configuration for the 1,3diarylpropane moiety. Similar diagnostic Cotton effects in the 210−240 nm regions of the ECD spectra of 1,1,3-triarylpropan2-ols and 4-arylflavan-3-ols also permitted assignment of the absolute configurations of the biphenylmethine chromophores in these similar classes of flavonoids.12,13 Since the configurational assignments were confirmed by TDDFT calculations, it was deemed unnecessary to repeat the calculations for molecules of type 3 with their highly flexible conformational

itineraries. On the basis of these data, the structure of compound 3, mansoin C, was defined as (2S)-8-[(1S)-(2-Oβ-D-glucopyranosyl-4-hydroxyphenyl)-3-(4-hydroxyphenyl)propyl]eriodictyol-3′-O-β-D-glucopyranoside. The HRESIMS data of mansoins D−F (4−6) showed sodium adduct ions at m/z 1281.4049, 1281.4042, and 1281.4048 [M + Na]+, respectively, which, in conjunction with the 13C NMR data, indicated molecular formulas of C63H70O27 for all three compounds, the same as for mansoin A.7 Compounds 4−6 showed 1D and 2D NMR spectra similar to those obtained for mansoin A (Tables 1 and 2). Analyses of the 13C NMR and DEPT-135 spectra of 4−6 disclosed signals of 36 aromatic carbons, comprising 17 methine, 11 oxygenated tertiary, and eight quaternary carbons, consistent with the presence of six aromatic rings. Altogether, 17 aromatic protons were evident in the 1H NMR spectra, with splitting patterns similar to those observed for mansoin A, indicating the presence of two p-disubstituted and three 1,2,4-trisubstituted aromatic rings. The presence of one fully substituted aromatic ring in each compound was inferred, as this was consistent with all other data. The presence of a flavanone moiety was indicated by the presence of proton resonances and corresponding carbons with chemical shifts typical for flavanone methine and methylene C



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Figure 1. ECD spectra of compounds (a) 1, (b) 3, (c) 4, (d) 5, and (e) 6.

groups. The flavanone units of compounds 4−6 were identified as eriodictyol, the same as in mansoins A, B, and C. Two C6− C3−C6 moieties were also indicated for each of compounds 4− 6 by the resonances of two methine and four methylene groups, with HMBC correlations similar to those observed for compound 1. Collectively, these data indicated that compounds 4−6 are heterotrimeric flavonoids isomeric with mansoin A (1). The linkage positions between the glucopyranosyl units and the constituent phenolic moieties of compound 4 were determined using correlations between the anomeric protons at δH 4.78 (2H, d, J = 7.6 Hz) and 4.69 (1H, m) and the carbons at δC 146.6 (C-3′; flavanone), 157.7 (C-2′; side chain 2), and 157.5 (C-2′; side chain 1), respectively. These data indicated linkages to the same phenolic oxygens as defined for mansoin A. The ECD spectrum of 4 (Figure 1) showed positive and negative CEs at ca. 355 and 280 nm, respectively, permitting definition of a 2S absolute configuration for the flavanone moiety.7 In addition, the ECD spectrum showed sequential negative and positive CEs at 241 and 207 nm, respectively, the opposite of the positive couplet observed for mansoin A. When taken in conjunction with the UV maxima at 225 nm, the split CE indicated M-helicity for both the C-1

biphenylmethine chromophores, permitting assignment of the R absolute configuration for C-1 of both 1,3-diarylpropyl moieties of mansoin D (4) (vide supra). The structure of compound 4 (mansoin D) was therefore defined as (2S)-6,8di[(1R)-(2-O-β- D -glucopyranosyl-4-hydroxyphenyl)-3-(4hydroxyphenyl)propyl]eriodyctiol-3′-O-β-D-glucopyranoside. Similar to mansoins A−D (1−4), the linkages of the glucopyranosyl moieties in mansoin E (5) were established by HMBC correlations between the anomeric protons at δH 4.80 (d, 7.8 Hz), 5.24 (d, 7.8 Hz), and 4.58 (d, 7.5 Hz) with the carbons at δC 146.6 (C-3′; flavanone), 156.5 (C-4′; side chain 1), and 157.3 (C-2′; side chain 2), respectively. Thus, mansoin E (5) was determined to be a regioisomer of mansoin A (1), with one glucopyranosyl unit attached to C-4′ instead of to C2′ of side chain 1. The ECD spectrum (Figure 1) of mansoin E (5) showed low-amplitude positive and negative CEs near 330 and 290 nm, respectively, indicating a 2S absolute configuration for the flavanone moiety. Comparison of the ECD spectra of mansoins E (5) and A (1) indicated that the positive CE near 230 nm likely represented the higher amplitude component of a positive couplet, with the lower amplitude component being D



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Table 2. 13C NMR Spectroscopic Data in Methanol-d4 of Mansoins A (1), C (3), D (4), E (5), and F (6)a position 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′

a

mansoin A (1)7

mansoin C (3)

mansoin D (4)

mansoin E (5)

mansoin F (6)

δC, type

δC, type

δC, type

δC, type

δC, type

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

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

1 2 3 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″/6″ 3″/5″ 4″

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,

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

1 2 3 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″/6″ 3″/5″ 4″

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,


1

103.5, CH

1

104.0, CH

1

103.6, CH

Flavanone Moiety 80.5, CH 80.6 CH 43.8, CH2 44.4, CH2 198.1, C 198.5, C 163.4, C 160.6, C 95.7, CH 112.3, C 167.3, C 163.1, C 111.8, C 111.5, C 162.6, C 161.1, C 103.6, C 104.6, C 131.8, C 132.0, C 116.4, CH 117.7, CH 146.6, C 146.6, C 148.7, C 148.7, C 117.1, CH 117.3, CH 123.8, CH 123.9, CH Side Chain 1 35.1, CH 34.9, CH2 35.2, CH2 125.1, C 157.5, C 104.1, CH 157.0, C 110.2, CH 131.0, CH 135.2, C 130.4, CH 115.9, CH 155.9, C Side Chain 2 34.1, CH 35.4, CH 36.1, CH2 34.9, CH2 34.8, CH2 35.2, CH2 125.3, C 125.7, C 156.9, C 157.7, C 103.0, CH 104.5, CH 156.9, C 157.2, CH 109.4, CH 110.7, CH 131.4, CH 131.0, CH 135.3, C 135.3, C 130.2, CH 130.3, CH 116.0, CH 116.1, CH 155.9, C 156.0, C glc-1 103.3, CH 104.4, CH glc-2 103.9, CH glc-3 102.0, CH 104.1, CH

80.6, 44.5, 198.9, 160.8, 112.0, 163.5, 111.6, 162.1, 106.4, 132.0, 117.3, 146.6, 148.8, 116.7, 123.7,

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

80.9, 44.8, 198.9, nd 112.6, nd 111.9, nd 104.0, 132.2, 117.0, 146.7, 148.6, 116.3, 123.3,

CH CH2 C

C C CH C C CH CH

34.8, 35.0, 35.0, 124.4, 156.5, 109.3, 157.2, 110.8, 131.2, 135.0, 130.3, 116.0, 156.0,


34.9, 34.4, 34.3, 124.5, 156.8, 104.5, 157.2, 110.7, 130.8, 134.9, 130.4, 116.1, 155.9,


34.9, 35.2, 37.0, 127.1, 156.9, 109.3, 157.3, 110.7, 131.0, 135.1, 130.5, 116.0, 156.0,


35.4, 34.8, 36.4, 126.8, 157.1, 104.5, 157.5, 109.6, 130.8, 134.9, 130.4, 116.0, 156.0,


C C

104.2, CH

103.7, CH

102.6, CH

103.2, CH

103.0, CH

103.4, CH

Data acquired on a Bruker AVIII NMR spectrometer, at 100 MHz (nd, not determined).

obscured by the CEs of the 1La electronic transitions of the aromatic chromophores. The presence of the positive couplet was strongly supported by the UV absorption maximum at 225 nm. Therefore, both 1,3-diarylpropyl moieties in 5 were assigned with a C-1 (S) absolute configuration. The differential location of one of the glucopyranosyl moieties presumably affects the overall conformation of the molecule and therefore

affects the shape of the positive CE couplet. Collectively, these data permitted identification of the structure of compound 5 (mansoin E) as (2S)-6-[(1S)-(4-O-β-D-glucopyranosyl-2-hydroxyphenyl)-3-(4-hydroxyphenyl)propyl]-8-[(1S)-(2-O-β-Dglucopyranosyl-4-hydroxyphenyl)-3-(4-hydroxyphenyl)propyl]eriodictyol-3′-O-β-D-glucopyranoside. E



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dins inhibited NF-κB at different levels in the activation pathway.17 Mackenzie and co-workers reported the inhibition of the binding of NF-κB proteins to its κB DNA consensus sequence in whole cells, nuclear fractions, and purified chemical systems for the procyanidin dimers A1, A2, B1, and B2.18 They established a structure−activity relationship for the dimers evaluated, where the presence of select OH groups in the B2 dimer was required for inhibition. They proposed that the orientation of the hydroxy groups in the folded structure of B2, in which ring B′ stacks onto ring A, mimics the guanine pairs in the κB DNA sequence that specifically interact with NF-κB proteins. The A-type dimers, which have different spatial arrangements, did not, however, inhibit the binding of NF-κB proteins to their κB DNA sequence.16,17 Whether mansoin A (1) or F (6) can also act by inhibiting NF-κB binding to κB sites is currently being investigated. In summary, four new hetero-oligomeric glucosylated flavonoids, mansoins C−F (3−6), were obtained from M. hirsuta fruits. Mansoin F (6) inhibited TNF-α release by LPSstimulated THP-1 cells and reduced the phosphorylation levels of p65-NFκB, when assayed at 50 μM. These results indicate the potential anti-inflammatory properties of compound 6, probably due to inhibition of the NF-κB pathway.

The linkage positions for the two glucopyranosyl units in compound 6 were determined from correlations between the anomeric protons at δH 4.76 (d, 7.4 Hz) and 4.65 (d, 7.5 Hz) and the carbons at δC 146.8 (C-3′; flavanone) and 157.5 (C-2′; side chain 1), respectively. The linkage position for the third glucopyranosyl unit of mansoin F (6) could not be defined due to the absence of HMBC correlations between the anomeric proton (δH 5.16) and the flavanyl substructure. In the ECD spectrum of mansoin F (6), the positive and negative CEs near 330 and 290 nm, respectively, indicated a 2S configuration for the eriodictyol moiety. The positive CE at ca. 220 nm indicated that both mansoins F (6) and A (1) have the same S absolute configurations at the C-1 stereogenic centers of the 1,3diarylpropyl moieties. The positive CE at ca. 220 nm in the spectrum of 6 showed the same sign and shape as those of 5, indicating that mansoin F (6) is a regioisomer of mansoins A (1) and E (5). The potential anti-inflammatory activity of compound 6 was evaluated by measuring TNF-α release in LPS-stimulated THP1 cells. Mansoin F (6) reduced TNF-α release in comparison with untreated cells, with an IC50 value of 19.3 ± 1.3 μM (Figure 2). The low amounts of mansoins C−E precluded their biological evaluation.

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EXPERIMENTAL SECTION

General Experimental Procedures. UV and ECD spectra were obtained on a JASCO J-815 spectrometer (JASCO Inc., Easton, MD, USA) using high-purity 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 NMR spectrometer (Bruker Biospin AG, Switzerland) with tetramethylsilane as 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 was performed on a Waters 600 HPLC system equipped with a Delta 600 quaternary solvent pump, an in-line degasser, a 2996 photodiode array detector, and Empower 2 software (Waters, Milford, MA, USA). The isolation and purification of the mansoins were carried out using C18 Luna columns (250 × 21.2 mm i.d., 5.0 μm and 250.0 × 10 mm i.d., 5.0 μm) (Phenomenex, Torrance, CA, USA). TLC was performed on silica gel 60 F254 plates (0.20 mm thickness, Merck, Germany). The optical density 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 the TNF-α assay was purchased from R&D Systems (DY210 TNF-α 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. Júlio A. Lombardi (UFMG). A voucher specimen (BHCB 23862) was deposited at the herbarium of Instituto de Ciências Biológicas UFMG. 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 dried in a desiccator to give a light brown residue, coded MHFr (4.6 g). A portion of MHFr (1.0 g) was fractionated by preparative HPLC on an ODS column (Phenomenex Luna, 250 × 21.2 mm i.d., 5 μm) using a gradient elution of H2O (A) and MeCN (B) as described in a previous communication7 to give eight fractions. Fractions 1 (13− 15 min), 3 (16−17 min), 5 (19−20 min), and 6 (20−21 min) were additionally purified over a Phenomenex ODS column (10.0 × 250 mm i.d., 5.0 μm), using similar chromatographic conditions at a flow rate of 3 mL/min to afford mansoins C (3) (2.5 mg, tR = 14.2 min), D

Figure 2. Concentration−response inhibition of TNF-α release (%) in LPS-stimulated cells elicited by mansoin F (6). Levels of TNF-α were measured by ELISA. Each point represents the mean ± SEM of three replicates.

The effects of mansoins A (1) and F (6) on the phosphorylation levels of the main NF-κB subunit p65/RelA were also evaluated. This subunit is a transcription factor that regulates and is regulated by several proteins involved in inflammation.14 Both mansoins A (1) and F (6) reduced the levels of phosphorylated p65/RelA-NFκB (Figure 3). These mansoins also inhibited NF-κB nuclear translocation (data not shown), indicating that they can inhibit the proinflammatory signaling at different levels. Under resting conditions, NF-κB is inactive due to association with its inhibitors, IκBs. LPS triggers the activation of NF-κB through the classical pathway. The IKκB kinase complex is activated, leading to the phosphorylation of IκB-α. Following phosphorylation, the IκB-α is ubiquitinated and degraded by proteasomes, allowing NF-κB dimers to be phosphorylated. Phosphorylated NF-κB dimers are transported into the nucleus and promote the transcription of several genes, primarily those of pro-inflammatory cytokines, such as TNF-α and IL-1β.15 Several flavonoids have been reported to interfere with NFκB activation. Luteolin suppressed LPS-induced NF-κB signaling and pro-inflammatory gene expression in intestinal epithelial cells and dendritic cells.16 Epicatechin and procyaniF



Article

Figure 3. Effect of mansoins A (1) and F (6) on the phosphorylation levels of p65/RelA-NFκB. THP-1 cells were pretreated for 3 h with 1 or 6 before LPS addition to the cells. Western blot analyses were performed from whole cell extracts to detect phosphorylation levels of p65-NFκB (upper panel). Membranes were reprobed with anti β-actin for loading control. Densitometric analysis of Western blot membranes by ImageJ software is presented as arbitrary units. Results are expressed as mean ± SEM and include data from two independent experiments performed in triplicate. *p < 0.05, when compared to untreated cells (Unt), and #p < 0.05, when compared to cells challenged with LPS only. (4) (4.7 mg, tR = 16.7 min), E (5) (6.5 mg, tR = 18.3 min), and F (6) (23.3 mg tR = 20.8). Mansoin C (3): pale yellow powder; UV (MeOH) λmax 225, 284, 325 nm; ECD (c 0.06, MeOH) λ (θ) 211 (−4.4 × 103); 238 (1.9 × 103), 292 (−2.0 × 102), 316 (1.3 × 102), 342 (1.0 × 102); 1H and 13C NMR data, Tables 1 and 2; HRESITOFMS m/z 877.2547 [M + Na]+ (calcd for C42H70O27Na 877.2531). Mansoin D (4): pale yellow powder; UV (MeOH) λmax 225, 284, 325 nm; ECD (c 0.02, MeOH) λ (θ) 207 (2.2 × 105), 241 (−1.01 × 105), 295 (−2.8 × 104), 349 (4.9 × 103); 1H and 13C NMR data, Tables 1 and 2; HRESITOFMS m/z 1281.4049 [M + Na]+ (calcd for C63H70O27Na 1281.4104). Mansoin E (5): pale yellow powder; UV (MeOH) λmax 225, 284, 325 nm; ECD (c 0.02, MeOH) λ (θ) 215 (1.8 × 105), 230 (1.4 × 105), 285 (−2.6 × 104), 295 (−2.5 × 104), 353 (1.6 × 104); 1H and 13C NMR data, Tables 1 and 2; HRESITOFMS m/z 1281.4042 [M + Na]+ (calcd for C63H70O27Na 1281.4104). Mansoin F (6): pale yellow powder; UV (MeOH) λmax 225, 284, 325 nm; ECD (c 0.04, MeOH) λ (θ) 220 (8.3 × 104), 284 (−2.4 × 104), 295 (−2.5 × 104), 318 (9.6 × 103), 341 (7.6 × 103); 1H and 13C NMR data, Tables 1 and 2; HRESITOFMS m/z 1281.4048 [M + Na]+ (calcd for C63H70O27 [M + Na]+ 1281.4104). TNF-α Production Assay. The assay was performed using THP-1 cells (ATCC TIB-202) as described previously.7 The cells were pretreated with mansoin F (6) at concentrations from 3.9 to 250 μM for 3 h. LPS (100 ng/mL, Sigma-Aldrich, St. Louis, MO, USA) was employed as the inflammatory stimulus. TNF-α release was measured using an ELISA assay according to the manufacturer’s instructions (TNF-α duo set, DY210, R&D Systems). Cell viability was evaluated by the MTT method19 using untreated cells as the reference for viability. Samples were considered nontoxic for the THP-1 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). IC50 values were determined by nonlinear regression using GraphPad Prism, version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Experiments were performed in triplicate. Evaluation of NF-κB Inhibition by Western Blot Analysis. THP-1 cells (ATCC TIB-202) were transferred to a 12.5 cm3 culture flask at a concentration of 5 × 106 cells per flask and incubated for 18 h

with RPMI medium supplemented with 1% SFB to initiate serum starvation, which was kept throughout the experiment. The cells were pretreated with mansoins A (1) and F (6) at 50 μM for 3 h. The pretreated cells were then stimulated with LPS (100 ng/mL), incubated for 18 h, and washed with cold PBS, and whole cell extracts were prepared as described previously.20 Briefly, cells were lysed on ice with cold lysis buffer [1% (v/v) Triton X-100, 100 mmol/ L Tris/HCl, pH 8.0, 10% (v/v) glycerol, 5 mmol/L EDTA, 200 mmol/L NaCl, 1 mmol/L dithiothreitol, 1 mmol/L phenylmethylsulfonyl fluoride, 25 mmol/L NaF, 2.5 μg/mL leupeptin, 5 μg/mL aprotinin, and 1 mmol/L sodium orthovanadate]. Lysates were centrifuged at 13000g for 10 min at 4 °C, and protein amounts were quantified using the Bradford assay reagent from Bio-Rad (Bio-Rad, Hercules, CA, USA). Extracts (30 μg) were separated by electrophoresis on a denaturing 10% polyacrylamide-SDS gel and electrotransferred to nitrocellulose membranes, as described previously.18 Membranes were blocked overnight at 4 °C with PBS containing 5% (w/v) nonfat dry milk and 0.1% Tween-20, washed three times with PBS containing 0.1% Tween-20, and then incubated with specific Pp65/RelA primary antibody (#3031, Cell Signaling Technology, Boston, MA, USA) or anti β-actin (A5316, Sigma) using a dilution of 1:1000 in phosphate-buffered saline containing 5% (w/v) BSA and 0.1% Tween-20. After washing, membranes were incubated with proper horseradish peroxidase-conjugated secondary antibody (1:3000). Immunoreactive bands were visualized by ECL detection, as described by the manufacturer (GE Healthcare, Wauwatosa, WI, USA). The levels of P-p65/RelA were quantified using a densitometric analysis software (ImageJ, Image Processing and Analysis in Java, NIH, Bethesda, MD, USA), and the values were normalized to the values of β-actin in the same sample. Changes in protein levels were estimated, and the results were expressed as P-p65/RelA/β-actin ratio, measured in arbitrary units.

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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.6b00390. HPLC profile, sugar derivatization results, 1D and 2D NMR, and HRESIMS spectra of compounds 3−6 (PDF) G



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AUTHOR INFORMATION

Corresponding Author

*Tel (F. C. Braga): +55 31 3409-6951. Fax: +55 31 3409-6935. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by CNPq, Brazil (SWE grant for P.R.V.C., 200972/2011-01; research grant 475365/2013-5), Fapemig (fellowship for P.R.V.C., BIP-0224-15), and, in part, by the United States Department of Agriculture, ARS, Specific Cooperative Agreement No. 58-6408-2-009, and from the European Community’s Seventh Framework Programme [FP72007-2013] under grant agreement no. HEALTH-F4-2011281608. We thank Dr. D. Rosado and the Department of Chemistry and Biochemistry at Mississippi College, Clinton, MS, USA, for the use of the JASCO J-815 spectrometer.

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REFERENCES

(1) De Miranda, C. S. A.; Reinhard, K. J. Mem. Ins. Oswaldo Cruz 2003, 98, 207−211. (2) Silva, D. M. Perfil Metabolômico e Farmacológico de Mansoa hirsuta DC. (Bignoniaceae) por RMN 1H. Ph.D. Thesis, Universidade Federal de Alagoas, Maceio, AL, Brazil, 2010; p 133. (3) Rocha, A. D.; Oliveira, A. B.; Souza-Filho, J. D.; Lombardi, J. A.; Braga, F. C. Phytother. Res. 2004, 18, 463−467. (4) Braga, F. C.; Wagner, H.; Lombardi, J. A.; Oliveira, A. B. Phytomedicine 2000, 7, 245−250. (5) Campana, P. R. V.; Braga, F. C.; Cortes, S. F. Phytomedicine 2009, 16, 456−461. (6) Endringer, D. C.; Valadares, Y. M.; Campana, P. R. V.; Campos, J. J.; Guimarães, K. G.; Pezzuto, J. M.; Braga, F. C. Phytother. Res. 2010, 24, 928−933. (7) Campana, P. R. V.; Coleman, C. M.; Teixeira, M. M.; Ferreira, D.; Braga, F. C. J. Nat. Prod. 2014, 77, 824−830. (8) Himmelreich, U.; Masaoud, M.; Adam, G.; Ripperger, H. Phytochemistry 1995, 39, 949−951. (9) Zheng, Q. A.; Xu, M.; Yang, C. R.; Wang, D.; Li, H. Z.; Zhu, H. T.; Zhang, Y. Nat. Prod. Bioprospect. 2012, 2, 111−116. (10) Moawad, A.; Hetta, M.; Zjawiony, J. K.; Jacob, M. R.; Hifnawy, M.; Marais, J. P. J.; Ferreira, D. Planta Med. 2010, 76, 796−802. (11) Friebolin, H. Basic One and Two-Dimensional NMR Spectroscopy; Wiley-VCH Verlag GmbH: Weinheim, 2005. (12) Ding, Y.; Li, X. C.; Ferreira, D. J. Nat. Prod. 2010, 73, 435−440. (13) Wilhelm-Mouton, A.; Bonnet, S. L.; Ding, Y.; Li, X. C.; Ferreira, D.; Van der Westhuizen, J. H. J. Photochem. Photobiol., A 2012, 227, 18−24. (14) Hayden, M. S.; Ghosh, S. Semin. Immunol. 2014, 26, 253−266. (15) Pasparakis, M. Nat. Rev. Immunol. 2009, 9, 778−788. (16) Kim, J. S.; Jobin, C. Immunology 2005, 115, 375−387. (17) Fraga, C. G.; Oteiza, P. I. Free Radical Biol. Med. 2011, 51, 813− 823. (18) Mackenzie, G. G.; Delfino, J. M.; Keen, C. L.; Fraga, C. G.; Oteiza, P. I. Biochem. Pharmacol. 2009, 78, 1252−1262. (19) Mosmann, T. J. Immunol. Methods 1983, 65, 55−63. (20) Sousa, L. P.; Lopes, F.; Silva, D. M.; Tavares, L. P.; Vieira, A. T.; Rezende, B. M.; Carmo, A. F.; Russo, R. C.; Garcia, C. C.; Bonjardim, C. A.; Alessandri, A. L.; Rossi, A. G.; Pinho, V.; Teixeira, M. M. J. Leukocyte Biol. 2010, 87, 895−904.

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