Flavonol glycosides from Costus spicatus

Article abstract of DOI:10.1016/S0031-9422(99)00441-0

Two flavonol diglycosides, tamarixetin 3-O-neohesperidoside, kaempferide 3-O-neohesperidoside and the known quercetin 3-O-neohesperidoside, together with six other known flavonoids were isolated from the leaves of Costus spicatus and their structures were elucidated by a combination of spectroscopic and chemical methods. The flavonol diglycosides were evaluated for inhibitory activity of nitric oxide production by activated macrophages (Fig. 1).

Full text of DOI:10.1016/S0031-9422(99)00441-0

Phytochemistry 53 (2000) 87±92
www.elsevier.com/locate/phytochem
Flavonol glycosides from Costus spicatus
Bernadete P. da Silva, Robson R. Bernardo, Jose P. Parente*
NuÂcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, Brazil
Received 17 March 1999; received in revised form 14 July 1999
Abstract
Two ¯avonol diglycosides, tamarixetin 3-O-neohesperidoside, kaempferide 3-O-neohesperidoside and the known quercetin 3-
O-neohesperidoside, together with six other known ¯avonoids were isolated from the leaves of Costus spicatus and their
structures were elucidated by a combination of spectroscopic and chemical methods. The ¯avonol diglycosides were evaluated
for inhibitory activity of nitric oxide production by activated macrophages (Fig. 1). # 2000 Elsevier Science Ltd. All rights
reserved.
Keywords: Costus spicatus; Costaceae; Leaves; Flavonol glycosides; Nitric oxide; Macrophage activation
1. Introduction
2. Results and discussion
Costus spicatus Swartz (Costaceae), commonly called
`cana do brejo' in Brazil, is a medicinal plant found in
wet coastal forests. The rhizome of this plant is used
for the treatment of complaints of the bladder and ure-
thra and to expel kidney stones (Manfred, 1947). An
infusion of the aerial parts is taken to treat colds, sore
throats, dysentery and diarrhea (Cruz, 1965). To our
knowledge, no phytochemical studies on the aerial
parts have been reported. Here, we describe the iso-
lation and structure elucidation of two new ¯avonol
diglycosides from the leaves of C. spicatus, named
tamarixetin 3-O-neohesperidoside (1), kaempferide 3-
O-neohesperidoside (2) and the known quercetin 3-O-
neohesperidoside (3), together with tamarixetin 3-O-b-
D-glucopyranoside, kaempferide 3-O-b-D-glucopyrano-
side, quercetin 3-O-b-D-glucopyranoside, tamarixetin,
kaempferide and quercetin. Compounds 1±3 here
found to exhibit anti-in¯ammatory properties.
A methanolic extract of the leaves of C. spicatus was
puri®ed by Amberlite XAD-7 column chromatog-
raphy. Compounds 1±3 were isolated from the puri®ed
leaf extract by column chromatography over silica gel
and Sephadex LH-20 gel ®ltration followed by pre-
parative HPLC. The pure ¯avonol diglycosides (1±3)
were checked for homogeneity by analytical HPLC
which a€orded Rt (min) for 1 (18.83), 2 (17.23) and 3
(15.30).
Compound 1 was assigned the molecular formula
C28H32O16 by analysis of LSIMS (neg. ion mode) m/z
623 (M-H) and 13C-NMR spectral data (Table 1).
The IR spectrum showed bands of hydroxyl groups
(3400 cm 1), an a,b-unsaturated ketone (1651 cm 1),
1
1
and aromatic rings (1600, 1560, 1506 cm ). H-NMR
spectral data taken in DMSO-d6, revealed H-2-H-6
meta-coupling (1.8 Hz) at d 7.54, H-6-H-5 ortho-coup-
ling (8.4 Hz) at d 7.54 and 6.56, respectively, and H-8-
H-6 meta-coupling (1.8 Hz) at d 6.48 and 6.22, respect-
ively. As far as the disaccharide moiety is concerned,
the doublet (7.2 Hz) at d 5.80 has assigned to the
* Corrsponding author. Tel.: +21-270-2683; fax: +21-270-2683.
E-mail address: parente@nppn.ufrj.br (J.P. Parente).
0031-9422/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00441-0

88
B.P. da Silva et al. / Phytochemistry 53 (2000) 87±92
Fig. 1. Inhibitory activity on nitric oxide production of activated macrophages by compounds 1 (*), 2 (q) and 3 (r). Data points presented as
mean2SD, Ãp < 0.01, Ãp < 0.05. Signi®cant di€erence from control group.
anomeric proton of 3-O-b-D-glucopyranoside and a
broad singlet at 5.06, together with the doublet (6.2
Hz) at d 0.68, to the anomeric and methyl group pro-
tons of a-L-rhamnopyranose, respectively (Barrero,
sides. D-glucose and L-rhamnose were identi®ed by
GC±EIMS of the pertrimethylsilylated methylglyco-
sides. The UV spectrum of 1 in MeOH was indicative
of a 3-O-glycosidated ¯avonol (Markham, 1972) and
UV spectrometry using shift reagents (NaOMe,
NaOMe/H3BO3, AlCl3 and AlCl3/HCl) con®rmed the
structure of 1.
On acid hydrolysis, compound 1 yielded tamarixetin,
glucose and rhamnose. Mp, UV, IR, 1H- and 13C-
NMR spectral data, and EIMS were consistent with
the structure of tamarixetin (Barrero et al., 1998;
Struck & Kirk, 1970). Hence, 1 was established as 3-
[[2-O-(6-deoxy-a-L-mannopyranosyl)-b-D-glucopyrano-
syl]oxy]-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-
4H-1-benzopyran-4-one, named tamarixetin 3-O-neo-
hesperidoside.
Haidour, Munoz-Dorado, Akssira, Sedqui
&
Mansour, 1998). In NOE di€erence experiments, sep-
arate saturation of the methoxyl signal at d 3.83
resulted in the enhancement of H-5 ꢀd 6.86, d, J ˆ 8:4
Hz), indicating that this methoxyl group was at C-4.
The correlation peaks between the methoxyl proton
signal, and its corresponding quaternary carbon reson-
ance in the COLOC spectrum, allowed the carbon res-
onance at
Furthermore, the correlation peaks between H-2 and
d
149.38 to be assigned to C-4.
C-3 con®rmed these assignments.
The 13C-NMR spectrum showed a singlet which
resonated at d 55.74, and was assigned to the carbon
of the methoxyl-substituent at C-4. The signal at d
177.30 was attributed to the carbonyl carbon. The sig-
Compound 2 was assigned the molecular formula
C28H32O15 by analysis of its LSIMS (neg. ion mode)
m/z 607 (M-H) and 13C-NMR data (Table 1). The IR
spectrum was consistent with the proposed structure of
nals of the aglycone were assigned by DEPT, H±13C
1
COSY and H±13C COLOC and by comparison with
2. The H spectrum displayed, in addition to a signal
1
1
data from the literature (Barrero et al., 1998; Kim,
Higuchi, Kitamura & Komori, 1991). In the 13C-NMR
spectrum, signals of a b-glucopyranosyl and an a-
rhamnopyranosyl moiety were detected. The down®eld
shift of the C-2 and the up®eld shift of C-1 suggested
the position of attachment of the rhamnosyl moiety to
be C-2 of glucose. This linkage was proven by methyl-
ation analysis of 1, which showed a 2-linked glucopyr-
anose and a terminal rhamnopyranose. The molar
carbohydrate composition of 1 indicated the presence
of two neutral monosaccharides, glucose : rhamnose
(1.0 : 0.9). Their absolute con®gurations were deter-
mined by GC analysis of their TMSi (-)-2-butylglyco-
for a methoxyl group, two doublets at d 6.92 and 8.06
for H-3, H-5 and H-2, H-6, respectively. Two doublets
at d 6.22 and 6.46 integrating for single protons were
assigned to H-6 and H-8, respectively. A doublet at d
5.68 (J =7.2 Hz) and a broad singlet at d 5.08 inte-
grating for single protons were attributed to H-1 of a
b-glucosyl unit and a-rhamnosyl unit, respectively
(Table 1).
The 13C-NMR spectrum showed peaks for 28 car-
bon (Table 1) singlet which resonated at d 55.95 was
assigned to the carbon of a methoxyl-substituent at C-
4. The signal at d 177.29 was attributed to the carbo-
nyl carbon. The resonance of the aromatic moiety was

B.P. da Silva et al. / Phytochemistry 53 (2000) 87±92
89
Table 1
1H- and 13C-NMR spectral data for compounds 1±3 in DMSO-d6
a,b,c
1
2
3
Attribution
d
13C
d 1H
d
13C
d 1H
d
13C
d 1H
2
156.34
132.60
177.30
104.05
161.24
98.77
156.08
132.74
177.29
104.03
161.23
98.72
156.41
133.38
177.31
103.89
161.21
98.63
3
4
4a
5
6
6.22 d (C-5, C-7)
J ˆ 1:8 Hz
6.22 d (C-5, C-7)
J ˆ 1:8 Hz
6.21 d (C-5, C-7)
J ˆ 1:8 Hz
7
8
164.19
93.71
164.20
93.70
164.12
93.55
6.48 d (C-7, C-8)
J ˆ 1:8 Hz
6.46 d (C-7, C-8)
J ˆ 1:8 Hz
6.42 d (C-7, C-8)
J ˆ 1:8 Hz
8a
1'
2'
156.32
121.08
113.56
156.06
120.93
130.77
156.45
121.61
115.33
7.98 d (C-3')
J ˆ 1:8 Hz
8.06 d
7.70 d (C-3')
J ˆ 1:8 Hz
J ˆ 8:4 Hz
6.92 d (C-4')
J ˆ 8:4 Hz
3'
146.88
115.13
144.68
4'
5'
149.38
115.59
159.91
115.13
148.38
116.21
6.86 d
J ˆ 8:4 Hz
7.54 dd
6.92 d (C-4')
J ˆ 8:4 Hz
8.06 d
6.80 d
J ˆ 8:5 Hz
7.72 dd
6'
121.89
130.77
121.21
J ˆ 1:8, 8:4 Hz
12.52 s
J ˆ 8:4 Hz
12.53 s
J ˆ 1:8, 8:5 Hz
12.52 s
5-OH
4'-OCH3
Glc-10
55.74
98.77
3.83 s (C-4')
5.80 d (C-3)
J ˆ 7:2 Hz
55.95
98.37
3.87 s (C-4')
5.68 d (C-3)
J ˆ 7:2 Hz
98.48
5.75 d (C-3)
J ˆ 7:2 Hz
20
30
40
50
76.56
77.34
69.73
77.13
60.60
100.75
70.20
70.62
71.90
68.29
17.00
76.54
77.32
69.71
77.13
60.58
100.60
70.18
70.62
71.88
68.28
17.25
76.56
77.34
69.72
77.13
60.60
100.68
70.20
70.62
71.90
68.28
17.20
60
Rha-1000
2000
3000
4000
5000
6000
5.06 bs (C-20)
5.08 bs (C-20)
5.06 bs (C-20)
0.68 d
J ˆ 6:2 Hz
0.76 d
J ˆ 6:2 Hz
0.68 d
J ˆ 6:2 Hz
a
1H±13C COLOC correlations in parentheses.
b Proton and carbon signals were assigned by 2D-COSY, 1H 13C COSY and 1H and 13C COLOC experiments.
c Multiplicities were determined by DEPT experiments.
assigned by DEPT, 1H±13C COSY and 1H±13C
COLOC and by comparison with data from the litera-
ture (Dauguet, Bert, Dolley, Bekaert & Lewin, 1993,
con®gurations of the sugars, the methylation analysis
and 1H- and 13C-NMR spectral data indicated that
compound 2 possessed the same sugar moiety as 1.
Hence 2 was established as 3-[[2-O-(6-deoxy-a-L-man-
nopyranosyl)-b-D-glucopyranosyl]oxy]-5,7-dihydroxy-2-
Ko®nas, Chinou, Loukis, Harvala, Maillard
Hostettmann, 1998; Wenkert & Gottlieb, 1997).
&
The UV spectral data in MeOH and with shift re-
agents were indicative of a 3-O-glycosidated ¯avonol
(Markham, 1972). On acid hydrolysis, compound 2
yielded a ¯avonol aglycone, glucose and rhamnose.
Mp, UV, IR, 1H- and 13C-NMR spectral data, and
EIMS of the aglycone were in accordance with those
reported in the literature for kaempferide (Majumder
& Chattopadhyay, 1985; Tiwari & Srivastava, 1979).
The molar carbohydrate composition, the absolute
(4-methoxyphenyl)-4H-1-benzopyran-4-one,
kaempferide 3-O-neohesperidoside.
named
Flavonoid 3 was isolated and identi®ed by spectral
1
analyses and chemical reactions. UV, IR and H-NMR
spectral data were similar to reported values for quer-
cetin 3-O-neohesperidoside (Bacon, Mabry
&
Harborne, 1975; Williams, Harborne & Cli€ord, 1971).
In the 13C-NMR spectrum (Table 1) the chemical
shifts of the glycosidic moiety resonances were very

90
B.P. da Silva et al. / Phytochemistry 53 (2000) 87±92
similar to those of 1 and 2, indicating a neohesperido-
side sequence. The LSIMS exhibited a [M-H] at m/z
607.
were obtained on a Varian Gemini 200 NMR spec-
trometer operating at 200 MHz for dH and 50 MHz
for dC, in DMSO-d6, TMS as international standard.
GC was carried out with FID, using a glass capillary
column (0.31 mm  25 m) SE-30. EIMS and GC±MS:
recorded at 70 eV. Negative LSIMS was carried out
using HMPA-glycerol as matrix, 35 kV anodic voltage,
8 kV accelerating voltage using Cs ions.
Silica gel column (230±400 mesh ASTM, Merck),
Amberlite XAD-7 nonionic polymeric adsorbent (20±
60 mesh, Aldrich) and Sephadex LH-20 were used for
CC. TLC was performed on silica gel coated plates
(Merck) using the following solvent systems: (a)
CHCl3±MeOH±H2O (65 : 35 : 10, lower phase) for ¯a-
vonol diglycosides and (b) CHCl3±MeOH (19 : 1) for
iso¯avone aglycones, and (c) n-BuOH±pyridine±H2O
(6 : 4 : 3) for sugars. Compounds 1±3 were detected
under UV (254 and 366 nm) and by spraying with
orcinol±H2SO4, sugars were detected by spraying with
Six other known ¯avonoids were isolated from the
leaves of C. spicatus and their structures were estab-
lished by comparison with data from the literature as
tamarixetin 3-O-b-D-glucopyranoside (Ishak, El Sissi,
El Sherbieny & Nawwar, 1972), kaempferide 3-O-b-D-
glucopyranoside (Tiwari & Srivastava, 1979), quercetin
3-O-b-D-glucopyranoside (Kim et al., 1991), tamarixe-
tin (Struck & Kirk, 1970), kaempferide (Majumder &
Chattopadhyay, 1985) and quercetin (Kim et al.,
1991).
In order to investigate the anti-in¯ammatory proper-
ties of the ¯avonol diglycosides, compounds 1±3 were
evaluated for their inhibitory activity on nitric oxide
production by activated macrophages (Dirsch,
Stuppner & Vollmar, 1998). Compound 3, which was
already enhance protein formation by endothelial cells
and inhibition of endothelial tissue injury (Chen, Fang,
Gu, Zhang & Zhao, 1990), exhibited an IC50 value of
55 mM, showing moderate inhibitory activity on nitric
oxide production. Nonetheless, compounds 1 and 2
showed IC50 values higher than 100 mM, indicating
slight inhibitory activity. The above results suggest
that these compounds may be the potential therapeutic
agents involved in in¯ammatory disorders, justifying
the use of C. spicatus in Brazilian traditional medicine
(Cruz, 1965).
aniline±diphenylamine-85
MeOH (1 : 1 : 5 : 43).
orthophosphoric
acid±
3.2. Plant material
Leaves of C. spicatus Benth. were collected at Ilha
do Fundao, Rio de Janeiro in September 1996, and
identi®ed by Luci S. Valle. A voucher specimen (no.
R192950) is deposited at the herbarium of the
National Museum, Rio de Janeiro, Brazil.
3.3. Extraction and isolation
Dried and powdered leaves of C. spicatus (250 g)
were extracted with cold MeOH (2 l). Evaporation of
the MeOH gave a residue (10 g), which was chromato-
graphed on Amberlite XAD-7 (100 g). Fractions eluted
with MeOH yielded a mixture of ¯avonol glycosides
(1.75 g), which was submitted to CC (120 Â 3 cm) on
silica gel eluted with CHCl3MeOH mixtures, of
increasing polarity (up to 35% MeOH) to a€ord a
mixture of three compounds (350 mg, CHCl3±MeOH,
66 : 34) which were isolated by Sephadex LH-20 gel ®l-
tration followed by preparative HPLC to a€ord tamar-
ixetin 3-O-neohesperidoside (1, 125 mg, Rf 0.28),
kaempferide 3-O-neohesperidoside (2, 70 mg, Rf 0.41)
and quercetin 3-O-neohesperidoside (3, 45 mg, Rf
0.19).
3. Experimental
3.1. General
3.4. High-performance liquid chromatography
Melting
points
were
determined
by
an
The analytical HPLC system (Shimadzu, LC-10AD)
was equipped with a diode-array detector, a 20 ml loop
and a 200 Â 4.6 mm ODS Hypersil column, 5 mm. For
the preparative separation, a 250 Â 10 mm Econosil
C18 column, 10 mm, was used. Analytical and prepara-
tive HPLC systems were operated at room temperature
Ellectrothermal 9200 micro melting point apparatus
and are uncorrected. OR were measured on a Perkin
Elmer 243B polarimeter. UV and IR spectra were
measured on a Shimadzu UV-1601 and on a Perkin
Elmer 599B, respectively. 1H- and 13C-NMR spectra

B.P. da Silva et al. / Phytochemistry 53 (2000) 87±92
91
1
using the same solvents: (A) HCOOH±H2O (1 : 9) and
(B) HCOOH±H2O±MeOH (1 : 4 : 5). The elution pro-
®le for analytical HPLC consisted of isocratic elution
(90% A, 10% B) for 10 min followed by a linear gra-
dient from 10 to 100% B for 20 min and then isocratic
elution (100% B) for 10 min followed by linear gradi-
ent from 100 to 10% B for 10 min. The ¯ow rate was
Melting point and UV, IR, H- and 13C-NMR spectral
data and EIMS of kaempferide were in accordance
with those reported in the literature (Tiwari
Srivastava, 1979; Majumder & Chattopadhyay, 1985).
&
3.7. Quercetin 3-O-neohesperidoside (3)
1
1.0 ml min and aliquots of 15 ml were injected. The
Physical constants, UV, IR and 1H-NMR spectral
data of compound 3 were in accordance with those
reported in the literature (Williams et al., 1971; Bacon
et al., 1975). 13C-NMR spectral data are shown in
Table 1. Negative LSIMS, m/z (rel. int.): 609 [M-H]
(72), 301 [M-309] (100). Compound 3 (40 mg) was
hydrolyzed by the procedure described for 1 and 2 to
a€ord quercetin (14 mg), glucose and rhamnose.
Identity of quercetin was established by comparison
with an authentic sample through analysis of mp, IR,
1H- and 13C-NMR, and EIMS.
elution pro®le for preparative separations consisted of
an isocratic elution (90% A, 10% B) for 10 min, a lin-
ear gradient from 10 to 100% B for 20 min, an iso-
cratic elution for 15 min, followed by linear gradient
from 100 to 10% B for 10 min. The ¯ow rate was 4.0
1
ml min
.
3.5. Tamarixetin 3-O-neohesperidoside (1)
Yellow amorphous powder from MeOH, mp 180±
20
MeOH
max
1908 dec, ‰aŠ
788 (DMSO, c 0.001). UV l
nm
D
ꢀlog e): 254 (3.3), 286 (1.0), 373 (3.5); + AlCl3: 268,
298, 386; + AlCl3/HCl: 270, 300, 376; + NaOMe:
271, 328, 414; + NaOMe/H3BO3: 252, 282, 370. IR
3.8. Molar carbohydrate composition and D, L
con®gurations
nmax cm 1: 3400 (OH), 1651, 1600, 1560, 1506, 1289,
Monosaccharides were analyzed as their TMSi
methylglycosides obtained after methanolysis (0.5 M
HCl in MeOH, 24 h, 808) and trimethylsilylation
(Kamerling, Gerwig, Vliegenthart & Clamp, 1975).
The con®gurations of the glycosides were established
by capillary GC and GC±MS of their TMSi ( )-2-
butylglycosides (Gerwig, Kamerling & Vliegenthart,
1978).
KBr
1205, 1166, 1129, 1050, 1030, 980. H- and 13C-NMR
1
spectral data shown in Table 1. Negative LSIMS, m/z
(rel. int.): 623 [M-H] (30), 315 [M-309] (100).
Compound 1 (40 mg) was heated in 2 M HCl (5 ml)
until re¯ux began, this being continued for 2 h.
Aglycone was extracted with EtOAc and evaporated to
dryness in vacuo. The residue was dissolved in MeOH
and the solution on concentration yielded a yellow
compound which on further crystallization gave tamar-
ixetin (18 mg). Melting point and UV, IR, 1H- and
13C-NMR spectral data and mass spectral analysis
were in accordance with those reported in the litera-
ture (Struck & Kirk, 1970; Barrero et al., 1998).
Aqueous layer was adjusted to pH 6 by addition of
NaHCO3. After lyophilization, sugars were dissolved
in pyridine and analyzed by silica gel-TLC in the
above-described system. After spraying, rhamnose
gave a green spot at Rf 0.75, and glucose gave a blue
spot at Rf 0.70.
3.9. Methylation analysis
Compounds 1±3 were methylated with DMSO-
lithium methylsulphinyl carbanion-CH3I (Parente,
Cardon, Leroy, Montreuil, Fournet & Ricart, 1985).
The methyl ethers were obtained either (a) after hy-
drolysis (4 N TFA, 2 h, 1008) and analyzed as partially
poliol-acetates by GC±MS (Fournet, Dhalluin, Leroy,
Montreuil & Mayer, 1978) or (b) after methanolysis
(0.5 M HCl in MeOH, 24 h, 808) and analyzed as par-
tially methylated methylglycosides by GC±MS
(Fournet, Strecker, Leroy & Montreuil, 1981).
3.6. Kaempferide 3-O-neohesperidoside (2)
3.10. Measurement of nitric oxide production
Yellow amorphous powder from MeOH, mp 170±
20
MeOH
max
1808 dec, ‰aŠ
85 (DMSO, c 0.001). UV l
nm
Male BALB/c mice weighing from 20 to 25 g were
used. Cells were collected from the peritoneal cavities
by washing with culture medium, and were inoculated
into ¯at-bottomed 96-well tissue culture plates (1 Â 106
cells/ml) in RPMI 1640 medium containing 10% fetal
calf serum. After incubation for 24 h at 378C in a CO2
incubator, nonadherent cells were removed by washing
with fresh culture medium. The cell suspensions were
incubated with 1 mg/ml Escherichia coli lipopolysac-
charide as stimulant and compounds 1±3 at di€erent
D
ꢀlog e): 252 sh, 266 (3.0), 353 (3.5); +AlCl3: 254, 270,
418; +AlCl3/HCl: 256, 270, 355; +NaOMe: 280, 323,
KBr
1
411; +NaOMe/H3BO3: 254, 268, 355. IR nmax cm
3438 (OH), 1650, 1600, 1558, 1504, 1287, 1203, 1163,
:
1127, 1048, 1029, 975. H- and 13C-NMR spectral data
1
shown in Table 1. Negative LSIMS, m/z (rel. int.): 607
[M-H] (27), 299 [M-309] (100). Compound 1 (40 mg)
was hydrolyzed by the procedure described above to
a€ord kaempferide (15 mg), glucose and rhamnose.

92
B.P. da Silva et al. / Phytochemistry 53 (2000) 87±92
analysis of glycoprotein glycans. Analytical Biochemistry, 116,
489±502.
concentrations for 48 h at 378C. After this period, 100
ml of cell culture supernatant were removed and com-
bined with 90 ml 1% sulfanilamide in 5% H3PO4 and
90 ml 0.1% N-(1-naphthyl)-ethylenediamine dihy-
drochloride followed by spectrophotometric measure-
ment at 550 nm (Green, Wagner, Glogowski, Skipper,
Wishnok & Tannenbaum, 1982).
Gerwig, G. J., Kamerling, J. P., & Vliegenthart, J. F. G. (1978).
Determination of the D and L con®guration of neutral monosac-
charides by high-resolution capillary G. L. C. Carbohydrate
Research, 62, 349±357.
Green, L. C., Wagner, D. A., Glogowski, J., Skipper, P. L.,
Wishnok, J., & Tannenbaum, S. (1982). Analysis of nitrate,
nitrite, and
Biochemistry, 126, 131±138.
‰
15NŠ nitrate in biological ¯uids. Analytical
Ishak, M. S., El Sissi, H. I., El Sherbieny, A. E. A., & Nawwar, M.
A. M. (1972). Tannins and polyphenolics of the galls of Tamarix
aphylla. Planta Medica, 21(4), 374±381.
Acknowledgements
Kamerling, J. P., Gerwig, G. J., Vliegenthart, J. F. G., & Clamp, J.
R. (1975). Characterization by gas±liquid chromatography±mass
spectrometry and proton-magnetic-resonance spectroscopy of per-
trimethylsilyl methyl glycosides obtained in the methanolysis of
glycoproteins and glycopeptides. Biochemical Journal, 151, 491±
495.
The authors are grateful to Eduardo M. B. da Silva
and Maria C. P. Lima for recording the spectra. They
also express their thanks to the Brazilian Research
Council for ®nancial support.
Kim, Y. C., Higuchi, R., Kitamura, Y., & Komori, T. (1991).
Degradation of ¯avonoid glycosides. Liebigs Annalen der
Chemie, 1285-1289.
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