J. Agric. Food Chem. 2001, 49, 3796−3801
3796
Isolation and HPLC Quantitative Analysis of Flavonoid Glycosides
from Brazilian Beverages (Maytenus ilicifolia and M. aquifolium)
Joao Paulo V. Leite,† Luca Rastrelli,*,‡ Giovanni Romussi,§ Alaide B. Oliveira,†
Janete H. Y. Vilegas,| Wagner Vilegas,⊥ and Cosimo Pizza‡
Departamento de Quimica, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, Dipartimento di
Scienze Farmaceutiche, Università degli Studi di Salerno, Via Ponte don Melillo, 84084, Fisciano (SA),
Italy, Dipartimento di Chimica e Tecnologie Farmaceutiche, Universitá degli Studi di Genova, Genoa, Italy,
Instituto de Quimica de São Carlos, Universidade de São Paulo, São Carlos, Brazil, Instituto de Quimica de
Araraquara, Universidade Estadual Paulista, C. Postal 355, 14801-970, Araraquara, SP, Brazil
Aqueous infusions of Brazilian Maytenus leaves are used as beverages, foodstuffs, and phytomedicines. Previously, we isolated two new flavonoid tetrasaccharides from the infusion of Maytenus
aquifolium leaves that showed antiulcer activity. In this investigation a new flavonoid tetrasaccharide, kaempferol-3-O-R-L-rhamnopyranosyl (1f6)-O-[R-L-arabinopyranosyl (1f3)-O-R-L-rhamnopyranosyl (1f2)]-O-β-D-galactopyranoside (3), was isolated, together with kaempferol tri- and
disaccharides and quercetin trisaccharides from the aqueous infusion of Maytenus ilicifolia leaves.
All structures were elucidated by ES-MS and NMR spectroscopic methods. The quantitative analysis
of the flavonoid glycosides from Maytenus ilicifolia and M. aquifolium has been performed by HPLC.
Keywords: Maytenus ilicifolia; M. aquifolium Martius; Celastraceae; espinheira santa; infusion;
beverage; flavonoids glycosides; 1D and 2D NMR; quantitative determination
INTRODUCTION
Maytenus ilicifolia Martius ex Reiss. and M. aquifolium Mart. (Celastraceae) are plant species widely used
in Brazilian folk medicines in the form of aqueous
infusions as antiulcers and against stomach diseases,
and also as beverages in daily life instead of green tea.
They are found in the local commerce as capsules,
powders, dried leaves, fresh leaves, or as aqueous or
aqueous-alcoholic preparations. Maytenus ilicifolia and
M. aquifolium are populary known as “espinheirasanta” (holly spines) because their leaves are spined.
Many other species also have similar morphology (e.g.,
Sorocea bomplandii Baill., Moraceae and Zolernia ilicifolia, Leguminosae) and therefore many adulterations
are commonly found in the Brazilian local commerce.
M. aquifolium was also reported as being an adulterant
of the “yerba-mate” (Ilex paraguariensis St. Hil., Aquifoliaceae) in Paraguay (1). This beverage is also widely
used in South Brazil. In a previous work we isolated
two new flavonoid tetra-glycosides from the infusion of
the leaves of M. aquifolium: quercetin-3-O-R-L-rhamnopyranosyl(1f6)-O-[β-D-glucopyranosyl(1f3)-O-R-Lrhamnopyranosyl(1f2)]-O-β-D-galactopyranoside (1), and
kaempferol-3-O-R-L-rhamnopyranosyl (1f6)-O-[β-D-glucopyranosyl(1f3)-O-R-L-rhamnopyranosyl(1f2)]-O-β-Dgalactopyranoside (2) (Figure 1); and we evaluated
the antiulcer activity of the infusion (2, 3). In this
investigation of the aqueous infusion of the leaves of
M. ilicifolia a new flavonoid tetra-glycoside, kaempferol* To whom correspondence should be addressed. Phone:
0039 89 964356. Fax: 0039 89 964356. E-mail: rastrelli@unisa.it.
†
Universidade Federal de Minas Gerais.
‡ Università degli Studi di Salerno.
§
Universitá degli Studi di Genova.
|
Universidade de São Paulo.
⊥
Universidade Estadual Paulista.
3-O-R-L-rhamnopyranosyl (1f6)-O-[R-L-arabinopyranosyl (1f3)-O-R-L-rhamnopyranosyl(1f2)]-O-β-D-galactopyranoside (3) (Figure 1), was isolated, together with
kaempferol tri- and disaccharides (4 and 5) and quercetin trisaccharides (6) (Figure 2). The quantitative
analysis of flavonoids from M. aquifolium and M.
ilicifolia extracts has been performed by HPLC, as a
potential method for quality control purposes. The
chemical information obtained is important not only for
the correct understanding of this folk utilization but
also for the future validation of these compounds as
markers for the assessment of Brazilian Maytenus
infusions (4).
MATERIALS AND METHODS
Biological Material. The leaves of Maytenus ilicifolia and
M. aquifolium were furnished by Ana Maria Soares Pereira,
UNAERP, Ribeirao Preto, SP, Brazil. Voucher samples are
deposited at the Herbario of the Universidade Estadual
Paulista (UNESP). Commercial samples were also purchased
in the local market and submitted to the same experimental
procedures.
Apparatus. The ES-MS spectra were determined on a
Fisons Platform spectrometer both in the positive (90 V) and
negative (100 V) mode. The sample was dissolved in MeOH
and injected directly.
UV spectra were measured in a HP 8472-A spectrometer
(MeOH, c ) 1). IR spectra: Nicolet Impact 400, KBr.
A Bruker DRX-600 spectrometer, operating at 599.19 MHz
for 1H and 150.858 for 13C, with the UXNMR software package,
was used for NMR experiments measured in CD3OD. The
DEPT experiments were performed using transfer pulse of
135° to obtain positive signals for CH and CH3 and negative
signals for CH2. Polarization transfer delays were adjusted to
an average CH coupling of 135 Hz. ROESY (5), 1H-1H DQFCOSY (6, 7), 1H-13C HSQC, and HMBC (8) experiments were
obtained using the conventional pulse sequences as described
in the literature, and 1D TOCSY (9) was acquired using
10.1021/jf010294n CCC: $20.00 © 2001 American Chemical Society
Published on Web 07/18/2001
Flavanoid Glycosides from Brazilian Maytenus Beverages
J. Agric. Food Chem., Vol. 49, No. 8, 2001 3797
Figure 1. Flavonoid tetra-glycosides isolated from Maytenus aquifolium and Maytenus ilicifolia leaves: (1) quercetin-3-O-R-Lrhamnopyranosyl(1f6)-O-[β-D-glucopyranosyl(1f3)-O-R-L-rhamnopyranosyl(1f2)]-O-β-D-galactopyranoside, (2) kaempferol-3-OR-L-rhamnopyranosyl(1f6)-O-[β-D-glucopyranosyl(1f3)-O-R-L-rhamnopyranosyl(1f2)]-O-β-D-galactopyranoside, and (3) kaempferol3-O-R-L-rhamnopyranosyl(1f6)-O-[R-L-arabinopyranosyl(1f3)-O-R-L-rhamnopyranosyl(1f2)]-O-β-D-galactopyranoside. Gal ) β-Dgalactopyranosyl, Rha ) R-L-rhamnopyranosyl, Glc ) β-D-glucopyranosyl, and Ara ) R-L-glucopyranosyl.
waveform, generator-based GAUSS shaped pulse, a mixing
time ranging from 80 to 100 ms, and a MLEV-17 spin-lock
field of 10 kHz preceded by a 2-ms trim pulse.
HPLC separations were performed on a Waters 590 series
pumping system equipped with a Waters R401 refractive index
detector and with a Waters µ-Bondapak C-18 column and a
U6K injector.
GC analyses were run using a Hewlett-Packard 5890 gas
chromatograph equipped with a 5970 mass-selective detector
and a HP-5 fused-silica column (25 m × 0.2 mm i.d.; 0.33 µm
film).
Quantitative HPLC analyses were performed on a Shimadzu
liquid chromatograph, equipped with a LC-10AD pump, a
Rheodyne injector valve (fitted with a 20-µL loop), a SPD 10AV
UV-vis spectrophotometric detector (set at λ 254 nm), and a
Ultrasphere ODS 5 µm (Altex) column (250 × 4.6 mm i.d.).
Peak areas were calculated by a Shimadzu C-R6A integrator.
Extraction and Isolation. Leaves of M. ilicifolia were airdried and milled. A 200-g portion of the powdered plant was
boiled in water (1 L) for 8-9 min. The mixture was allowed to
cool, filtered through filter paper, and evaporated to dryness
affording 18 g of crude extract. An aliquot (2.0 g) was dissolved
in 10 mL of MeOH and fractionated on a Sephadex LH-20
column (1 m × 3 cm i.d.) with a flow rate of 0.5 mL/min. A
total of 100 fractions, of 8 mL each, was collected. After TLC
analysis (Si-gel, n-BuOH-AcOH-H2O 65:15:25, CHCl3-
MeOH-H2O 70:30:3, v/v), fractions with similar Rfs were
combined, giving 6 major fractions named A-F which were
further purified by HPLC (C-18 µ-Bondpak column (30 cm ×
7.8 mm, flow rate 2.5 mL/min). Fraction E (150 mg) was
purified using MeOH-H2O (40:60, v/v) as the eluent to yield
pure compound 3 (21.2 mg, Rt ) 16 min). Fraction D (200 mg)
was purified using MeOH-H2O (50:50, v/v) as the eluent to
yield pure compounds 4 (30.0 mg, Rt ) 14 min) and 5 (9.2 mg,
Rt ) 12 min). Fraction C (150 mg) was purified using MeOHH2O (60:40, v/v) as the eluent to yield pure compound 6 (18.1
mg, Rt ) 13 min).
Quantitative Analysis. Quantitative HPLC analyses were
carried out on an Ultrasphere ODS 5-µm (250 × 4.6 mm)
column, using isocratic elution with MeOH-H2O (40:60, v/v)
at a flow rate of 1.0 mL/min. Compounds 1 and 3 were used
as external standards. For sample preparation, 500 mg of
pulverized plant material was extracted twice with 80 mL of
methanol-water (80:20, v/v) at 60 °C for 15 min. After
filtration, the filtrate was adjusted to a final volume of 200
mL in a volumetric flask. A standard solution containing 50
µg/mL of flavonoid tetragycosides 1 and 3 in methanol-water
(80:20, v/v) was also prepared, and 1 mL of this solution was
diluted to 10 mL with the same solvent. A linear relationship
between peak area and concentration (1-10 µg/mL) was
observed with a correlation coefficient r ) 0.9997 for each
glycoside. The relationship between peak areas (y) and con-
3798 J. Agric. Food Chem., Vol. 49, No. 8, 2001
Figure 2. Known flavonoid glycosides isolated from Maytenus
ilicifolia leaves: (4) kaempferol-3-O-R-L-rhamnopyranosyl(1f6)-O-[ L -rhamnopyranosyl(1f2)]-O-β- D -galactopyranoside, (5) kaempferol-3-O-R-L-rhamnopyranosyl(1f2)]-O-β-Dgalactopyranoside, and (6) quercetin-3-O-R-L-rhamnopyranosyl
hf(1f6)-O-[R-L-rhamnopyranosyl(1f2)]-O-β-D-galactopyranoside.
centrations in µg/mL (x) was y ) 10240x - 984 (1) and y )
9593x - 842 (3). The minimum detection limit was 0.2 ng,
which resulted in a signal-to-noise ratio of 3:1. Reproducibility
was verified with 5 extracts of an identical sample. Relative
standard deviations (%) were 4.79 and 4.95, respectively; for
retention times the standard deviation was less than 1%.
Acid Hydrolysis of Compounds 3-6. A solution of each
compound (3.0 mg) in 6% HCl (3.5 mL) was refluxed for 2 h.
The reaction mixture was diluted with H2O and then extracted
with EtOAc. The resulting products were identified by their
Rf values on TLC and also by their 1H NMR spectra.
Methanolysis of Compounds 3-6. Each compound (1.0
mg) was heated in a vial for 24 h at 80 °C in MeOH-2% HCl
(2 mL). After MeOH and HCl evaporation in a N2 stream,
Ag2CO3 and MeOH were added until CO2 production stopped.
The centrifugate was dried over P2O5. The resulting monosaccharides were treated with Trisil-Z (Pierce) and analyzed by
GC-MS.
Compound 3. Yellowish amorphous solid. mp 269-271 °C.
[R]D25 -63° (c 0.1, CH3OH); UV λ max (nm, MeOH): 265, 352;
+ KOH: 271, 327, 396; +AlCl3: 270, 301 sh, 350; +AlCl3 +
HCl: 272, 301 sh, 348, 393; + NaOAc: 271, 363; + NaOAc +
H3BO3: 265, 352. IR (KBr): 3376 (OH), 1650 (CdO) cm-1. ESMS, m/z (rel int.) (100 V, negative mode): 871 [M - H]- (100),
725 [M - H - rha]- (6), 749 [M - H - ara]- (2), 285 [M - H
- ara - 2 rha - gal]- ) [A - H]- (79); (90 V, positive mode):
911 [M + K]+ (40), 895 [M + Na]+ (47), 873 [M + H]+ (18),
595 [M + H - ara - rha]+ (20), 449 [M + H - ara - 2 rha]+
(16), 287 [A + H]+ ) [M + H - glu - 2 rha - gal]+ (100).
Anal. Calcd for C38H48O23: C 52.29%, H 5.54%, O 42.16%.
Found: C 52.11%, H 5.48%, O 41.03%. 1H and 13C NMR data
are provided in Tables 1 and 2.
Compound 4. Colorless amorphous solid. UV λ max (nm,
MeOH): 265, 352; +KOH: 270, 326, 395; +AlCl3: 269, 300
sh, 350; +AlCl3 + HCl: 270, 300 sh, 347, 392; + NaOAc: 270,
362; +NaOAc + H3BO3: 267, 352. IR (KBr): 3374 (OH), 1653
(CdO) cm-1. ES-MS, m/z (rel int.) (100 V, negative mode): 739
[M - H]- (100), 593 [M - H - rha]- (7), 447 [M - H - 2 rha](7), 285 [M - H - 2 rha - gal]- ) [A - H]- (69); (90 V, positive
mode): 779 [M + K]+ (39, 763 [M + Na]+ (53), 741 [M + H]+
(14), 595 [M + H-rha]+ (18), 449 [M + H - 2 rha]+ (15), 287
[A + H]+ ) [M + H - 2 rha - gal]+ (100); Anal. calcd for
C33H40O19: C 53.51%, H 5.44%, O 41.04%. Found: C 52.38%,
H 5.41%, O 40.89%. 1H and 13C NMR data are presented in
Tables 1 and 2.
Leite et al.
Compound 5. Colorless needles. UV λ max (nm, MeOH):
267, 351; +KOH: 270, 325, 395; +AlCl3: 271, 302 sh, 352;
+AlCl3 + HCl: 270, 302 sh, 346, 393; + NaOAc: 270, 362;
+NaOAc + H3BO3: 264, 353. IR (KBr): 3374 (OH), 1653 (Cd
O) cm-1. ES-MS, m/z (rel int.) (100 V, negative mode): 593
[M - H]- (100), 447 [M - H - rha]- (5), 285 [M - H - rha gal]- ) [A - H]- (83); (90 V, positive mode): 633 [M + K]+
(30), 627 [M + Na]+ (60), 595 [M + H]+ (14), 449 [M + H rha]+ (15), 287 [A + H]+ ) [M + H - rha - gal]+ (100). Anal.
calcd for C27H30O15: C 54.51%, H 5.09%, O 40.37%. Found: C
54.38%, H 4.81%, O 40.19%. 1H and 13C NMR data are
presented in Tables 1 and 2.
Compound 6. Colorless needles. UV λ max (nm, MeOH):
267, 352; +KOH: 272, 330, 392; +AlCl3: 271, 305 sh, 349;
+AlCl3 + HCl: 273, 305 sh, 346, 393; + NaOAc: 271, 363;
+NaOAc + H3BO3: 269, 353. IR (KBr): 3380 (OH), 1655 (Cd
O) cm-1. ES-MS, m/z (rel int.) (100 V, negative mode): 755
[M - H]- (100), 609 [M - H -rha]- (10), 301 [M - H - 2 rha
- gal]- ) [A - H]- (80); (90 V, positive mode): 795 [M + K]+
(40), 779 [M + Na]+ (45), 757 [M + H]+ (11), 611 [M + H rha]+ (18), 465 [M + H - 2 rha]+ (13), 303 [A + H]+ ) [M + H
- 2 rha - gal]+ (100); Anal. calcd for C33H40O20,: C 52.38%, H
5.33%, O 42.29%. Found: C 52.19%, H 5.21%, O 42.03%. 1H
and 13C NMR data are provided in Tables 1 and 2.
RESULTS AND DISCUSSION
The infusion from the leaves of M. ilicifolia was
prepared as described in the previous section. Carlini
(10) has previously established the antiulcer activity
of the aqueous infusion and did not detect any toxic
effect.
The infusion was fractionated by GPC to investigate
its chemical constituents as described above. Fractions
were further purified on reversed-phase HPLC to yield
pure flavonoid glycosides 3-6 as the major constituents
of this infusion. Acid hydrolysis of 3-6 released
kaempferol for 3-5 and quercetin for 6. The aglycons
were identified by 1H and 13C NMR spectra. The gaschromatographic analysis of the methanolysis products
showed the presence of galactose, rhamnose, and arabinose, in the ratio 1 gal:2 rha:1 ara for 3, 1 gal:2 rha
for 4 and 6, and 1 gal:1 rha for 5. Retention times were
identical to those of the authentic Trisil sugars.
The ES-MS (100 V, negative ion) mass spectrum of 3
gave as base peak the [M - H]- ion at m/z 871. The
fragment at m/z 285 corresponds to the deprotonated
aglycon [A - H]-. Fragment ions occurred at m/z 725
[(M - H) - 146]- and at m/z 739 [(M - H) - 132]-,
which were interpreted as independent losses of terminal deoxyhexose and pentose units. In the ES-MS
spectrum in the positive-ion mode (90 V) we observed
the pseudomolecular ion [M + H]+ at m/z 873. The
adducts [M + Na]+ at m/z 895 and [M + K]+ at m/z
911 were also observed. The fragment at m/z 595 [M +
H - 146 - 132]+ corresponds to the loss of the two
terminal sugars. The fragments at m/z 449 [M + H 146 - 132 - 146]+ and the base peak at at m/z 287 [M
+ H - 146 - 132 - 146 - 162]+ ) [A + H]+ correspond
to the subsequent losses of deoxyhexose and hexose
moieties.
The complete structure of 3 was elucidated by 1D and
2D NMR experiments at 600 MHz. The 1H NMR
spectrum of 1 (Table 1) displayed signals for two metacoupled protons at δ 6.18 (d, J ) 1.5 Hz, H-6) and δ
6.35 (d, J ) 1.5 Hz, H-8) and also for an ortho-coupled
system at δ 8.08 (d, J ) 8.5 Hz, H-2′ and H-6′) and δ
6.92 (d, J ) 8.5 Hz, H-3′ and H-5′) indicating a
kaempferol derivative (11).
The 13C NMR shifts of the aglycon part of 3 (Table 2)
corresponded well with the shifts for kaempferol, with
Flavanoid Glycosides from Brazilian Maytenus Beverages
Table 1.
1H
proton
6
8
2′
3′
5′
6′
3-Gal
1′′
2′′
3′′
4′′
5′′
6a′′
6b′′
(6-1) Rha
1′′′′
2′′′′
3′′′′
4′′′′
5′′′′
6′′′′
(3-1) Ara
1′′′′′
2′′′′′
3′′′′′
4′′′′′
5′′′′′
(2-1) Rha
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
J. Agric. Food Chem., Vol. 49, No. 8, 2001 3799
NMR Assignments (δH in CD3OD) of Compounds 3-6a
3
4
5
6
6.18 (d, 1.5)
6.35 (d, 1.5)
8.08 (d, 8.5)
6.92 (d, 8.5)
6.92 (d, 8.5)
8.08 (d, 8.5)
6.20 (d, 1.5)
6.40 (d, 1.5)
8.09 (d, 8.5)
6.92 (d, 8,5)
6.92 (d, 8.5)
8.09 (d, 8.5)
6.17 (d, 1.5)
6.35 (d, 1.5)
8.10 (d, 8.5)
6.91 (d, 8.5)
6.91 (d, 8.5)
8.10 (d, 8.5)
6.17 (d, 1.5)
6.35 (d, 1.5)
7.72 (d, 1.5)
-----6.90 (d, 8.5)
7.61 (dd, 1.5, 8.5)
5.57 (d, 7.5)
3.96 (dd, 7.5, 9.7)
3.73 (dd, 7.5, 3.6)
3.51 (dd, 3.5, 1.5)
3.67 (ddd, 1.5, 5.0, 7.0)
3.45 (dd, 12.0, 7.0)
3.76 (dd, 12.0, 5.0)
5.63 (d, 7.5)
3.95 (dd, 7.5, 9.6)
3.73 (dd, 7.5, 3.5)
3.51 (dd, 3.5, 1.5)
3.66 (ddd, 1.5, 5.0, 7.0)
3.45 (dd, 12.0, 7.0)
3.77 (dd, 12.0, 5.0)
5.55 (d, 7.5)
3.57 (dd, 7.5, 9.5)
3.74 (dd, 7.5, 3.6)
3.49 (dd, 3.5, 1.5)
3.44(ddd, 1.5, 5.0, 7.0)
3.58 (dd, 12.0, 7.0)
3.73 (dd, 12.0, 5.0)
5.63 (d, 7.5)
3.94 (dd, 7.5, 9.5)
3.75 (dd, 7.5, 3.5)
3.50 (dd, 3.5, 1.5)
3.66 (dd,5.0,7.0)
3.45 (dd, 12.0, 7.0)
3.76 (dd, 12.0, 5.0)
4.55 (d, 1.5)
3.60 (dd, 3.5, 1.5)
3.55 (dd, 9.5, 3.5)
3.31 (t, 9.5, 9.5)
3.57 (dq, 9.5, 6.0)
1.20 (d, 6.6)
4.55 (d, 1.5)
3.60 (dd, 3.5, 1.5)
3.53 (dd, 9.5, 3.5)
3.29 (t, 9.5, 9.5)
3.56 (dq, 9.5, 6.0)
1.20 (d, 6.6)
4.53 (d, 1.5)
3.61 (dd, 3.5, 1.5)
3.54 (dd, 9.5, 3,5)
3.34 (t, 9.5, 9.5)
3.58 (dq, 9.5, 6.0)
1.22 (d, 6.0)
4.52 (d, 5.2)
3.68 (dd, 5.2, 8.5)
3.59 (dd, 8.5, 3.0)
3.86 (m)
3.93 (dd 12.0, 3.0)
3.67 (dd 12.0, 2.0)
5.27 (d, 1.5)
4.26 (dd, 3.0, 1.5)
3.95 (dd, 9.5, 3.0)
3.58 (t, 9.5, 9.5)
4.17 (dq, 9.5, 6.0)
1.02 (d, 6.0)
5.24 (d, 1.5)
4.02 (dd, 3.0, 1.5)
3.82 (dd, 9.5, 1.5)
3.36 (t, 9.5, 9.5)
4.09 (dq, 9.5, 6.0)
1.01 (d, 6.0)
5.24 (d, 1.5)
4.01 (dd, 3.0, 1.5)
3.80 (dd, 9.5, 1.5)
3.33 (dd, 9.5, 9.5)
4.05 (dq, 9.5, 6.0)
0.97 (d, 6.0)
5.27 (d, 1.5)
4.26 (dd, 3.0, 1.5)
3.81 (dd, 9.5, 1,5)
3.34 (dd, 9.5, 1.5)
4.15 (dq, 9.5, 6.0)
1.00 (d, 6.0)
a Chemical shift values are in ppm and J values in Hz are presented in parentheses. All signals were assigned by 2DHOHAHA, DQF-COSY, HSQC, and HMBC studies. Gal ) β-D-galactopyranosyl, Rha ) R-L-rhamnopyranosyl, and Ara ) R-Larabinopyranosyl.
the only significant difference being those corresponding
to C-2 and C-3. These shifts are analogous to those
reported when the 3-hydroxy group is glycosylated in a
flavonol glycoside (12). Four anomeric protons were
easily identified in the spectra of 3. They resonated at
δ 5.57 (d, J ) 7.5 Hz), δ 5.27 (d, J ) 1.5 Hz), δ 4.55 (d,
J ) 1.5 Hz), and δ 4.52 (d, J ) 5.2 Hz), and they
correlated to carbons at δ 101.1, δ 102.1, δ 101.8, and δ
106.6, respectively. From the assigned aglycon and
sugar values (Tables 1 and 2), it was apparent that a
tetrasaccharide unit was attached to C-3 of the aglycone.
The structure of tetrasaccharide chain has been determined by a combination of 1D TOCSY, 2D DFQ-COSY,
HSQC, and HMBC experiments. The isolated anomeric
signals resonating at uncrowded regions of the spectrum, between 5.57 and 4.64 ppm were the starting
points for the 1D-TOCSY experiments. Because of the
selectivity of multistep coherence transfer, the 1DTOCSY subspectra of the single monosaccharide unit
could be extracted from the overlapping region of the
spectrum (between 3.0 and 4.0 ppm). Each subspectra
could be attributed to one set of coupled protons such
as H-C (1) to H-C (4) for arabinose or H-C (1) to H-C
(4) for galactose of a carbohydrate moiety.
The irradiation of the signal at δ 5.57 showed a set
of coupled resonances in a sugar ascribable from H-1 to
H-4 of a galactose unit linked at C-3 of the aglycone.
1D TOCSY subspectra obtained by irradiating at δ 4.55
and 1.20 ppm allowed the identification of one rhamnose
moiety, whereas 1D TOCSY subspectra obtained by
irradiating at δ 5.27 and 1.02 ppm led to the identifica-
tion of the second rhamnose unit. The irradiation at δ
4.52 showed connectivities to three methine protons (δ
3.68, 3.59, and 3.86 ppm). The coherence transfer to H-5
was not obtained because of the small JH4-H5 value of
this arabinose unit. The sequential assignments of these
sugar protons as shown in Table 1 derived from their
distinctive DQF-COSY patterns. The assignments of
all proton resonances for the sugar moieties immediately allowed assignment of the resonances of the linked
carbon atoms by HSQC (Table 2).
Information about the sequence of the tetrasaccharide
chain was deduced from an HMBC experiment. Key
correlation peaks were observed between the anomeric
proton of the galactose (δ 5.57) and the C-3 of the
kaempferol (δ 134.4), the anomeric proton signal of the
inner rhamnose (δ 5.27) and the C-2 of galactose (δ
77.2), the anomeric proton of the arabinose (δ 4.52) and
the C-3 of the inner rhamnose (δ 82.7), and the anomeric
proton of the outer rhamnose (δ 4.55) and the C-2 of
the galactose (δ 77.2).
The β-configuration at the anomeric position for the
galactopyranosyl unity (JH1-H2 ) 7.5 Hz) was easily seen
from their relatively large 3JH1-H2 coupling constants
(7-8 Hz). The R-configuration in the rhamnose residues was clear from their H-1 unsplitting pattern
and their distinct C-3 and C-5 chemical shift differences from that of methyl β-L-rhamnopyranoside (12),
whereas the R-configuration for the arabinopyranosyl
unit (JH1-H2 ) 5.2 Hz) was established by the results of
ROESY experiments as previously reported (13). These
data suggested that the structure of 3 is kaempferol-3-
3800 J. Agric. Food Chem., Vol. 49, No. 8, 2001
Leite et al.
Table 2. 13C NMR Assignments (δC in CD3OD) of
Compounds 3-6a
carbon
2
3
4
5
6
7
8
9
10
1′
2′
3′
4′
5′
6′
3-Gal
1′′
2′′
3′′
4′′
5′′
6′′
(6-1) Rha
1′′′′
2′′′′
3′′′′
4′′′′
5′′′′
6′′′′
(3-1) Ara
1′′′′′
2′′′′′
3′′′′′
4′′′′′
5′′′′′
(2-1) Rha
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
3
4
5
6
C
C
C
C
CH
C
CH
C
C
C
CH
CH
C
CH
CH
DEPT
158.5
134.4
179.7
163.1
101.0
167.5
95.5
158.5
106.6
122.9
132.2
116.2
161.0
116.2
132.2
158.4
134.6
179.5
163.1
101.7
167.5
95.6
158.7
106.6
123.1
132.3
116.3
161.0
116.3
132.3
158.5
134.4
179.7
163.2
101.8
167.5
95.7
158.6
106.6
122.9
132.2
116.4
161.1
116.4
132.2
158.2
134.3
179.0
163.4
99.7
165.8
94.4
158.2
105.6
123.1
115.9
145.6
149.4
117.1
122.8
CH
CH
CH
CH
CH
CH2
101.1
77.2
75.7
70.7
75.3
67.1
102.5
77.8
75.4
70.5
75.6
67.2
104.3
72.8
75.0
70.1
75.3
67.3
102.4
77.3
75.1
70.7
75.5
66.8
CH
CH
CH
CH
CH
CH3
101.8
72.0
72.2
73.9
69.7
17.9
101.8
72.1
72.2
73.7
69.6
17.8
101.8
72.0
72.3
73.8
69.7
17.8
101.6
71.9
72.1
73.7
69.6
17.1
CH
CH
CH
CH
CH2
106.6
73.1
74.0
69.7
67.1
CH
CH
CH
CH
CH
CH3
102.1
72.0
82.7
72.9
69.5
17.6
100.3
72.3
72.4
73.9
69.7
17.9
100.8
72.1
72.2
73.9
69.6
17.8
a All signals were assigned by 2D-HOHAHA, DQF-COSY,
HSQC, and HMBC studies. Gal ) β-D-galactopyranosyl, Rha )
R-L-rhamnopyranosyl, and Ara ) R-L-arabinopyranosyl.
O-R-L-rhamnopyranosyl (1f6)-O-[R-L-arabinopyranosyl(1f3)-O-R-L-rhamnopyranosyl(1f2)]-O-β-D-galactopyranoside.
Compounds 4 and 5 presented 1H and 13C NMR
spectra almost superimposable to those of compound 3.
The main differences were the absence of the arabinose
signal at δ 4.52 for compound 4 and the absence of
signals of arabinose at δ 4.52 and of rhamnose at δ 4.55
for compound 5 in the 1H NMR spectra, together with
the absence of their respective signals in the 13C NMR
spectra (see Tables 1 and 2). From these considerations
the structure of kaempferol-3-O-R-L-rhamnopyranosyl(1f6)-O-[R-L-rhamnopyranosyl(1f2)]-O-β-D-galactopyranoside was assigned to 4, and the structure of
kaempferol-3-O-R-L-rhamnopyranosyl(1f2)-O-β-D-galactopyranoside was assigned to 5 (14). Minor amounts
of the quercetin-3-O-R-L-rhamnopyranosyl(1f6)-O-[RL-rhamnopyranosyl(1f2)]-O-β-D-galactopyranoside 6 were
also detected by its NMR data (Tables 1 and 2) and ESMS spectra (14).
The quantitative analysis of the flavonoids glycosides
from M. ilicifolia (1 and 2) and M. aquifolium (3-6)
extracts was performed by HPLC. The concentrations
of each compound in the extracts, calculated from the
experimental peak areas by interpolation to standard
calibration curves, were 1.75% for compound 1, 1.50%
for 2, 1.18% for compound 3, 1.62% for 4, 0.98% for 5,
and 0.52% for 6, corresponding to 157.5 mg/100 g dried
weight for compound 1, 135.0 mg for 2, 106.2 mg for 3,
145.8 mg for 4, 88.2 mg for 5, and 46.8 mg for 6. Relative
standard deviations were in the range of 3.76-4.12%
calculated as mean of five replications, whereas for
retention times the relative standard deviation was less
than 1%.
Similar quercetin and kaempferol tetra-glycosides
were isolated from the aqueous infusion of M. aquifolium leaves (2, 3), but with a terminal glucose unit
instead of arabinose. There are few other reports of the
isolation of flavonoid tetra-saccharides from plant infusions. Kijima et al (15) reported the presence of
kaempferol-3-O-R-L-rhamnopyranosyl(1f2)-O-[R-L-rhamnopyranosyl(1f6)]-O-β-D-glucopyranoside-7-O-β-D-glucopyranoside from the water soluble fraction of the
extract of Alangium premnifolium, whereas Hu et al
(16) described the occurrence of anhydroicaritin-3-O-RL-rhamnopyranosyl-(1f2)-R-L-rhamnopyranoside-7-Oβ-D-glucopyranosyl-(1f2)-β-D-glucopyranoside from the
aerial parts of Epimedium acuminatum.
Polyphenolic compounds, including flavonoids, have
been the subject of increasing interest since in vitro and
in vivo biological assays indicated that flavonoids can
mediate a range of mechanisms related to anticancer,
antitumor, and anti-oxidant activities, among others
(17). The contribution of the flavonoids to the dietary
intake of polyphenolic compounds is considerable. In
fact, cereals, legume seeds, fruits, wine, and tea contain
significant amounts of flavonoids and their derivatives
(18). The 3-O-glycosides of quercetin and kaempferol are
the most common group of flavonoids. The sugar moiety
is an important factor for the bioavailability of the
flavonoid derivatives (19).
The occurrence of tetra-glycosylated flavonoids in
these two species may confirm their strong botanical
correlation and afford a valuable chemical marker for
the quality control of the Brazilian Maytenus marketed as phytomedicines, foodstuffs, or beverages in
daily life.
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Received for review March 6, 2001. Revised manuscript
received June 1, 2001. Accepted June 4, 2001. The authors
are grateful to FAPESP (Fundaçao de Amparo à Pesquisa do
Estado de Sao Paulo) that has sponsored part of this work and
to CNPq (Conselho Nacional de Desenvolvimento Cientifico e
Tecnologico) for fellowships to W.V. and J.H.Y.V.
JF010294N