Phenylpropanoid glucosides from leaves of Coussarea hydrangeifolia (Rubiaceae)

Article abstract of DOI:10.1016/j.phytochem.2005.06.019

Phenylpropanoid glycosides, 1′-O-benzyl-α-l-rhamnopyranosyl- (1″ → 6′)-β-d-glucopyranoside (1) and α-l- xylopyranosyl-(4″ → 2′)-(3-O-β-d-glucopyranosyl)-1′- O-E-caffeoyl-β-d-glucopyranoside (2), together with the known derivatives, 1,6-di-O-caffeoyl-β-d-glucopyranoside (3), 1-O-(E)-caffeoyl-β-d- glucopyranoside (4) and 1-O-(E)-feruloyl-β-d-glucopyranoside (5), were isolated from leaves of Coussarea hydrangeifolia. Their structures were determined by IR, HRESIMS, and 1D and 2D NMR experiments, and their antioxidant activities, evaluated by assaying the free radical scavenging capacity using the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical as substrate. The antioxidant activities of 3 and 4 (IC₅₀ values of 15.0 and 19.2 μM, respectively) were comparable to that of the standard positive control caffeic acid, whilst 2 and 5 were only weakly active and 1 was inactive.

Full text of DOI:10.1016/j.phytochem.2005.06.019

PHYTOCHEMISTRY
Phytochemistry 66 (2005) 1927–1932
www.elsevier.com/locate/phytochem
Phenylpropanoid glucosides from leaves of
Coussarea hydrangeifolia (Rubiaceae)
a
a
a
Lidilhone Hamerski , Mauro Dionei Bomm , Dulce Helena Siqueira Silva ,
b
a
c
´
Maria Claudia Marx Young , Maysa Furlan , Marcos Nogueira Eberlin ,
a
a
a,
*
´
Ian Castro-Gamboa , Alberto Jose Cavalheiro , Vanderlan da Silva Bolzani
a
NuBBE, Nu´cleo de Bioensaios, Bioss´ıntese e Ecofisiologia de Produtos Naturais, Instituto de Qu´ımica,
Universidade Estadual Paulista, CP-355, CEP 14800-900 Araraquara, SP, Brazil
b
ˆ
Sec¸a˜o de Fisiologia e Bioqu´ımica de Plantas, Instituto de Botanica, CP 4005, 01061-970, Sa˜o Paulo, SP, Brazil
Thomson Mass Spectrometry Laboratory, Instituto de Quı´mica, Universidade Estadual de Campinas-UNICAMP,
CP 6154, CEP 13083-970 Campinas, SP, Brazil
c
Received 18 November 2004; received in revised form 2 May 2005
Available online 10 August 2005
Abstract
Phenylpropanoid glycosides, 10-O-benzyl-a-L-rhamnopyranosyl-(100 ! 60)-b-D-glucopyranoside (1) and a-L-xylopyranosyl-
(400 ! 20)-(3-O-b-D-glucopyranosyl)-10-O-E-caffeoyl-b-D-glucopyranoside (2), together with the known derivatives, 1,6-di-O-caf-
feoyl-b-D-glucopyranoside (3), 1-O-(E)-caffeoyl-b-D-glucopyranoside (4) and 1-O-(E)-feruloyl-b-D-glucopyranoside (5), were isolated
from leaves of Coussarea hydrangeifolia. Their structures were determined by IR, HRESIMS, and 1D and 2D NMR experiments, and
their antioxidant activities, evaluated by assaying the free radical scavenging capacity using the DPPH (1,1-diphenyl-2-pic-
rylhydrazyl) radical as substrate. The antioxidant activities of 3 and 4 (IC50 values of 15.0 and 19.2 lM, respectively) were comparable
to that of the standard positive control caffeic acid, whilst 2 and 5 were only weakly active and 1 was inactive.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Coussarea hydrangeifolia; Rubiaceae; Phenylpropanoid glycosides; Antioxidant activity
1. Introduction
Brazil, where it is considered to be a pioneer species (Cor-
rea, 1974). In the course of our studies concerning the
bioactive principles of Brazilian plants, with special
Although Coussarea is considered to be only a small
genus within the taxon Rubiaceae, it is well represented
in the main Brazilian biomes including the Amazoˆnia
and the Cerrado. Coussarea hydrangeifolia, commonly
know as ‘‘falsa-quina’’, is characteristic of the vegetation
of the Cerrado and of the semi-deciduous forests of
emphasis on the vegetation of the State of Sao Paulo
˜
(Pauletti et al., 2003; Hamerski et al., 2003), we found
that the aqueous extract of the leaves of C. hydrangeifolia
showed antioxidant activity, as determined by a prelimin-
ary test with b-carotene, and strong free radical scaveng-
ing activity against 1,1-diphenyl-2-picrylhydrazyl radical
(DPPH). Fractionation of this extract provided five phe-
nylpropanoid glycosides including two new compounds,
1 and 2, which had their structures determined by spec-
troscopic methods.
*
Corresponding author. Tel.: +55 16 33016660; fax: +55 16
33227932.
E-mail address: bolzaniv@iq.unesp.br (V. da Silva Bolzani).
0031-9422/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2005.06.019

1928
L. Hamerski et al. / Phytochemistry 66 (2005) 1927–1932
2. Results and discussion
J = 6.0 Hz, H-600). Additionally, the signals at d 4.85
(d, J = 11.5 Hz) and 5.15 (d, J = 11.5 Hz) could be as-
cribed to benzylic oxymethylene hydrogens, thus cha-
racterising the aromatic portion of 1 as a benzylic
system. The 13C NMR spectrum (Table 1) showed sig-
nals at d 137.8, 128.2 (2·), 127.9 (2·), 127.2 and 70.9,
which were consistent with the presence of a monosub-
stituted benzylic ring. Two anomeric methine carbons
at d 103.7 and 102.6, several hydroxymethines, one
hydroxymethylene carbon at d 68.4 and one methyl
group at d 18.7 resembled those of glucose and rham-
nose, respectively, and clearly suggested that 1 possessed
glucopyranosyl and rhamnopyranosyl units. Data from
COSY and TOCSY experiments revealed the hydrogen
sets belonging to the glucosyl and rhamnosyl moieties.
The complete assignment of hydrogen and carbon sig-
nals and the positions of the bonds of the sugar moieties
in 1 were determined by an HMBC experiment which
showed long-range correlations between H-60 and C-40
and the anomeric C-100, between H-100 and the methylene
C-60, and between H-7 of the benzylic system and C-10
Compound 1 was isolated from the aqueous extract
of C. hydrangeifolia as an amorphous powder with an
30
½aꢀD of ꢁ44.3ꢁ. A quasimolecular ion peak [M ꢁ H]ꢁ
for 1 was observed at m/z 415.1610 in the high-resolu-
tion electrospray ionisation mass spectrum (HRESIMS;
negative mode) suggesting a molecular formula of
C19H28O10. The IR spectrum exhibited strong absorp-
tion bands at 3430 and 1042 cmꢁ1 suggesting the pres-
ence of hydroxyl groups. The 1H NMR spectrum
(Table 1) displayed signals that could be attributed to
a monosubstituted benzene ring [d 7.54 (d, J = 7.5 Hz,
H-2,6), 7.30 (m, H-3,5), 7.23 (m, H-4)], a b-D-glucosyl
moiety [d 4.90 (d, J = 7.5 Hz, H-10), 4.06 (dd, J = 7.5,
8.0 Hz, H-20), 4.19 (t like, J = 8.0 Hz, H-30), 4.12 (m,
H-4), 4.20 (ddd, J = 9.5, 7.5, 2.5 Hz, H-50), 4.18 (dd,
J = 11.0, 7.5 Hz, H ꢁ 60a), 4.64 (dd, J = 11.0, 2.5 Hz,
H ꢁ 60b), and a rhamnosyl group [d 5.55 (d, J = 1.0 Hz,
H-100), 4.62 (m, H-200), 4.55 (dd, J = 3.5, 9.0 Hz, H-300),
4.26 (t like, J = 9.0 Hz, H-400), 4.97 (m, H-5), 1.62 (d,
Table 1
1H and 13C NMR spectroscopic data of hydrangeifolin I (1), hydrangeifolin II (2) and 1,6-di-O-caffeoyl-b-D-glucopyranose (3) in pyridine-d5
Position
1
d
–
2
d
–
3
d
–
1H
d
13C
1H
d
13H
1H
d
13C
1
2
3
4
5
6
7
137.8 (s)
128.2 (d)
127.9 (d)
127.2 (d)
127.9 (d)
128.2 (d)
70.9 (t)
126.6 (s)
115.8 (d)
147.0 (s)
148.7 (s)
116.7 (d)
122.2 (d)
145.5 (d)
127.6 (s)
111.7 (d)
148.8 (s)
149.5 (s)
116.2 (d)
124.1 (d)
114.8 (d)
7.54 d (7.5)
7.30 m
7.23 m
7.53 d (2.0)
–
–
7.16 d (8.0)
7.10 dd (2.0; 8.0)
7.97 d (15.5)
7.05 m
–
–
6.80 m
6.99 m
6.64 d (16.0)
7.30 m
7.54 d (7.5)
4.85 d (11.5)
5.15 d (11.5)
8
9
6.56 d (15.5)
–
6.20 d (8.0)
4.27 t (9.0)
4.34 m
114.4 (d)
165.3 (s)
96.1 (d)
78.8 (d)
78.5 (d)
71.1 (d)
79.5 (d)
62.3 (t)
7.93 d (16.0)
–
147.4 (d)
169.6 (s)
104.3 (d)
74.4 (d)
76.9 (d)
71.0 (d)
77.5 (d)
66.5 (t)
10
20
30
40
50
60
4.90 d (7.5)
4.06 dd (7.5)
4.19 t (8.0)
103.7 (d)
75.1 (d)
77.2 (d)
71.9 (d)
78.5 (d)
68.4 (t)
4.48 d (7.4)
4.04 t (9.0)
3.95 t (9.0)
3.93 t (9.0)
4.10 m
4.29 m
4.39 m
–
4.12 m
4.36 m
4.08 m
4.20 ddd (9.5, 7.5, 2.5)
4.18 dd (11.0, 7.5)
4.64 d (11.0, 2.5)
5.55 d (1.0)
4.62 m
4.55 dd (3.5; 9.0)
4.26 t (9.0)
4.40 d (12.0)
4.60 d (12.0)
5.90 d (3.5)
4.20 dd (3.5; 9.0)
4.08 m
4.75 t (9.0)
4.54 dd (2.5; 11.5)
4.42 dd (5.5; 11.5)
100
200
300
400
500
102.6 (d)
72.3 (d)
72.8 (d)
74.1 (d)
69.8 (d)
94.2 (d)
74.6 (d)
72.7 (d)
75.5 (d)
63.1 (t)
127.5 (s)
111.7 (d)
148.8 (s)
149.4 (s)
116.2 (d)
7.05 m
–
–
4.97 m
6.80 m
600
1.62 d (6.0)
18.7 (d)
6.99 m
124.1 (d)
114.7 (d)
147.1 (d)
169.7 (s)
700
6.52 d (15.5)
7.87 d (15.5)
–
800
900
1000
2000
3000
4000
5000
6000
5.35 d (7.5)
4.29 t (8.5)
4.14 t (8.5)
4.25 t (8.5)
4.03 ddd (2.5; 5.5; 9.0)
4.39 d (11.5)
4.58 d (11.5)
99.6 (d)
74.3 (d)
76.9 (d)
72.2 (d)
78.6 (d)
63.3 (t)

L. Hamerski et al. / Phytochemistry 66 (2005) 1927–1932
1929
of the glucosyl moiety and C-2/C-6 of the aromatic
system. Consequently, the structure of 1 was established
to be 10-O-benzyl-a-L-rhamnopyranosyl-(100 ! 60)-b-D-
glucopyranoside, and this new compound has been
named hydrangeifolin I.
Compound 2 exhibited a quasimolecular ion peak
[M ꢁ H]ꢁ at m/z 635.1828 in the HRESIMS (negative
mode) suggesting a molecular formula C26H36O18.
MS/MS analysis of the quasimolecular ion revealed a
significant fragment ion at m/z 502.1321 assigned to
[M ꢁ H ꢁ deoxyhexose]ꢁ. The 13C NMR spectrum
(Table 1) clearly showed the presence of three sugar
moieties together with nine additional signals that
could be attributed to the aglycone moiety. Data from
the 1H NMR spectrum (Table 1) indicated the pres-
96.1) of the b-glucopyranosyl unit also suggested that
the a-xylopyranosyl unit was attached to C-20 of the
glucopyranosyl moiety. The cross peak in the HMBC
experiment between d 4.27 (t, J = 9.0 Hz, H-20) and
d 75.5 (C-400), and the correlation in the NOESY
experiment between the signals at d 4.27 (t, J = 9.0,
H-20) and at d 4.75 (t, J = 9.0, H-400), confirmed the
(400 ! 20) linkage of the disaccharide moiety. Acid
hydrolysis of 2 afforded glucose and xylose, which
were identified by TLC by comparison with authentic
samples. Consequently, the structure of 2 was estab-
lished as a-L-xylopyranosyl-(400 ! 20)-(3-O-b-D-gluco-
pyranosyl)-10-O E-caffeoyl-b-D-glucopyranoside, and
this new compound has been named hydrangeifolin
II (see Fig. 1).
ence of an (E)-caffeoyl system [1H,
d
7.97 (d,
Compound 3 was identified as 1,6-di-O-caffeoyl-b-D-
glucopyranose from physical and spectroscopic data
(Akhmedov and Litvinenko, 1968). Although this com-
pound was first isolated from Scrophularia grossheimi (a
traditional medicinal plant used in Russia for the treat-
ment of jaundice, hepatitis and rheumatism), to the best
of our knowledge its 13C NMR spectroscopic data re-
main unreported. The assignment of the 13C NMR sig-
nals of 3, based mainly on 1D and 2D NMR
experiments, is provided in Table 1. Compounds 4 and
5 were identified as 1-O-(E)-caffeoyl-b-D-glucopyranose
and 1-O-(E)-feruloyl-b-D-glucopyranose, respectively
(Birkofer et al., 1961, 1969).
Preliminary TLC autographic test using b-carotene as
revealing agent (Silva et al., 2001) evidenced several anti-
oxidant compounds in the aqueous extract from leaves of
C. hydrangeifolia. Compounds 1–5, isolated from this ex-
tract, were evaluated for their free radical scavenging
activity towards the stable free radical DPPH (Pauletti
et al., 2003), and compared to standard antioxidants
J = 15.5 Hz, H-7), 6.56 (1H, d, J = 15.5 Hz, H-8),
7.53 (1H, d, J = 2.0 Hz, H-2), 7.16 (1H, d,
J = 8.0 Hz, H-5), 7.10 (1H, dd, J = 2.0, 8.0 Hz, H-6)]
together with three anomeric protons, two of which
[5.35 (d, J = 7.5 Hz) and 6.20 (d, J = 8.0 Hz)] were as-
signed to the two b-glucose units, and one [d 5.90 (d,
J = 3.5 Hz)], to an a-xylose moiety. From the 13C
NMR spectrum it could be established that the aro-
matic unit was an ester of (E)-caffeic acid [d 165.3
(C-9), 145.5 (C-7), 114.4 (C-8), 126.6 (C-1), 115.8 (C-
2), 147.0 (C-3), 148.7 (C-4), 116.7 (C-5), 122.2 (C-6)].
Complete assignment of the protons in each sugar sys-
tem was achieved by considering the 1D and 2D
1
TOCSY, 1D and 2D NOESY, and the H–1H COSY
spectra, while the carbons were assigned from HMQC
and HMBC data. A 1D TOCSY experiment obtained
by irradiating at the well-resolved anomeric proton at
d 5.35 (1H, d, J = 7.5 Hz) revealed a set of coupled
oxymethine protons (d 4.29, 4.14, 4.25 and 4.03) and
a methylene proton (d 4.39). These data, when ana-
lysed with the correlated 13C NMR signals in the
HMQC spectrum, led to the identification of a b-D-
glucosyl moiety. Its position at C-3 of the caffeic acid
moiety was established unambiguously by HMBC cor-
relations observed between the anomeric proton H-1000
at d 5.35 and C-3 (d 147.0), and from the proton at d
7.10 (H-6) to C-2 (d 115.8) and C-4 (d 148.7). A 1D
TOCSY experiment obtained by irradiating at the
well-resolved anomeric proton at d 6.20 (1H, d,
J = 8.0 Hz) showed a set of coupled oxymethine pro-
tons (d 4.27, 4.34, 4.36 and 4.08) and oxymethylene
protons (d 4.40 and 4.60). Analysis of the correlated
13C NMR signals in the HMQC spectrum led to the
identification of an additional b-D-glucosyl moiety.
From the HMBC correlation observed between the
signals at d 6.20 (H-10) and d 165.3 [the carbonyl of
the (E)-caffeoyl group], the site of esterification with
the (E)-caffeoyl acid unit was determined to be at C-
10 of the glucopyranosyl. The downfield shift of C-20
(d 78.8) together with the upfield shift of C-10 (d
O
O
O
HO
OH
OH
O
HO
OH
1
OH
OH
H
H
O
H
O
H
H
H
H
H
O
HO
HO
H
O
OH
OH
O
O
OH
H
O
H
H
HO
H
H
HO
H
OH
H
HO
H
H
H
OH
2
HMBC
NOESY
Fig. 1. Selected HMBC correlations (H ! C) for hydrangeifolin I (1)
and hydrangeifolin II (2), and NOESY correlations for 2.

1930
L. Hamerski et al. / Phytochemistry 66 (2005) 1927–1932
Table 2
3. Experimental
Antioxidant activities of phenylpropanoids isolated from leaves of C.
hydrangeifolia and standard compounds
3.1. General
Compound
DPPH (IC50, lM)
Hydrangeifolin I (1)
Hydrangeifolin II (2)
n.a.a
33.2
15.0
19.2
35.5
13.7
27.7
IR and UV spectra were obtained using Perkin–El-
mer model 1600 series FTIR and Lambda 14P UV/
VIS spectrometers, respectively. ES-MS were measured
on a VG Platform Fisons instrument and HRESIMS
on a VG Autospec (40 eV) spectrometer. 1H NMR
(500 MHz), 13C NMR (125 MHz) and 2D NMR spectra
were obtained using a Varian Inova model 500 spec-
trometer. The NMR data were measured in pyridine-
d5 or DMSO-d6 and chemical shifts are expressed in d
(ppm) values with reference to TMS. Preparative HPLC
was conducted using a Phenomenex Luna ODS column
(250 · 21.20 mm i.d.; 10 lm) coupled to a Varian Prep-
Star S0-1 pump with flow rate of the mobile phase set
at 10 mL/min; and a Varian ProStar 320 UV/VIS detec-
tor set at 254 nm. Sephadex LH-20 (Pharmacia), C-18
(Merck) and Si-gel (Merck) were used as stationary
phases in CC fractionations. A Varian Star 9090 electro-
chemical detector (ElCD) composed of a glassy carbon
1,6-Di-O-caffeoyl-b-D-glucopyranose (3)
1-O-(E)-caffeoyl-b-D-glucopyranose (4)
1-O-(E)-feruloyl-b-D-glucopyranose (5)
Caffeic acidb
p-Coumaric acidb
a
n.a., not active.
Standard compounds employed as positive controls.
b
caffeic and p-coumaric acids, used as positive controls.
As shown on Table 2, compounds 3 and 4 exhibited
strong antioxidant activities, comparable with that of
caffeic acid, probably due to the presence of the 3,4-dihy-
droxy (catechol) moiety in their structures. Compounds
2 and 5, which bear only one free hydroxy group, showed
weak free radical scavenging activities towards DPPH
and compound 1 was inactive.
1'
5'
7
O
6'
1
5"
O
O
1"
HO
OH
OH
O
4
HO
OH
2"
OH
1
OH
5"'
O
9
O
1'
5'
HO
O
3
4
HO
1
OH
OH
7
O
2"'
O
1"'
OH
O
2'
HO
HO
OH
O
4"
HO
2"
1"
2
OH
O
5"
OH
OH
O
7
1'
5'
3"
HO
HO
9"
3
1
O
1"
O
7"
HO
OH
3'
O
HO
5
3
O
7
1'
5'
9
RO
OH
3
1
O
O
HO
OH
3'
HO
HO
5
R
4 H
5 Me

L. Hamerski et al. / Phytochemistry 66 (2005) 1927–1932
1931
working-electrode and an Ag/AgCl reference electrode
was employed in the DPPHÅ assay.
and purified by HPLC on an ODS column eluted with
MeCN/H2 O (1:9) to yield 1 (12.0 mg; tR 12.54 min).
Fractions 13–14 from the first Sephadex column were
combined (500 mg) and subjected to CC over C-18
eluted with a MeOH/H2O step gradient (in ratios 1:19,
3:17 and 3:2, successively) to yield fractions A, B and
C. Fraction A (132.7 mg) was further purified by HPLC
on an ODS column eluted with MeCN/H2O (7:93) to
yield 2 (35.8 mg: tR 10.25 min). Fraction B (67.5 mg)
was subjected to HPLC on an ODS column eluted with
MeCN/H2O (1:9) to yield, 3 (6.1 mg; tR 22.38 min) and
4 (16.9 mg; tR 14.64 min). Fraction C (145.9 mg) was
purified under the same conditions and afforded 5
(8.4 mg, tR 18.91 min).
3.2. Plant material
Leaves of C. hydrangeifolia were collected in Mogi-
Guac¸u (Sao Paulo State, Brazil) in December 1997
˜
and identified and authenticated by Dr. Ines Cordeiro
(Instituto de Botaˆnica, Sao Paulo, SP, Brazil). A vou-
˜
cher specimen was deposited in the Herbarium of the
Botanic Garden, Sao Paulo (number SP 167423).
˜
3.3. Antioxidant assay
The b-carotene test was performed on TLC plates
according to a previously described method (Pratt and
Miller, 1984;Silva et al., 2001). The stable radical DPPHÅ
was used for the determination of free radical scaveng-
ing activity: a methanolic solution (200 lM, 2 mL) of
DPPH was added to methanol (1 mL) containing an
appropriate amount of each test compound in order to
yield a final concentration of 100, 80, 40, 20, 10 or
5 lM. Caffeic and p-coumaric acids were used as stan-
dard positive controls, whilst a solution of DPPH
(200 lM, 2 mL) in methanol (1 mL) served as blank.
All samples were evaluated as triplicates (n = 3). Each
reaction mixture was shaken thoroughly and incubated
for 30 min at room temperature in the dark. The
amount of the reduced form of the radical (DPPH-H)
generated in situ was determined from the measured de-
crease in current (I) in the electrochemical cell of the
ElCD (Son and Lewis, 2002). The area measured on
each chromatogram was normalised and compared with
that obtained for the blank. The radical scavenging
activity of each sample was expressed in terms of IC50
(the concentration in lM of test compound required
to produce a 50% decrease in I caused by the formation
of DPPH-H) (Pauletti et al., 2003).
3.4.1. Hydrangeifolin I. 10-O-benzyl-a-L-
rhamnopyranosyl-(10 ! 60)-b-D-glucopyranoside (1)
30
Amorphous powder, ½aꢀD ꢁ 44.3ꢂ
(c = 0.003,
MeOH). UV kMeOH 264, 204. IR (film) cmꢁ1 3450,
2920, 1600, 1550, 1460, 1100, 1020. ES-MS [M+Na]+
m/z 439. HRESIMS [M ꢁ H]ꢁ m/z 415.1610 (Calcd.
1
for C19H28O10, 415.1604). H and 13C NMR (pyridine-
d5), see Table 1.
3.4.2. Hydrangeifolin II. a-L-xylopyranosyl-(40 0 ! 20)-
(3-O-b-D-glucopyranosyl)-10-O-E-caffeoyl-b-D-
glucopyranoside (2)
30
Amorphous powder, ½aꢀD þ 6.5ꢂ
(c = 0.0039,
MeOH). UV kMeOH 282, 209. IR (film) cmꢁ1 3445,
2940, 1650, 1560, 1520, 1098, 1010. ES-MS [M + Na]+
m/z 659. HRESIMS [M ꢁ H]ꢁ m/z 635.1858 (Calcd.
1
for C26H36O19, 635.1821). H and 13C NMR (pyridine-
d5), see Table 1.
3.4.3. 1,6-Di-O-caffeoyl-b-D-glucopyranose (3)
30
Amorphous powder, ½aꢀD ꢁ 24.6ꢂ (c = 0.0058,
MeOH). UV kMeOH 334, 299, 265, 209. ES-MS
[M+Na]+ m/z 529. HRESIMS [M ꢁ H]ꢁ m/z 505.1355
(Calcd. for C24H24O13, 505.1345).1 H and 13C NMR
(pyridine-d5) (see Table 1).
3.4. Extraction and isolation
Dried and powdered leaves (2.70 kg) of C. hydrangei-
folia were extracted with EtOH (3.0 L) for 24 h at room
temperature, followed by solvent evaporation under re-
duced pressure to yield the crude extract (65.80 g). This
extract was suspended in water (3.0 L) and partitioned
with n-BuOH (3.0 L): the aqueous solution was evapo-
rated to dryness and the resulting residue (2.0 g) sub-
jected to gel filtration on a Sephadex LH-20 column
(3.0 · 120 cm) eluted with MeOH. Fractions (10 mL
each) were collected and analysed by TLC (CHCl3/
MeOH 6:4). On the basis of their TLC profiles, fractions
6–10 were combined (380.8 mg) and further subjected to
over Sephadex LH-20 CC with MeOH as eluent. Frac-
tions 8–12 from this second column fractionation were
combined (54.3 mg) on the basis of their TLC profiles
3.4.4. 1-O-(E)-caffeoyl-b-D-glucopyranose (4)
30
Amorphous powder, ½aꢀD ꢁ 16.5ꢂ
(c = 0.003,
MeOH). UV kMeOH 332, 300, 264, 211. The IR, 1H
and 13C NMR, and ES-MS data were in agreement with
literature values (Birkofer et al., 1961).
3.4.5. 1-O-(E)-feruloyl-b-D-glucopyranose (5)
30
Amorphous powder, ½aꢀD ꢁ 13.5ꢂ (c = 0.0028,
1
MeOH). UV kMeOH 281, 201. IR, H and 13C NMR,
and ES-MS data were in agreement with the literature
values (Birkofer et al., 1961).
3.4.6. Acid hydrolysis of 2
A solution of 2 (10 mg) in 2 M HCl (10 mL) was
heated at 90 ꢁC for 30 min and the mixture separated

1932
L. Hamerski et al. / Phytochemistry 66 (2005) 1927–1932
Nauk Azerbajdzhanskoj SSR 24, 89–92 [Chemical Abstracts, 70,
235, 1969, 118028p].
Birkofer, L., Thomas, U., Nouvertn, W., Kaiser, C., 1961. Naturlich
¨
by CC over Si-gel eluted with 15–70% MeOH to yield
caffeic acid (3.5 mg), glucose and xylose. The sugars
were identified by TLC using authentic standards for
comparison.
vorkommende Zuckerester von Phenolcarbonsa¨uren. Z. Natur-
forsch. Pt B 16, 249–251.
Birkofer, L., Kaiser, C., Hillges, B., Becker, F., 1969. NMR spectro-
scopic studies of acyl glycoses. Liebigs Ann. Chem. 725, 196–202.
Acknowledgements
´
Correa, M.P., 1974. Diciona´rio de Plantas Uteis do Brasil. Ministe´rio
da Agricultura, Rio de Janeiro.
This work was supported by Programa Biota-FA-
PESP (Project 98/05074-0). L.H. acknowledges FA-
Hamerski, L., Furlan, M., Silva, D.H.S., Cavalheiro, A.J., Eberlin,
M.N., Tomazela, D.M., Bolzani, V.S., 2003. Iridoid glucosides
from Randia spinosa Rubiaceae. Phytochemistry 63, 397–400.
Pauletti, P.M., Castro-Gamboa, I., Silva, D.H.S., Tomazela, D.M.,
Eberlin, M.N., Young, M.C.M., Bolzani, V.da S., 2003. New
antioxidant C-glucosylxanthones from stems of Arrabidaea sam-
ydoides. J. Nat. Prod. 66, 1384–1387.
Pratt, D.E., Miller, E.E., 1984. A flavonoid antioxidant in Spanish
peanuts Arachia hypogoea. J. Am. Oil Chem. Soc. 61, 1064–1067.
Silva, D.H.S, Pereira, F.C., Zanoni, M.V.B., Yoshida, M., 2001.
Lipophyllic antioxidants from Iryanthera juruensis fruits. Phyto-
chemistry 57 (3), 437–442.
PESP for
a Ph.D. fellowship: V. da S.B. and
M.C.M.Y acknowledge CNPq for research fellowships.
The authors thank Universidade de Campinas (UNI-
CAMP), Campinas SP, Brazil for providing high-resolu-
tion mass spectra.
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