HPLC-ESIMSn profiling, isolation, structural elucidation, and evaluation of the antioxidant potential of phenolics from Paepalanthus geniculatus

Article abstract of DOI:10.1021/np200604k

The methanol extract of the flowers of Paepalanthus geniculatus Kunth. showed radical-scavenging activity in the TEAC assay. An analytical approach based on HPLC-ESIMSⁿ was applied to obtain the metabolite profile of this extract and led to the rapid identification of 19 polyphenolic compounds comprising flavonoids and naphthopyranones. The new naphthopyranone (10, 16), quercetagetin (1, 5, 7, 13), and galetine derivatives (9, 11, 17, 19), and a flavonol glucoside cyclodimer in the truxillate form (12), were identified. Compounds 2, 6, and 7 showed the highest antioxidant capacity and ability to affect the levels of intracellular ROS in human prostate cancer cells (PC3).

Full text of DOI:10.1021/np200604k

Article
pubs.acs.org/jnp
n
HPLC-ESIMS Profiling, Isolation, Structural Elucidation, and
Evaluation of the Antioxidant Potential of Phenolics from
Paepalanthus geniculatus
†
‡
‡
†
Fabiano Pereira do Amaral, Assunta Napolitano, Milena Masullo, Lourdes Campaner dos Santos,
‡
†
‡
,‡
Michela Festa, Wagner Vilegas, Cosimo Pizza, and Sonia Piacente*
‡Dipartimento di Scienze Farmaceutiche e Biomediche, Universita
SA), Italy
̀
degli Studi di Salerno, Via Ponte Don Melillo, 84084 Fisciano
(
†
Institute of Chemistry, Organic Chemistry Department, UNESP, Sao
Sao Paulo, Brazil
̃
Paulo State University, CP 355, CEP 14800-900 Araraquara,
̃
*
S Supporting Information
ABSTRACT: The methanol extract of the flowers of
Paepalanthus geniculatus Kunth. showed radical-scavenging
activity in the TEAC assay. An analytical approach based on
n
HPLC-ESIMS was applied to obtain the metabolite profile of
this extract and led to the rapid identification of 19 polyphenolic
compounds comprising flavonoids and naphthopyranones. The
new naphthopyranone (10, 16), quercetagetin (1, 5, 7, 13), and
galetine derivatives (9, 11, 17, 19), and a flavonol glucoside
cyclodimer in the truxillate form (12), were identified.
Compounds 2, 6, and 7 showed the highest antioxidant capacity
and ability to affect the levels of intracellular ROS in human
prostate cancer cells (PC3).
Paepalanthus, the largest genus of the Eriocaulaceae family,
includes approximately 500 species distributed in Africa and
Central and South America. Most of the known species grow in
of the intracellular reactive oxygen species (ROS) levels in
human prostate cancer cells (PC3) through flow cytometry.
1
RESULTS AND DISCUSSION
Brazil and contain bioactive naphthopyranones and flavo-
■
2
−6
noids.
Glycosides of the naphthopyranone paepalantine are
A preliminary metabolite fingerprint of the MeOH extract of
the flowers of P. geniculatus was obtained by negative HPLC-
reported to exert a cytotoxic activity comparable to that of
7
8
9
n
n
cisplatin and a lower mutagenic activity than paepalantine.
ESIMS . Analysis of the ESIMS of the 19 compounds revealed
the presence of glycosides of flavonoids (1−9, 11−13, 17−19)
and naphthopyranones (10, 14−16) (Supporting Information).
The MeOH extract was fractionated over Sephadex LH-20,
and the fractions were rechromatographed by RP-HPLC-UV to
yield compounds 1−19. The known patuletin 3-O-β-D-
Some naphthopyranones interfere with DNA repair systems,
showing beneficial properties as potential anticancer drugs.
1
0
11
Paepalantine displays antibiotic, mutagenic, in vivo and in
1
2
13
vitro cytotoxicity, and intestinal anti-inflammatory effects.
The remarkable antioxidant activity of paepalantine
1
4
17
prompted us to evaluate the antioxidant capacity of the
methanol extract of the flowers of P. geniculatus Kunth., a
species not previously investigated.
rutinoside (2), 6-methoxykaempferol-3-O-α-L-rhamnopyra-
18
nosyl-(1→2)-β-D-glucopyranoside (3), patuletin 3-O-β-D-
17
glucoside (4), 6-methoxykaempferol-3-O-α-L-rhamnopyrano-
1
9
The good antioxidant activity in the Trolox Equivalent
syl-(1→6)-β-D-glucopyranoside (6), 6-methoxykaempferol 3-
1
5,16
20
Antioxidant Capacity (TEAC) assay
(TEAC value = 1.479
O-β-D-glucoside (8), paepalantine-9-O-α-D-glucopyranosyl-
4
mM) encouraged us to investigate its constituents. An
(1→6)-α-D-glucopyranoside (14), paepalantine-9-O-β-D-
allopyranosyl(1→6)-α-D-glucopyranoside (15), and 6-methox-
ykaempferol-3-O-β-D-6″-(p-coumaroyl)glucopyranoside (18)
3
n
analytical approach, based on HPLC-ESIMS , was applied to
21
rapidly obtain a metabolite profile of the MeOH extract of the
flowers of P. geniculatus. This guided the isolation of 19
polyphenolic compounds. The new compounds comprise two
naphthopyranone (10, 16), four quercetagetin (1, 5, 7, 13), and
four galetine derivatives (9, 11, 17, 19) and one flavonol
glucoside cyclodimer (12).
were identified by comparison of their observed and reported
NMR and ESIMS data.
The positive HR-MALDI-TOFMS spectrum of 12 showed
+
an [M + H] ion at m/z 1249.3037, suggesting the molecular
The antioxidant activities of the isolated metabolites were
evaluated by the TEAC assay and by measuring the reduction
Received: July 19, 2011
Published: April 17, 2012
©
2012 American Chemical Society and
American Society of Pharmacognosy
547
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Journal of Natural Products
Article
two signals at δ 3.87 and 3.83, corresponding to two methoxy
1
groups (Table 1). Moreover, the H NMR spectrum displayed
two signals at δ 5.33 (d, J = 8.8 Hz) and 5.03 (d, J = 8.8 Hz),
which, on the basis of 1D-TOCSY, DQF-COSY, HSQC, and
HMBC experiments, were assigned to the anomeric protons of
two β-glucopyranosyl units. A detailed analysis of the NMR
data confirmed the presence of two 6-methoxykaempferol
moieties. Significant downfield shifts of the C-6 methylene
protons of the glucose units suggested that both C-6 hydroxy
groups of the glucosyl units in 12 were acylated. The presence
of a di(4-hydroxyphenyl)cyclobutanedicarboxylic lignan-type
moiety was indicated by the aromatic proton signals at δ 6.60
(
8
2H, d, J = 8.5 Hz), 6.56 (2H, d, J = 8.5 Hz), 6.85 (2H, d, J =
.5 Hz), and 6.66 (2H, d, J = 8.5 Hz) and four methine proton
signals at δ 4.07, 3.69, 3.47, and 3.44 (each ddd, J = 10.2, 8.6,
2
.3 Hz) (Table 1). In the 13C NMR spectrum, signals due to
3
the acyl moieties at δ 173.2 and 171.4 and four sp carbons at δ
4
8.5, 47.2, 41.9, and 40.9 were observed. The COSY
experiment showed the sequence δ 4.07, 3.47, 3.69, 3.44, and
this last signal correlated with the signal at δ 4.07, confirming
the occurrence of a cyclobutane ring. The HSQC spectrum
22
showed correlations between the proton signals at δ 4.07, 3.47,
3
4
.69, and 3.44 and the corresponding carbons at δ 40.9, 48.5,
1.9, and 47.2, respectively. On this basis, the acyl units of 12
could be either truxinyl type ([7.7′, 8.8′]-lignan) or truxillyl type
23
(
[7.8′, 8.7′]-lignan). HMBC correlations showed that the
signals at δ 48.5 and 47.2 were attributable to the carboxyl-
bearing carbons, while the signals at δ 41.9 and 40.9 were due
to carbons linked to the 4-hydroxyphenyl moieties. In
particular, correlations of the proton signal at δ 4.07 (H-7‴)
with the carbon resonance at δ 128.8 (H-2‴/H-6‴), the proton
signal at δ 3.69 (H-7‴) with the carbon resonance at δ 129.4
(
H-2‴/H-6‴), and both protons at δ 4.07 and 3.69 with both
formula C H O (calcd for C H O , 1249.3031). The
carboxyl carbons at δ 173.2 and 171.4 suggested the placement
of the acyl units as in a truxillyl derivative (Figure 1), as
confirmed by the absence in the ROESY spectrum of
correlations between the aromatic signals H-2‴/H-6‴ of a 4-
hydroxyphenyl group and H-2‴/H-6‴ of the other 4-
hydroxyphenyl group. The HMBC correlations of both H-7‴
with both carboxylic carbons would not be expected in a
truxinyl derivative (e.g., monochaetin in Figure 1). The
orientation of the cyclobutane substituents was established by
a ROESY experiment, which showed correlations of the
equivalent H-2‴/H-6‴ at δ 6.85 with both H-7‴ and H-8‴
at δ 3.47. Further correlations were observed between H-2‴/H-
6‴ (δ 6.60) and both H-7‴ and H-8‴ at δ 3.44. The presence
62
56 28
62 57 28
ESIMS spectrum of 12 in the negative ion mode displayed an
−
2
[
M − H] ion at m/z 1247. The ESIMS spectrum showed an
−
ion [(M − 316) − H] at m/z 931, originating from the neutral
loss of a galetine moiety, and a minor ion at m/z 623, due to
loss of a 308 amu fragment. The ESIMS spectrum of the ion at
3
m/z 623 yielded a major ion at m/z 315, due to the loss of a
3
08 amu fragment, thus suggesting a dimeric structure of 12
comprising two galetine units linked to a 308 amu moiety.
1
The H NMR spectrum of compound 12 showed signals
ascribable to ortho-coupled protons at δ 8.11 (2H, d, J = 8.6
Hz), 7.97 (2H, d, J = 8.5 Hz), 6.80 (2H, d, J = 8.6 Hz), and
6.76 (2H, d, J = 8.5 Hz), two singlets at δ 6.32 and 6.27, and
5
48
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Table 1. 13C and H NMR Data (J in Hz) of Compounds 12 and 19 (600 MHz, δ ppm, in methanol-d )
1
4
1
2
19
δH (J in Hz)
δC
160.1
δH (J in Hz)
δC
2
3
4
5
6
7
8
9
1
1
2
3
4
5
6
159.6
135.5
179.0
150.7
132.5
159.0
95.5
133.6
178.0
150.0, 150.1
131.0
157.1
94.9, 95.3
152.0
6.32, 6.27, s
6.47, s
153.4
105.6
123.2
131.9
116.0
161.3
116.0
131.9
60.8
0
′
104.6
121.0
′
132.1, 131.9
115.8
8.11, d (8.6), 7.97 d (8.5)
6.80, d (8.6), 6.76, d (8.5)
8.03, d (8.5)
6.85, d (8.5)
′
′
157.1
′
115.8
6.80, d (8.6), 6.76, d (8.5)
8.11, d (8.6), 7.97, d (8.5)
3.87, 3.83, s
6.85, d (8.5)
8.03, d (8.5)
3.85, s
′
132.1, 131.9
60.7
OCH (C6)
3
β-D-Glc at C-3
103.1
β-D-Glc at C-3
1″
2″
3″
4″
5″
6″
5.33, d (8.8), 5.03, d (8.8)
103.5
75.5
77.8
71.5
75.5
63.8
5.26, d (8.8)
75.4
3.54, dd (8.8, 9.0), 3.48, dd (8.8, 9.0)
3.49, dd (8.8, 9.0)
3.47, dd (9.0, 9.0)
3.37, dd (9.0, 9.0)
3.47, m
77.4
3.46, dd (9.0, 9.0), 3.46, dd (9.0, 9.0)
71.5
3.15, dd (9.0, 9.0), 3.05, dd (9.0, 9.0)
76.1, 75.7
65.5, 64.5
3.25, m, 2.75, m
3.95, dd (2.5, 12.0), 4.41, dd (2.5, 12.0), 3.79, dd (4.5, 12.0), 3.37, dd (4.5, 12.0)
4.31, dd (2.5, 12.0)
4
.26, dd (4.5, 12.0)
Truxillyl moiety
114.6
Acyl moiety
127.5
1‴
2‴
3‴
4‴
5‴
6‴
7‴
128.8, 129.4
115.7
6.60, d (8.5), 6.85, d (8.5)
6.56, d (8.5), 6.66, d (8.5)
111.5
7.10, d (1.9)
149.5
156.9, 157.2
115.7
149.8
6.56, d (8.5), 6.66, d (8.5)
6.60, d (8.5), 6.85, d (8.5)
4.07, ddd (10.2, 8.6, 2.3)
116.3
6.84, d (8.5)
128.8, 129.4
40.9, 41.9
123.5
6.94, dd (1.9, 8.5)
6.15, d (16.0)
114.5
3
.69, ddd (10.2, 8.6, 2.3)
3.47, ddd (10.2, 8.6, 2.3)
.44, ddd, (10.2, 8.6, 2.3)
8
‴
‴
48.5, 47.2
146.4
7.43, d (16.0)
3.93, s
3
9
173.2, 171.4
168.5
56.2
OCH (C3‴)
3
2
of the two 6-methoxykaempferolglucoside moieties and the
probable puckering of the cyclobutane ring caused the upfield
shift of the cyclobutane proton resonances, compared to those
ESIMS spectrum highlighted the presence of the aglycone ion
at m/z 331, due to contemporary loss of two sugar units.
1
The H NMR spectrum of compound 1 was similar to that of
24
17
of the corresponding protons of a truxillate acid. Moreover,
the nonequivalence of NMR signals was due to the presence of
the chiral glucose moieties, as previously reported for
stachysetin, a dimer of apigenin-7-O-(6-p-coumaroyl)-β-D-
patuletin 3-O-β-D-rutinoside (2), except for the downfield
shift of H-8 (δ 6.79) and the different chemical shift of the
methoxy group (δ 4.02) (Table 2). On the basis of the
correlation between the proton signal at δ 4.02 and the carbon
resonance at δ 155.7 (C-7), the methoxy group was located at
C-7. Thus compound 1 is the new 6-hydroxy-7-methoxyquer-
cetin-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside.
The positive HR-MALDI-TOFMS spectra of compounds 5
and 7 suggested the molecular formula C H O . The
25
glucopyranoside in the truxinate arrangement.
Geniculatin (12) represents a head-to-tail dimer of
compound 18, formed via photoinduced [2+2]-cycloaddition
of two cinnamic acid moieties. Dimeric products including
cyclobutane rings, produced from various phenylpropanoid
30
34 18
26
acids, have been previously reported. However, stachysetin
negative ESIMS spectra of the two compounds showed an
[M − H] ion at m/z 681. Analysis of the ESIMS spectra of 5
and 7 showed the same fragmentation pattern and ascertained
24
−
2
from Stachys aegyptiaca and monochaetin from Monochaetum
2
6
multiflorum are flavone glycoside dimers in the truxinate form
Figure 1). Thus, this is the first report of a diflavonoid ester of
−
(
the presence of an acetyl moiety {[(M − 42) − H] , m/z 639},
−
dicarboxylic acid in the truxillate arrangement.
a deoxyhexose {[(M − 42 − 146) − H] , m/z 493}, and a
−
Compound 1 exhibited in the positive HR-MALDI-TOFMS
hexose {[(M − 42 − 146 − 162) − H] , m/z 331} moiety.
+
1
spectrum an [M + H] ion at m/z 641.1716, corresponding to
The H NMR spectrum of compound 5 was similar to that of
17
the molecular formula C H O . The negative ESIMS
patuletin 3-O-β-D-rutinoside (2), except for the downfield
shift observed for H-2rha (δ 4.80) and an additional signal at δ
2
8
32 17
−
spectrum displayed an [M − H] ion at m/z 639. The
5
49
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Journal of Natural Products
Article
2
.11 (s) corresponding to an acetyl function (Table 3). In the
HMBC spectrum, correlations between the carbon resonance at
δ 172.6 and the proton signals at δ 4.80 (H-2 ) and 2.11
rha
confirmed the presence of an acetyl group at C-2 . Thus,
rha
compound 5 is the new 6-methoxyquercetin-3-O-(2-O-acetyl)-
α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside.
The NMR data of compound 7, in comparison with those of
5
, suggested a different position of the acetyl group. In the
COSY experiment, the downfield shifted signal at δ 4.82 was
assigned to H-4rha (Table 3). HMBC correlations between the
carbon resonance at δ 172.8 and the proton signals at δ 4.82
(
H-4 ) and 2.04 confirmed the location of the acetyl function
rha
at C-4 . Thus, compound 7 is the new 6-methoxyquercetin-3-
rha
O-(4-O-acetyl)-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyrano-
side.
The molecular formula of compound 9 was established as
+
C H O by HR-MALDI-TOFMS (m/z 667.1874 [M + H] ,
30
34 17
calcd for C H O , 667.1869). The ESIMS spectrum of
3
0
35 17
−
compound 9 showed the [M − H] ion at m/z 665. The
ESIMS spectrum showed consecutive losses of 42, 146, and
2
1
62 amu, to afford the aglycone ion at m/z 315. The NMR data
of the aglycone moiety of compound 9 (Table 2) were similar
19
to those of 6, while the NMR data of the sugar region of 9
were closely related to those of 5, showing a 2-O-
acetylrhamnopyranosyl unit linked to C-6 of the glucopyranosyl
unit. Thus, compound 9 is the new 6-methoxykaempferol-3-O-
Figure 1. Diagnostic HMBC correlations and 13C and H NMR data
1
of the cyclobutane ring in the truxillate moiety (compounds 12) and in
26
the truxinate moiety (monochaetin ).
(
2-O-acetyl)-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyrano-
side.
HR-MALDI-TOFMS of 11 suggested the same molecular
formula as 9. The NMR data of 11 were similar to those
1
13
observed for 9, except for the H and C NMR values of the
rhamnopyranosyl unit. In this case, H-4rha (δ 4.82) instead of
H-2rha was downfield shifted due to the occurrence of the acetyl
Table 2. 13C and H NMR Data (J in Hz) of the Aglycone
Moieties of Compounds 1, 5, and 9 (600 MHz, δ ppm, in
1
a
methanol-d4)
group at C-4 , as indicated by the HMBC correlation between
rha
1
5
9
the proton signal at δ 2.04 and the carbon resonance at δ 74.9
(
C-4 ). Thus, compound 11 is the new 6-methoxykaempferol-
rha
δH (J in
Hz)
δH (J in
Hz)
δH (J in
Hz)
δC
δC
δC
3-O-(4-O-acetyl)-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyra-
noside.
2
3
4
5
6
7
8
9
1
1
2
159.7
135.5
179.0
150.5
131.4
155.7
91.3
159.7
135.5
179.0
150.7
132.4
158.6
95.0
159.4
134.9
179.4
150.8
132.7
158.6
95.0
+
The HR-MALDI-TOFMS of 13 showed the [M + H] ion at
m/z 725.1930 (calcd for C H O , 725.1924). The ESIMS
2
32 37 19
−
spectrum of the [M − H] ion at m/z 723 displayed an [(M −
−
3
4
2) − H] ion at m/z 681 whose ESIMS spectrum was similar
1
to that of compound 5. The H NMR spectrum of compound
3 was similar to that of 5 but showed a further signal at δ 2.04,
1
6.79, s
6.55, s
6.56, s
1
corresponding to an acetyl group (Table 3). The H NMR
resonances of the sugar moiety in 13 differed from those of 5 in
the downfield shift of H-4rha (δ 4.74); this observation together
with the HMBC correlation between the proton signal at δ 2.04
151.3
106.8
123.5
153.8
106.0
123.2
153.6
105.9
122.4
0
′
′
117.4 7.72, d
1.9)
117.4 7.68, d
(1.9)
132.0 8.09, d
(8.5)
(
and the carbon resonance at δ 74.9 (C-4 ) (Table 3)
rha
3′
146.0
146.0
115.9 6.91, d
permitted the placement of a second acetyl group at C-4rha.
Thus, compound 13 is the new 6-methoxyquercetin-3-O-(2,4-
di-O-acetyl)-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside.
The HR-MALDI-TOFMS spectrum of 17 showed an [M +
(
8.5)
4
′
′
149.9
149.9
161.3
5
115.7 6.92, d
8.5)
115.8 6.91, d
(8.5)
115.9 6.91, d
(8.5)
(
+
H] ion at m/z 709.1981, suggesting the molecular formula
6′
123.3 7.67, dd,
123.5 7.66, dd
132.0 8.09, d
(8.5)
(1.9, 8.5)
(1.9, 8.5)
C H O (calcd for C H O , 709.1974). The negative
32
36 18
32 37 18
−
OCH3
C7)
56.5
4.02, s
ESIMS spectrum displayed the [M − H] ion at m/z 707, 42
amu higher than those observed for 9 and 11, suggesting the
occurrence of a further acetyl unit, as confirmed by ESIMS
spectra. H NMR data of 17 in comparison with those of 9
showed a further signal at δ 1.99, corresponding to an
additional acetyl group (Table 3). COSY correlations between
the anomeric proton of rhamnose at δ 4.52 with the signal at δ
5.02, which in turn correlated with the signal at δ 4.91,
(
OCH3
C6)
60.7
3.93, s
60.8
3.91, s
n
(
1
a
The chemical shift values of the aglycone portion of 7 and 13 were
superimposable with those reported for 5; the chemical shift values of
the aglycone portion of 11 and 17 were superimposable with those
reported for 9.
5
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Journal of Natural Products
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Table 3. 13C and H NMR Data (J in Hz) of the Sugar Portions of Compounds 1, 5, 7, 13, and 17 (600 MHz, δ ppm, in
1
a
methanol-d4)
1
5
7
13
17
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
β-D-Glc at C-3
β-D-Glc at C-3
β-D-Glc at C-3
β-D-Glc at C-3
β-D-Glc at C-3
1
2
104.1 5.16, d (8.8)
104.3 5.14, d (8.8)
103.4 5.36, d (8.8)
103.3 5.36, d (8.8)
104.0 5.15, d (8.8)
75.3
77.8
71.8
3.51, dd (8.8,
.0)
75.5
78.0
71.3
3.51, dd (8.8,
9.0)
75.4
76.6
70.9
3.54, dd (8.8,
9.0)
75.3
76.9
70.8
3.54, dd (8.8,
9.0)
75.4
77.1
71.3
3.49, dd (8.8,
9.0)
9
3
4
3.45, dd (9.0,
.0)
3.44, dd (9.0,
9.0)
3.40, dd (9.0,
9.0)
3.40, dd (9.0,
9.0)
3.39, dd (9.0,
9.0)
9
3.30 dd (9.0,
.0)
3.27 dd (9.0,
9.0)
3.41 dd (9.0,
9.0)
3.42 dd (9.0,
9.0)
3.25, dd (9.0,
9.0)
9
5
6
77.0
68.4
3.35 m
77.2
68.6
3.38 m
77.8
67.8
3.47 m
77.7
68.0
3.46 m
77.7
68.2
3.44, m
3.83, dd (2.5,
3.88, dd (2.5,
12.0)
3.84, dd (2.5,
12.0)
3.82, dd (2.5,
12.0)
3.86, dd (2.5,
12.0)
1
2.0)
3
.41, dd (4.5,
2.0)
3.40, dd (4.5,
12.0)
3.54, dd (4.5,
12.0)
3.60, dd (4.5,
12.0)
3.43, dd (4.5,
12.0)
1
α-L-Rha at C-6glc
α-L-Rha at C-6glc
α-L-Rha at C-6glc
α-L-Rha at C-6glc
α-L-Rha at C-6glc
1
2
102.0 4.54, d (1.2)
102.1 4.53, d (1.2)
101.8 4.60, d (1.2)
99.4
73.7
4.62, d (1.2)
99.1
70.7
4.52, d (1.2)
71.8
3.63, dd (1.2,
.2)
75.5
4.80, dd (1.2,
3.2)
71.9
3.73, dd (1.2,
3.2)
4.97, dd (1.2,
3.2)
5.02, dd (1.2,
3.2)
3
3
71.8
3.56, dd (3.2,
.7)
69.4
3.75, dd (3.2,
9.7)
70.0
3.72, dd (3.2,
9.7)
68.0
3.91, dd (3.2,
9.7)
72.7
4.91, dd (3.2,
9.7)
9
4
5
6
73.7
69.4
17.6
3.31, t (9.7)
3.46, m
70.7
69.5
17.5
172.6
3.50, t (9.7)
3.58, m
74.9
67.2
17.4
4.82, t (9.7)
3.60, m
74.9
67.5
17.4
170.5
4.74, t (9.7)
3.62, m
70.9
69.5
17.7
171.9
3.40, t (9.7)
3.60, m
1.14, d (6.5)
1.17, d (6.5)
0.90 d (6.5)
0.90, d (6.5)
1.17, d (6.5)
COOCH (C-
3
2rha)
COOCH (C-
20.9
2.11, s
20.6
2.09, s
20.5
172.4
20.6
2.06, s
1.99, s
3
2rha)
COOCH (C-
3
3rha)
COOCH (C-
3
3rha)
COOCH (C-
172.8
20.8
171.0
20.7
3
4rha
)
COOCH (C-
2.04, s
2.04, s
3
4rha)
a
The chemical shift values of the sugar portion of 9 and 11 were superimposable with those reported for 5 and 7, respectively.
permitted location of the acetyl groups at C-2rha and C-3rha
signals at δ 4.31 and 4.26 with the carboxylic carbon at δ 168.5
confirmed this assumption. Thus, compound 19 is the new 6-
methoxykaempferol-3-O-(6-E-feruloyl)-β-D-glucopyranoside.
The positive HR-MALDI-TOFMS spectrum of compound
(Table 3). Thus, compound 17 is the new 6-methoxykaempfer-
ol-3-O-(2,3-di-O-acetyl)-α-L-rhamnopyranosyl-(1→6)-β-D-glu-
copyranoside.
+
The HR-MALDI-TOFMS spectrum of 19 showed an [M +
10 showed an [M + H] ion at m/z 613.1770, corresponding to
+
H] ion at m/z 655.1663, supporting the molecular formula
the molecular formula C H O . The tandem ESIMS
2
7
32 16
−
C H O (calcd for C H O , 655.1657). The ESIMS
spectrum of the [M − H] ion at m/z 611 showed two ions
at m/z 449 and 287, originating from the consecutive losses of
162 amu. The fragmentation pattern of the ion at m/z 287
suggested a naphthopyranone structure (Figure 2).
3
2
30 15
32 31 15
−
spectrum of 19 showed the [M − H] ion at m/z 653. The
ESIMS spectrum of this ion showed an ion at m/z 477
2
originating from the loss of a ferulic acid moiety (176 amu).
1
The H NMR spectrum of compound 19 showed two proton
The NMR data of 10 were similar to that of paepalantine-9-
4
signals at δ 8.03 (d, J = 8.5, 2H) and 6.85 (d, J = 8.5, 2H), a
singlet at δ 6.47 assigned to H-8, and a signal at δ 3.85,
corresponding to a methoxy group (Table 1). These data, along
with those derived from HSQC and HMBC experiments,
allowed the identification of the aglycone moiety of 19 as 6-
O-α-D-glucopyranosyl-(1→6)-α-D-glucopyranoside (14), dif-
fering in the absence of the methoxy group at C-7 (Table 4).
Consequently, compound 10 is the new 7,9,10-trihydroxy-5-
methoxy-3-methyl-1H-naphtho[2,3-c]pyran-1-one-9-O-β-D-glu-
copyranosyl-(1→6)-β-D-glucopyranoside.
1
methoxykaempferol. Further signals occurring in the H NMR
The HR-MALDI-TOFMS spectrum of 16 showed an [M +
+
spectrum at δ 7.10 (J = 1.9 Hz), 6.84 (J = 8.5 Hz), 6.94 (J = 1.9,
H] ion at m/z 451.1239, suggesting the molecular formula
8
.5 Hz), 6.15 (J = 16.0 Hz), 7.43 (J = 16.0 Hz), and 3.93 (3H,
C H O (calcd for C H O , 451.1235). The full negative
21
22 11
21 23 11
−
s) were attributed to an (E)-feruloyl moiety. This evidence,
together with the occurrence of a β-glucopyranosyl unit, was
confirmed by HSQC, HMBC, and COSY correlations. The
downfield shifts of H -6 and C-6 of the glucose unit (δ 4.31
mass spectrum of 16 displayed an [M − H] ion at m/z 449.
2
−
The ESIMS spectrum showed an [(M − 162) − H] ion at m/
z 287, and multistage mass spectra of this ion indicated its
naphthopyranone nature.
2
H
1
and 4.26; δ 63.8) suggested the location of the (E)-feruloyl
The H NMR spectrum of the aglycone moiety of compound
C
moiety at C-6 . HMBC correlation between the two proton
16 was similar to that of 10, but in the sugar region showed
glc
5
51
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Journal of Natural Products
Article
n
Figure 2. ESIMS fragmentation pathway of 10.
signals for only one β-glucopyranosyl unit with the anomeric
proton at δ 5.15 (d, J = 8.8 Hz) (Table 4). A detailed analysis of
NMR data permitted identification of 16 as the new 7,9,10-
trihydroxy-5-methoxy-3-methyl-1H-naphtho[2,3-c]pyran-1-
one-9-O-β-D-glucopyranoside.
effect on PC3 cancer cells. No significant antioxidant effects
were observed with the other analyzed compounds.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a JASCO DIP 1000 polarimeter. IR measurements
were obtained on a Bruker IFS-48 spectrometer. UV spectra were
obtained on a Beckman DU 670 spectrometer. NMR experiments
were performed on a Bruker DRX-600 spectrometer at 300 K. All 2D-
The antioxidant activity of compounds 1−19 was tested by
the TEAC assay and expressed as TEAC value, defined as the
concentration of Trolox solution with antioxidant potential
15,16
equivalent to a 1 mM concentration of the test sample.
The
TEAC values of 1−19 were compared to those of quercetin,
quercetin 3-O-glucoside, and kaempferol 3-O-glucoside (Table
NMR spectra were acquired in methanol-d (99.95%, Sigma-Aldrich),
4
and standard pulse sequences and phase cycling were used for DQF-
COSY, HSQC, HMBC, ROESY, and TOCSY spectra. The ROESY
spectra were acquired with tmix = 400 ms. Exact masses were measured
by an AB SCIEX Voyager DE mass spectrometer equipped with a 337
nm laser and delay extraction and operated in positive ion reflector
mode. Samples were analyzed by MALDI-TOF mass spectrometry. A
mixture of analyte solution and α-cyano-4-hydroxycinnamic acid
(Sigma) was applied to the metallic sample plate and dried. Mass
calibration was performed with the ions from adrenocorticotropic
hormone (ACTH) fragment 18−39 human at 2465.1989 Da and α-
cyano-4-hydroxycinnamic acid at 190.0504 Da as internal standard.
5
1
). The results showed that most flavonoids (1−3, 5−9, 11, 13,
7−19) exhibited higher free-radical-scavenging activity than
naphthopyranones (14−16). In particular, flavonoids 2, 6, and
7
were more active than the reference antioxidant compounds,
with compound 7 showing the highest antioxidant activity
2.160 mM). The head-to-tail dimer 12 showed antioxidant
(
activity lower than the related flavonoid monomer 18. Among
the naphthopyranone compounds, 10 and 16 displayed the
highest TEAC values, suggesting that a free hydroxy group at
C-7 improves the radical-scavenging capacity.
n
The MeOH extract was analyzed by online HPLC-ESIMS using a
ThermoFinnigan Spectra System HPLC coupled with an LCQ Deca
The ability of several antioxidant plant phenolics to reduce
the risk and slow the progression of prostate cancer by
́
ion trap mass spectrometer (ThermoFinnigan, San Jose, CA, USA).
HPLC separation was conducted on a C18 reversed-phase (RP)
column (5 μm, 3 mm × 150 mm; 100 A; Luna PFP(2), Phenomenex,
Torrance, CA, USA) at a flow rate of 0.2 mL/min. A gradient elution
27
prevention of cell oxidation has been widely reported.
Thus, the cytotoxic activity and the ability of all compounds
to affect the levels of intracellular ROS either in the control
prostate cancer PC3 cell line or in PC3 cells simultaneously
exposed to t-BOOH, a known pro-oxidant, were evaluated. In
agreement with TEAC results, the 24 h pretreatment of PC3
cells with 10 μM 2, 6, and 7, respectively, prevented elevation
of ROS induced by t-BuOOH, suggesting the potential activity
of these compounds to protect cells from oxidative stress
was performed by using H
O (A) and CH CN (B), both added with
2
3
0
.1% HCO H, as mobile phases. After a 3 min hold at 5% B, elution
2
was performed according to the following conditions: from 5% B to
5% B in 8 min; to 26% B in 3 min, hold at 26% B for 10 min; to 27%
2
B in 3 min, hold at 27% B for 5 min; to 38% B in 8 min. The column
effluent was analyzed by ESIMS in negative ion mode, and the mass
spectra were acquired and processed using the software provided by
the manufacturer. The capillary voltage was set at −32 V, the spray
voltage at 5 kV, and the tube lens offset at 30 V. The capillary
(
Figure 3). None of the compounds had a significant cytotoxic
5
52
dx.doi.org/10.1021/np200604k | J. Nat. Prod. 2012, 75, 547−556

Journal of Natural Products
Article
Table 4. 13C and H NMR Data (J in Hz) of Compounds 10
1
Table 5. Free Radical Scavenging Activities of Compounds
1−19 and the Methanolic Extract of P. geniculatus Flowers in
the TEAC Assay
and 16 (600 MHz, δ ppm, in methanol-d4)
1
0
16
compound
TEAC value (mM ± SD)
δC
δH (J in Hz)
δC
δH (J in Hz)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
1.545 ± 0.075
2.017 ± 0.111
1.529 ± 0.038
0.525 ± 0.025
1.373 ± 0.034
2.059 ± 0.027
2.160 ± 0.031
1.267 ± 0.075
1.000 ± 0.134
1.038 ± 0.017
0.968 ± 0.036
0.738 ± 0.009
1.214 ± 0.015
0.706 ± 0.049
0.570 ± 0.021
0.898 ± 0.030
1.397 ± 0.059
1.279 ± 0.191
0.984 ± 0.041
1.866 ± 0.008
1.780 ± 0.004
1.178 ± 0.010
TEAC value (mg/mL ± SD)
1.479 ± 0.0155
1
3
4
4
5
5
6
7
8
9
9
1
1
1
169.2
153.4
99.7
169.0
152.5
99.5
6.62, s
6.65, s
a
a
124.7
140.2
137.2
99.3
124.7
139.0
137.0
99.3
7.06, d (1.5)
7.07, d (1.5)
161.5
160.5
104.7 7.12, d (1.5)
104.7 7.00, d (1.5)
0
1
2
3
4
5
6
7
8
9
160.0
110.7
160.8
98.2
159.0
a
109.6
0
160.0
0a
1
97.0
19.3
61.8
2.31, s
3.85, s
19.30 2.31, s
OCH (C5)
61.5
3.86, s
3
β-D-Glc at C-9
β-D-Glc at C-9
1
2
3
4
5
6
103.7 5.03, d (8.8)
104.0 5.15, d (8.8)
74.6
77.1
71.2
77.7
69.6
3.69, dd (8.8, 9.0)
75.4
77.1
71.3
77.7
3.49, dd (8.8, 9.0)
3.55, dd (9.0, 9.0)
3.47 dd (9.0, 9.0)
3.81 m
3.39, dd (9.0, 9.0)
3.25, dd (9.0, 9.0)
3.44, m
quercetin
quercetin 3-O-glucoside
kaempferol 3-O-glucoside
4.24, dd (2.5, 12.0) 68.2
.89, dd (4.5, 12.0)
3.86, dd (2.5, 12.0)
3.43, dd (4.5, 12.0)
3
P. geniculatus MeOH extract
β-D-Glc at C-6glc
1
2
3
4
5
6
104.6 4.48, d (8.8)
74.9
77.7
71.2
77.4
62.4
3.31, dd (8.8, 9.0)
CA, USA), using H O + TFA (0.05%) and CH CN + TFA (0.05%) as
2 3
3.27, dd (9.0, 9.0)
3.34 dd (9.0, 9.0)
3.39 m
mobile phases, at a flow rate of 2.0 mL/min. Elution was performed
according to the following conditions: from 15% B to 25% B in 30
min, hold at 25% B for 20 min; to 35% B in 30 min; to 90% B in 20
min; and to 100% in 10 min.
3.89, dd (2.5, 12.0)
The chromatographic separation of Sephadex fractions 54−57
3
.70, dd (4.5, 12.0)
yielded 16 compounds: 1 (3.9 mg, t = 22.0 min), 2 (3.9 mg, t = 25.2
R
R
min), 3 (1.5 mg, t = 27.4 min), 5 (1.8 mg, t = 28.7 min), 6 (3.3 mg,
R
R
temperature was 280 °C. Data were acquired in MS and MSn
scanning modes. By using a syringe pump (flow rate 5 μL/min),
each pure compound dissolved in MeOH was infused in the ESI
1
t = 30.8 min), 7 (1.8 mg, t = 33.3 min), 8 (2.7 mg, t = 33.6 min),
R R R
10 (4.2 mg, t = 36.2 min), 9 (3.4 mg, t = 37.7 min), 11 (4.4 mg, t =
R
R
R
39.8 min), 13 (2.9 mg, t = 43.7 min), 14 (2.5 mg, t = 47.3 min), 15
R
R
n
source. Negative ESIMS analyses were performed using the same
conditions as those for HPLC-ESIMS analysis. CC was performed
(1.3 mg, t = 51.6 min), 17 (2.8 mg, t = 56.5 min), 18 (2.2 mg, t =
R R R
n
68.2 min), and 19 (1.0 mg, t = 69.6 min).
R
over Sephadex LH-20 (Pharmacia). HPLC-UV separations were
performed on an Agilent 1100 series liquid chromatograph, equipped
with a G-1312A binary pump, a G-1328B rheodyne injector, and a G-
The chromatographic separation of Sephadex fractions 64−68
yielded three compounds: 4 (1.6 mg, t = 30.2 min), 12 (3.5 mg, t =
R
R
41.0 min), and 16 (1.2 mg, t = 54.1 min).
R
25
1
3
365B multiple wavelength detector and detecting at λ = 254, 280, and
30 nm. GC analysis was performed on a Thermo Finnigan Trace GC
Compound 1: yellow powder; [α] −20.3 (c 0.1, MeOH); UV
D
(MeOH) λmax (log ε) 360 (4.20), 260 (4.15) nm; IR (KBr) νmax 3380
(br OH), 2915 (CH), 1680 (CO), 1656 (CC), 1618 (CC),
apparatus using a l-Chirasil-Val column (0.32 mm × 25 m). TLC was
performed on silica gel F254 (Merck) plates.
Plant Material. Flowers of P. geniculatus Kunth. were collected in
October 2007 at Santana do Riacho, State Minas Gerais, Brazil, and
identified by Professor Paulo Takeo Sano, from the IB-USP. A voucher
specimen is deposited at the Herbarium of the Departamento de
−
1
1
1580 (CC), 1460 (CC) cm ; H NMR (methanol-d , 600
4
MHz) and 13C NMR (methanol-d , 150 MHz) data of the aglycone
4
1
13
moiety, see Table 2; H NMR (methanol-d , 600 MHz) and C NMR
4
(methanol-d , 150 MHz) data of the sugar portion, see Table 3;
4
−
ESIMS m/z 639.4 [M − H] ; ESIMS/MS (collision energy 30%) m/z
639.1 (57.1), 493.1 (0.3), 457.1 (0.3), 331.1 (100.0), 316.1 (9.3);
ESIMS (collision energy 32%) of m/z 331.1 (17.3), 316.0 (100.0),
Botan
̂
ica do Instituto de Biocien
̂
cias, USP (SPF 139.580).
3
Extraction and Isolation. Flowers were dried in an oven at 40 °C
for a week and powdered. The material (300 g) was extracted by
percolation with MeOH (1.5 L) three times for three days, at room
temperature. After filtration and evaporation of the solvent to dryness
in vacuo, 14.64 g (4.88%) of crude extract was obtained. The dried
extract (4.0 g) was dissolved in MeOH (15 mL) and centrifuged for 10
min at 3500 rpm twice. The combined supernatants were fractionated
on a Sephadex LH-20 column (56 cm × 3 cm), using MeOH (1.5 L)
as mobile phase, affording 156 fractions (7 mL).
313.0 (7.4), 223.1 (0.7), 209.0 (10.5), 193.0 (0.1), 181.0 (9.8), 166.0
+
(0.7), 137.4 (0.2); HR-MALDI-TOFMS [M + H] m/z 641.1716
(calcd for C H O , 641.1712).
28
33 17
25
Compound 5: yellow powder; [α] −15.7 (c 0.15, MeOH); UV
D
(MeOH) λmax (log ε) 344 (4.70), 287 (3.85), 217 (4.60) nm; IR
(KBr) νmax 3380 (br OH), 2918 (CH), 1676 (CO), 1660 (CC),
−
1
1
1615 (CC), 1570 (CC), 1456 (CC) cm ; H NMR
13
(methanol-d , 600 MHz) and C NMR (methanol-d , 150 MHz)
4
4
1
The Sephadex fractions 54−57 (304 mg) and 64−68 (117 mg)
were analyzed by HPLC-UV, on a C18 reversed-phase column (Synergi
Hydro, 10 μm, 250 mm × 10.00 mm; 80 A; Phenomenex, Torrance,
data of the aglycone moiety, see Table 2; H NMR (methanol-d , 600
4
MHz) and 13C NMR (methanol-d , 150 MHz) data of the sugar
4
−
portion, see Table 3; ESIMS m/z 681.2 [M − H] ; ESIMS/MS
5
53
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Journal of Natural Products
Article
Figure 3. Effects of 2, 6, and 7 on ROS production in PC3 cells.
(
3
6
collision energy 30%) m/z 681.1 (23.4), 639.2 (100.0), 493.1 (1),
31.1 (25.4), 316.0 (2.8); ESIMS (collision energy 30%) of m/z
(KBr) νmax 3382 (br OH), 2922 (CH), 1684 (CO), 1658 (CC),
3
−1
1
1618 (CC), 1578 (CC), 1460 (CC) cm ; H NMR
13
39.1 (100.0), 493.2 (0.2), 457.2 (0.5), 331.1 (77.1), 316.1 (6.6);
(methanol-d , 600 MHz) and C NMR (methanol-d , 150 MHz)
4
4
4
ESIMS (collision energy 30%) of m/z 331.1 (43.9), 316.0 (100),
2
6
data of the aglycone moiety are superimposable with those reported
+
1
13
23.0 (2.5), 209.2 (1.2); HR-MALDI-TOFMS [M + H] m/z
for 5; H NMR (methanol-d , 600 MHz) and C NMR (methanol-d4,
4
83.1823 (calcd for C H O , 683.1818).
150 MHz) data of the sugar portion, see Table 2; ESIMS m/z 681.3
3
0
35 18
25
−
Compound 7: yellow powder; [α] −25.6 (c 0.1, MeOH); UV
[M − H] ; ESIMS/MS (collision energy 30%) m/z 681.1 (11.5),
D
3
(
MeOH) λmax (log ε) 344 (4.72), 285 (3.80), 215 (4.60) nm; IR
639.2 (100), 493.1 (1.6), 331.1 (30.3), 316.1 (4.3); ESIMS (collision
5
54
dx.doi.org/10.1021/np200604k | J. Nat. Prod. 2012, 75, 547−556

Journal of Natural Products
Article
energy 30%) of m/z 639.1 (51.1), 493.2 (0.4), 457.1 (0.3), 331.1
Compound 13: yellow powder; [α]25 −15.8 (c 0.1, MeOH); UV
D
4
(
(
100), 316.1 (11.5); ESIMS (collision energy 30%) of m/z 331.0
(MeOH) λmax (log ε) 335 (4.60), 278 (3.85), 218 (4.60) nm; IR
(KBr) νmax 3378 (br OH), 2922 (CH), 1680 (CO), 1660 (CC),
47.9), 316.1 (100), 222.9 (0.4), 209.0 (5.5), 180.9 (3.8); HR-
+
−1
1
MALDI-TOFMS [M + H] m/z 683.1825 (calcd for C H O ,
1612 (CC), 1580 (CC), 1460 (CC) cm ; H NMR
30
35 18
13
6
83.1818).
(methanol-d , 600 MHz) and C NMR (methanol-d , 150 MHz)
4
4
Compound 9: yellow powder; [α]25 −15.4 (c 0.1, MeOH); UV
data of aglycone moiety are superimposable with those reported for 5;
D
1
13
(
(
1
MeOH) λmax (log ε) 330 (4.70), 285 (3.85), 215 (4.60) nm; IR
KBr) νmax 3380 (br OH), 2918 (CH), 1684 (CO), 1658 (CC),
H NMR (methanol-d , 600 MHz) and C NMR (methanol-d , 150
4
4
MHz) data of the sugar portion, see Table 3; ESIMS m/z 723.3 [M −
−
1
1
−
615 (CC), 1580 (CC), 1465 (CC) cm ; H NMR
H] ; ESIMS/MS (collision energy 30%) m/z 723.1 (5.0), 681.2
13
3
(
methanol-d , 600 MHz) and C NMR (methanol-d , 150 MHz)
(100.0); ESIMS (collision energy 30%) of m/z 681.1 (25.1), 639.2
4
4
1
4
data of the aglycone moiety, see Table 2; H NMR (methanol-d , 600
(100.0), 493.0 (1.5), 331.0 (20.6); ESIMS (collision energy 32%) of
4
MHz) and 13C NMR (methanol-d , 150 MHz) data of the sugar
5
4
m/z 639.2 (18.7), 493.0 (0.1), 331.1 (100.0); ESIMS (collision
portion are superimposable with those reported for 5; ESIMS m/z
energy 32%) of m/z 331.1 (4.7), 223.3 (7.3), 180.9 (8.3); HR-
−
+
6
6
65.2 [M − H] ; ESIMS/MS (collision energy 32%) m/z 665.2 (4.7),
MALDI-TOFMS [M + H] m/z 725.1930 (calcd for C H O ,
3
2
37 19
23.1 (50.6), 477.1 (2.4), 315.1 (100.0), 300.0 (25.8); ESIMS3
725.1924).
Compound 16: yellow, amorphous powder; [α]25 −82.3 (c 0.1,
(
(
(
(
collision energy 32%) of m/z 623.1 (3.5), 477.1 (0.1), 315.1
D
3
100.0), 300.1 (25.5); ESIMS (collision energy 30%) of m/z 315.1
MeOH); UV (MeOH) λmax (log ε) 256 (4.60), 279 (4.72), 288
(4.80), 385 (3.78) nm; IR (KBr) νmax 3385 (br OH), 2918 (CH),
685(CO), 1662 (CC), 1617 (CC), 1581 (CC), 1465 (C
5.3), 300.0 (100.0), 195.4 (1.1), 181.1 (0.8), 166.3 (0.1); ESIMS4
collision energy 30%) of m/z 300.0 (0.1), 272.1 (100), 254.1 (9.5),
+
−1
1
13
1
66.2 (0.4); HR-MALDI-TOFMS [M + H] m/z 667.1874 (calcd for
C) cm ; H NMR (methanol-d , 600 MHz) and C NMR
4
C H O , 667.1869).
Compound 10: yellow, amorphous powder; [α]
(methanol-d , 150 MHz), see Table 4; ESIMS m/z 449.2 [M −
30
35 17
4
25
D
−
−70.5 (c 0.1,
H] ; ESIMS/MS (collision energy 30%) m/z 449.2 (1.5), 287.0
3
MeOH); UV (MeOH) λmax (log ε) 255 (4.60), 279 (4.80), 288
4.90), 385 (3.88) nm; IR (KBr) νmax 3385 (br OH), 2918 (CH),
(100.0), 272.1 (7.9); ESIMS (collision energy 30%) of m/z 287.3
4
(
(2.4), 272.2 (100.0); ESIMS (collision energy 40%) of m/z 272.1
1
685 (CO), 1660 (CC), 1614 (CC), 1582 (CC), 1465
(64.9), 271.1 (8.9), 256.9 (3.3), 244.2 (23.6), 243.1 (100.0), 229.0
−
1
1
13
(
CC) cm ; H NMR (methanol-d , 600 MHz) and C NMR
(7.5), 216.1 (81.5), 202.2 (3.2), 200.3 (10.7); HR-MALDI-TOFMS
4
−
+
(
methanol-d , 150 MHz), see Table 4; ESIMS m/z 611.3 [M − H] ;
[M + H] m/z 451.1239 (calcd for C H O , 451.1235).
4
21 23 11
Compound 17: yellow powder; [α]2 −32.0 (c 0.1, MeOH); UV
5
ESIMS/MS (collision energy 30%) m/z 611.2 (0.7), 448.9 (0.4), 287.0
D
3
(
(
(
(
100.0), 272.1 (47.8); ESIMS (collision energy 28%) of m/z 287.1
0.6): 272.1 (100.0); ESIMS (collision energy 40%) of m/z 272.2
40.7), 271.1 (25.9), 257.1 (16.1), 244.1 (30.6), 243.2 (100.0), 229.3
6.2), 216.1 (38.6), 202.3 (5.5), 200.0 (1.4); ESIMS (collision energy
(MeOH) λmax (log ε) 335 (4.70), 288 (3.85), 218 (4.60) nm; IR
(KBr) νmax 3380 (br OH), 2929 (CH), 1685 (CO), 1660 (CC),
4
−
1
1
1615 (CC), 1580 (CC), 1464 (CC) cm ; H NMR
4
13
(methanol-d
, 600 MHz) and C NMR (methanol-d
, 150 MHz)
4
4
4
3
8%) of m/z 271.2 (93.9), 243.0 (100.0); ESIMS (collision energy
data of aglycone moiety are superimposable with those reported for 9;
4
1
13
3
8%) of m/z 243.3 (1.5): 229.4 (8.7); ESIMS (collision energy 30%)
H NMR (methanol-d , 600 MHz) and C NMR (methanol-d , 150
4
4
4
of m/z 244.3 (15.4), 243.0 (33.4), 216.1 (13.5), 200.1 (12.7); ESIMS
MHz) data of the sugar portion, see Table 3; ESIMS m/z 707.3 [M −
collision energy 40%) of m/z 257.1 (5.2), 229.2 (100); ESIMS4
−
(
H] ; ESIMS/MS (collision energy 30%) m/z 707.2 (12.6), 665.2
3
(collision energy 40%) of m/z 202.1, 200.1 (26.7); HR-MALDI-
(100.0), 477.1 (4.2), 315.0 (87.5); ESIMS (collision energy 30%) of
+
4
TOFMS [M + H] m/z 613.1770 (calcd for C H O , 613.1763).
m/z 665.1 (16.2): 623.1 (41.1), 477.3 (1.2), 315.1 (100.0); ESIMS
27
33 16
Compound 11: yellow powder; [α]25 −40.2 (c 0.1, MeOH); UV
D
(collision energy 30%) of m/z 623.1 (9.7), 315.0 (100.0), 300.0
4
(
(
1
MeOH) λmax (log ε) 333 (4.70), 287 (3.85), 215 (4.70) nm; IR
KBr) νmax 3376 (br OH), 2920 (CH), 1685 (CO), 1660 (CC),
(39.9); ESIMS (collision energy 30%) of m/z 477.2, 314.9 (6.3),
4
281.9 (100.0), 181.3 (8.0); ESIMS (collision energy 30%) of m/z
−
1
1
612 (CC), 1578 (CC), 1460 (CC) cm ; H NMR
314.9 (0.8), 300.0 (100.0), 181.1 (1.3); HR-MALDI-TOFMS [M +
13
+
(
methanol-d , 600 MHz) and C NMR (methanol-d , 150 MHz)
H] m/z 709.1981 (calcd for C H O , 709.1974).
4
4
32 37 18
2
5
data of the aglycone moiety are superimposable with those reported
for 9; H NMR (methanol-d , 600 MHz) and C NMR (methanol-d4,
1
reported for 7; ESIMS m/z 665.2 [M − H] ; ESIMS/MS (collision
energy 30%) m/z 665.2 (15.7), 623.1 (57.1), 477.1 (1.2), 315.1
Compound 19: yellow powder; [α] −12.5 (c 0.1, MeOH); UV
(MeOH) λmax (log ε) 358 (4.10), 280 (sh), 265 (3.80) nm; IR (KBr)
D
1
13
4
−1
50 MHz) data of the sugar portion are superimposable with those
ν
3420 (br OH), 1660 (CO), 1615 (CC), 1600 (CC) cm ;
max
−
1
13
H NMR (methanol-d , 600 MHz) and C NMR (methanol-d , 150
MHz), see Table 1; ESIMS m/z 653.2 [M − H] ; ESIMS/MS
(collision energy 32%) m/z 653.2 (15.0), 477.0 (1.8), 315.1 (100.0);
ESIMS (collision energy 32%) of m/z 315.1 (26.7), 300.0 (100.0),
181.1 (2.9); ESIMS (collision energy 32%) of m/z 300.1 (3.9), 272.2
(100.0), 166.1 (8.5); HR-MALDI-TOFMS [M + H] m/z 655.1663
4
4
−
3
(
100.0), 300.1 (25.1); ESIMS (collision energy 30%) of m/z 623.1
3
3
(
10.1), 477.1 (0.1), 315.0 (100.0), 300.0 (24.0); ESIMS (collision
4
4
energy 30%) of m/z 315.1 (2.4), 300.0 (100.0), 180.9 (0.5); ESIMS
+
(collision energy 30%) of m/z 300.0 (0.4), 272.1 (100), 165.9 (0.4);
+
HR-MALDI-TOFMS [M + H] m/z 667.1876 (calcd for C H O ,
6
(calcd for C H O , 655.1657).
30
35 17
32 31 15
67.1869).
Acid Hydrolysis. The configuration of the sugar units was
established after hydrolysis of 1−19 with 1 N HCl, trimethylsilation,
and determination of the retention times by GC operating under the
Compound 12: white, amorphous powder; [α]25 −7.4 (c 0.1,
D
MeOH); UV (MeOH) λmax (log ε) 355 (4.10), 282 (sh), 265 (3.80)
28
nm; IR (KBr) ν 3422 (br OH), 1670 (CO), 1615 (CC), 1600
experimental conditions reported by De Marino et al., 2003.
max
−
1
1
13
(
(
CC) cm ; H NMR (methanol-d , 600 MHz) and C NMR
The peaks of the hydrolysate of 1 were detected at 10.72 (L-
rhamnose) and 14.73 min (D-glucose). For the hydrolysate of 10 a
peak at 14.73 min (D-glucose) was detected. Retention times for
authentic samples after being treated in the same manner with 1-
(trimethylsilyl)imidazole in pyridine were detected at 9.67 and 10.70
(L-rhamnose) and 14.71 min (D-glucose).
4
−
methanol-d , 150 MHz), see Table 1; ESIMS m/z 1247.4 [M − H] ;
4
ESIMS/MS (collision energy 30%) m/z 1247.2 (15.7), 931.1 (100.0),
3
8
29.2 (3.3), 623.2 (18.4), 477.0 (1.1); ESIMS (collision energy 30%)
of m/z 931.0 (5.5), 913.1 (7.4), 829.1 (31.3), 665.1 (8.6), 623.1
4
(
8
100.0), 477.3 (0.5); ESIMS (collision energy 30%) of m/z 913.3,
29.1 (100.0), 623.0 (43.0); ESIMS (collision energy 30%) of m/z
4
Antioxidant Activity. Pure compounds were tested by using the
3
17−19
829.1, 785.0 (35.3), 665.1 (100.0), 623.2 (57.8); ESIMS (collision
TEAC assay.
The TEAC value is based on the ability of the
antioxidant to scavenge the radical cation 2,2′-azinobis(3-ethyl-
4
•+
benzothiazoline-6-sulfonate) (ABTS ) with spectrophotometric anal-
•+
ysis. The ABTS cation radical was produced by the reaction between
7 mM ABTS in H O and 2.45 mM potassium persulfate, stored in the
2
•+
dark at room temperature for 12 h. ABTS is a blue-green chromogen
5
55
dx.doi.org/10.1021/np200604k | J. Nat. Prod. 2012, 75, 547−556

Journal of Natural Products
Article
with a characteristic absorption at 734 nm. The ABTS•+ solution was
then diluted with PBS (phosphate-buffered saline, pH 7.4) to an
absorbance of 0.70 at 734 nm and equilibrated at 30 °C. Samples were
diluted with MeOH to produce solutions of 0.3, 0.5, 1, and 1.5
concentration. The reaction was initiated by the addition of 1 mL of
diluted 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) to
(4) Piacente, S.; dos Santos, L. C.; Mahmood, N.; Zampelli, A.; Pizza,
C.; Vilegas, W. J. Nat. Prod. 2001, 64, 680−682.
(5) Santos, L. C.; Piacente, S.; Pizza, C.; Albert, K.; Dachtler, M.;
Vilegas, W. J. Nat. Prod. 2001, 64, 122−124.
(6) dos Santos, L. C.; Piacente, S.; Pizza, C.; Toro, R.; Sano, P. T.;
Vilegas, W. Biochem. Syst. Ecol. 2002, 30, 451−456.
(7) Devienne, K. F.; Raddi, M. S. G.; Varanda, E. A.; Vilegas, W. Z.
Naturforsch., C: J. Biosci. 2002, 57, 85−88.
10 μL of each sample solution. Determinations were repeated three
times for each sample solution. The percentage inhibition of
absorbance at 734 nm was calculated for each concentration relative
to a blank absorbance (MeOH) and was plotted as a function of
concentration of compound or standard, 6-hydroxy-2,5,7,8-tetrame-
thylcroman-2-carboxylic acid (Trolox, Aldrich Chemical Co., Gilling-
ham, Dorset, UK). The percentage inhibition was plotted as a function
of compound or standard concentration. The antioxidant activities of
MeOH extract and compounds 1−19 are expressed as TEAC values in
comparison with TEAC activity of the reported reference compounds,
quercetin, quercetin 3-O-glucoside, and kaempferol 3-O-glucoside.
The TEAC value is defined as the concentration of standard Trolox
solution with the same antioxidant capacity as a 1 mM concentration
of the investigated compound. In the case of the extract the TEAC
value is defined as the concentration of a standard Trolox solution with
the same antioxidant capacity as 1 mg/mL of the tested extract.
Cell Cultures. Human prostate cancer cells (PC3) were cultured in
DMEM medium supplemented with 2 mM L-glutamine, 10% heat-
inactivated fetal bovine serum, and 1% penicillin/streptomycin (all
from Cambrex Bioscience, Verviers, Belgium) at 37 °C in an
atmosphere of 95% O and 5% CO . Cells were plated at a density
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Davino, S. Phytochemistry 1990, 29, 2299−2301.
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11) Varanda, E. A.; Raddi, M. S. G.; de Luz Dias, F.; Araujo, M. C.
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(
(
́
M.R.; Raddi, M. S. G.; Vilegas, W.; Uyemura, S. A.; Santos, A. C.;
Curti, C. Phytochemistry 2007, 68, 1075−1080.
(15) Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.;
2
2
Rice-Evans, C. Free Radical Biol. Med. 1999, 26, 1231−1237.
5
of 1 × 10 cells/well (Falcon, BD Bioscience, Bedford, MA, USA) the
day before treatment. At the end of the incubation period, the cells
were processed for FACS analyses. The cells were used up to a
maximum of 10 passages.
(16) Piacente, S.; Montoro, P.; Oleszek, W.; Pizza, C. J. Nat. Prod.
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(
(
004, 67, 882−885.
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Flow Cytometry Estimation of Intracellular Redox State. The
effect of the compounds on intracellular reactive oxygen species was
evaluated by measuring dichlorofluorescein (DCF) fluorescence. Cells
were incubated for 24 h in presence and absence of compounds at
different concentrations (1−10 μM). At the end of the incubation
Sacchi, A.; Saija, A.; Tomaino, A.; Pellegrino, M. L.; de Caprariis, P. J.
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W. Biochem. Syst. Ecol. 2002, 30, 275−277.
21) de Andrade, F. D. P.; dos Santos, L. C.; Dokkedal, A. L.; Vilegas,
5
time, cells were washed and resuspended (2 × 10 cells/ml) in Hank’s
balanced salt solution containing 10 μM 2′,7′-dichlorodihydrofluor-
escein diacetate (DCFH-DA). Following a further 20 min incubation
at 37 °C, DCF fluorescence was monitored by flow cytometry (FL1-H
channel). In order to estimate the antioxidant potential of the
compounds, control and treated cells were exposed to 300 μM of the
oxidant t-BuOOH for 30 min at 37 °C before DCFH-DA loading.
Statistical Analysis. All the results are shown as mean ± SEM of
three experiments performed in triplicate. Statistical comparisons
between groups were made using ANOVA followed by the Bonferronu
paremetric test. Differences were considered significant if p < 0.05
(
W. Phytochemistry 1999, 51, 411−415.
(
(
22) Montaudo, G.; Caccamese, S. J. Org. Chem. 1973, 33, 710−716.
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2
364.
(
24) Chi, Y.-M.; Nakamura, M.; Zhao, X.-Y.; Yoshizawa, T.; Yan, W.-
M.; Hashimoto, F.; Kinjo, J.; Nohara, T.; Sakurada, S. Biol. Pharm. Bull.
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2
(
1
(
ASSOCIATED CONTENT
■
S
Supporting Information
Spectroscopic and spectrometric data for the new compounds
(
(
27) Shukla, S.; Gupta, S. Nutr. Cancer 2005, 53, 18−32.
28) De Marino, S.; Borbone, N.; Iorizzi, M.; Esposito, G.;
*
McClintock, J. B.; Zollo, F. J. Nat. Prod. 2003, 66, 515−519.
1
, 5, 7, 9−13, 16, 17, and 19. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
*
Corresponding Author
Tel: +39 089969763. Fax: +39 089969602. E-mail: piacente@
unisa.it.
Notes
The authors declare no competing financial interest.
REFERENCES
■
(
(
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dx.doi.org/10.1021/np200604k | J. Nat. Prod. 2012, 75, 547−556