TNF-α inhibition elicited by mansoins A and B, heterotrimeric flavonoids isolated from mansoa hirsuta

Article abstract of DOI:10.1021/np400929g

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 1,3-diarylpropane C₆-C₃-C₆ units. Their structures and absolute c

Full text of DOI:10.1021/np400929g

Article
pubs.acs.org/jnp
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 Fernao C. Braga*,†
̃
†Faculty of Pharmacy, Universidade Federal de Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil
‡Division of Pharmaceutical Sciences, Fundaca
̧ o 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 hetero-
trimeric flavonoids with a flavanone core linked to two 1,3-
diarylpropane 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).
and inflammatory bowel disease.9 Presently, there is consid-
ignoniaceae species are distributed primarily in neotropical
erable interest in finding new TNF-α inhibitors that may be
Bregions, with Brazil being regarded as the diversity center
Brazil and from Argentina to southeast Mexico. Mansoa species
́
found in the Amazonian forest are popularly named “cipo-de-
for this large botanical family.1 The genus Mansoa of this family
utilized as templates for new anti-inflammatory drugs, as
comprises 11 species that occur in the dry and wet forests of
current biologically derived agents in clinical use require intra-
articular 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.
alho” (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 constitu-
ents 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
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
© 2014 American Chemical Society and
American Society of Pharmacognosy
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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.
Figure 1. Important 1H−13C HMBC (via 3J) correlations for mansoin
A (1).
1
Altogether, 17 aromatic protons were evident in the H NMR
spectra, with splitting patterns characteristic of two para-
disubstituted 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).
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 phloroglu-
cinolysis,15,16 compound 1 afforded the diglucosylated hetero-
dimers 3 and 4, formed by cleavage of the side chain bonds to
C-8 and C-6 of the flavanone moiety, respectively (Table 2).
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′, C-
2′, 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.
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 1,3-
diarylpropane 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.
3
Owing to the lack of appropriate J 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 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
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,
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1
Table 1. H and 13C NMR Data in Methanol-d4 for Mansoins A (1) and B (2)
mansoin A
mansoin B
δH (J in Hz)
a
position
δH (J in Hz)
Flavanone Moiety
δC
HMBC
δC
Flavanone Moiety
2
5.27 dd (13.3, 2.6)
80.7, CH
44.3, CH2
198.2, C
162.4, C
112.6, C
164.0, C
111.8, C
160.9, C
104, C
3, 1′, 2′, 6′
2, 4, 1′
5.20 dd (13.5, 1.9)
80.9, CH
44.6, CH2
198.9, C
161.1, C
112.3, C
164.0, C
111.5, C
162.3, C
104.9, C
131.9, C
115.0, CH
146.4, C
146.8, C
116.3, CH
119.7, CH
3
3.18 dd (16.9, 13.3); 2.64 dd (16.9, 2.6)
3.12 dd (16.8, 13.5); 2.64 m
4
5
6
7
8
9
10
1′
2′
3′
4′
5′
6′
132.1, C
116.4, CH
146.6, C
148.6, C
117.0, CH
123.6, CH
7.31 d (1.4)
2, 1′, 3′, 4′, 6′
6.95 bs
6.99 d (8.2)
6.99 dd (8.2, 1.4)
Side Chain 1
4.78 m
1, 3′, 4′, 6′
1′, 2′, 4′, 5′
6.80 m
6.80 m
Side Chain 1
4.69
b
1
35.0, CH
36.6, CH2
35.2, CH2
127.0, C
5, 1′, 2
35.0, CH
36.1, CH2
35.1, CH2
126.8, C
b
2
2.65 m; 2.15 m
2.45 m
6, 1′, 1, 3, 1″
2.66 m; 2.39 m
2.39 m
3
1, 2, 1″, 2″, 6″
1′
2′
3′
4′
5′
6′
1″
2″
3″
4″
5″
6″
157.2, C
156.9, C
6.64 d (1.4)
104.2, CH
156.8, C
1′, 2′, 4′, 5′
6.63 d (1.7)
104.9, CH
157.3, CH
110.8, CH
131.4, CH
135.0, C
6.46 dd (8.3, 1.4)
7.47 d (8.3)
110.5, CH
131.3, CH
135.1, C
1′, 3′, 4′, 6′
1, 1′, 2′, 4′, 5′
6.41 dd (8.4, 1.7)
7.22 d (8.4)
6.84 d (8.1)
6.55 d (8.1)
130.3, CH
115.9, CH
155.7, C
3, 1′, 3′/5′, 4′
1′, 2′/6′, 4′
6.82 d (7.7)
6.54 d (7.7)
130.3, CH
115.9, CH
155.7, C
6.55 d (8.1)
6.84 d (8.1)
Side Chain 2
4.69 m
115.9, CH
130.3, CH
1′, 2′/6′, 4′
3, 1′, 3′/5′, 4′
6.54 d (7.7)
6.82 d (7.7)
Side Chain 2
4.63
115.9, CH
130.3, CH
b
b
1
35.2, CH
36.2, CH2
34.8, CH2
126.7, C
7, 9, 1′, 2
35.2, CH
36.3, CH2
34.7, CH2
126.5, C
b
2
2.37 m; 2.07 m
2.37 m
8, 1′, 1, 3, 1″
2.15 m; 2.07 m
2.39 m
3
1, 2, 1″, 2″, 6″
1′, 2′, 4′, 5′
1′
2′
3′
4′
5′
6′
1″
2″
3″
4″
5″
6″
157.2, C
156.8, C
6.62 d (1.4)
104.8, CH
157.1, C
6.66 d (1.6)
104.5, CH
157.3, C
6.39 dd (8.4, 1.4)
7.13 d (8.4)
110.6, CH
131.3, CH
135.2, C
1′, 3′, 4′, 6′
1, 1′, 2′, 4′, 5′
6.46 dd (8.5, 1.6)
7.46 d (8.5)
110.7, CH
131.2, CH
134.9, C
6.71 d (8.0)
6.52 d (8.0)
130.3, CH
115.9, CH
155.8, C
3, 1′, 3′/5′, 4′
1′, 2′/6′ 4′
6.71 d (7.7)
6.52 d (7.7)
130.3, CH
115.9, CH
155.7, C
6.52 d (8.0)
6.71 d (8.0)
glc-1
115.9, CH
130.3, CH
1′, 2′/6′, 4′,
3, 1′, 3′/5′, 4′,
6.52 d (7.7)
6.71 d (7.7)
glc-1
115.9, CH
130.3, CH
b
1
1
1
4.84 d (7.3)
glc-2
103.6, CH
104.0, CH
3′
glc-2
c
4.88 d (7.4)
glc-3
2′
4.86 d (7.4)
glc-3
103.7 CH
103.4 CH
d
4.92 d (7.2)
103.5, CH
2′
4.89 d (7.3)
a
b
c
d
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.
2). Similar Cotton effects were observed for eriodictyol (data
not shown), obtained by hydrolysis of 1 (vide supra), and are
consistent with those of (2S)-flavanones.17 This configurational
assignment is consistent with P-helicity of the heterocycle
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1
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.
Table 2. H NMR Data (400 MHz, Methanol-d4) for
Compounds 3 and 4
3
4
position
δH (J in Hz)
δH (J in Hz)
Flavonoids are secondary metabolites involved in several
aspects of plant development and defense. Antioxidant,
antihypertensive, anti-inflammatory, and antiproliferative activ-
ities have been described for these compounds.20,21 The anti-
inflammatory 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
Flavanone Moiety
Flavanone Moiety
2
3
5.25 dd (13.1, 3.2)
5.24 dd (12.5, 2.5)
3.09 dd (16.9, 13.1); 2.63 dd
(16.9, 3.2)
3.03 dd (16.9, 12.5); 2.60 dd
(16.9, 2.5)
6
5.86 s
8
5.87 s
2′
5′
6′
7.34 d (1.6)
6.94 d (8.4)
7.04 dd (8.4, 1.6)
Side Chain 1
4.78 m
7.22 d (1.4)
7.08 d (8.3)
6.94 dd (8.3, 1.4)
Side Chain 2
4.91 m
1
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.
2
2.56 m; 2.11 m
2.43 m
2.66 m; 2.39 m
2.39 m
3
3′
5′
6′
6.56 d (2.4)
6.37 dd (8.4, 2.4)
7.49 d (8.4)
6.63 d (1.7)
6.41 dd (8.4, 1.7)
7.22 d (8.4)
6.82 d (7.7)
6.54 d (7.7)
2″/6″ 6.86 d (8.4)
3″/5″ 6.65 d (8.4)
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 1,3-
diarylpropane moieties. This assignment is based on compar-
ison 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-glucopyranosyleryodyc-
tiol and (2S)-6,8-di[(1S)-(2′-O-β-D-glucopyranosyl-4′-hydrox-
yphenyl)-3-(4″-hydroxyphenyl)propyl]eryodyctiol, respec-
tively.
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 LPS-
stimulated 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
EXPERIMENTAL SECTION
■
General Experimental Procedures. UV and ECD absorbance
spectra were obtained on a JASCO J-815 spectrometer 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. H and 13C NMR spectra were
1
recorded in deuterated solvents on a Bruker AVIII 400 Ultra Shield
Figure 2. ECD spectra for compounds 1 (A) and 2 (B).
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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).
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
1
C63H70O27 [M + Na]+ 1281.4104); H 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
1
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
C63H70O27 [M + Na]+ 1119.3576); H 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
herbarium of the Instituto de Cien
number BHCB 23862.
̂ ́
cias Biologicas UFMG under the
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
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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 RP-
HPLC (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.
ACKNOWLEDGMENTS
■
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 Agree-
ment number 58-6408-2-009, and from the European
Community’s Seventh Framework Programme [FP7-2007-
2013] under grant agreement number HEALTH-F4-2011-
281608. 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.
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 (Sigma-
Aldrich), 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 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). 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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ASSOCIATED CONTENT
* Supporting Information
1D- and 2D-NMR spectra and HRMS of compounds 1 and 2
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AUTHOR INFORMATION
Corresponding Author
*Tel: +55 31 3409-6951. Fax: +55 31 3409-6935. E-mail:
fernao@netuno.lcc.ufmg.br.
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Notes
The authors declare no competing financial interest.
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