Article
With regard to stilbenes, HSCCC has been applied to the
J. Agric. Food Chem., Vol. 58, No. 11, 2010 6755
apparatus was rotated at a revolution speed of 850 rpm while a flow rate of
3 mL/min was used. For the separation, 630 mg of the biotransformed
mixture dissolved in the upper and lower phases (1:1) was injected via an
injection loop (20 mL). Elution was monitored with a Knauer K-2501 UV
detector at λ = 280 nm and recorded using a Servogor 120 plotter (Georz
Metrawatt). Fractions were collected with a fraction collector (Pharmacia
LKB Super Frac).
TLC Analysis. Evaluation of the HSCCC fractions was performed
by thin-layer chromatography on normal phase silica gel plates 60 F254
(Merck) with chloroform/ethyl acetate/methanol/water (25:55:5:1, v/v/v/v)
as solvent system. Visualization of the stilbenes and derivatives was
achieved by means of an anisaldehyde spray reagent according to the
method of Stahl (23).
HPLC-PDA. HPLC-PDA measurements were carried out on a
HPLC system from Jasco (Gross-Umstadt, Germany), consisting of a
pump PU-980, a degasser DG-98050, a gradient unit LG-980-02, and
a photodiode array detector MD-910. An analytical C18 column (Luna
C18, 250 ꢀ 4.6 mm, 5 μm, Phenomenex, Aschaffenburg, Germany) at a
flow rate of 0.8 mL/min [solvent system of 1% acetic acid (A), acetonitrile
(B)] was used. The applied gradient started over 1 min at 15% B, 10 min at
24% B, 45 min at 90% B, 50 min at 100% B, and resulted in 55 min at 15%
B. Each of the obtained fractions was analyzed by HPLC-PDA and
HPLC-ESI-MS/MS.
isolation of trans-resveratrol and piceid from Polygonum cuspida-
tum using different solvent systems (18-20). Furthermore,
HSCCC isolations of quadrangularin A and parthenocissin A
from Parthenocissuslaetevirens (21) and ofhopeaphenol, vitisinA,
and amurensin G from Vitis chunganeniss have been reported (22).
A great variety of stilbenoids are known to occur in grapevine,
but the structures of many minor stilbene oligomers remain to be
elucidated. Because it is rather difficult to isolate these minor
constituents in sufficient amounts for subsequent structural
characterization, we were searching for a more promising way
to prepare them. It is known that the natural biotransformation
of stilbene oligomers in grapevine can be simulated by in vitro
biotransformation with horseradish peroxidase (3, 6, 10-15).
Used in combination with HSCCC, larger amounts of the minor
compounds can be accumulated, thus allowing their unambig-
uous structure determination. Using this approach, two novel
minor components were isolated and characterized for the first
time. As these two new compounds could occur naturally as
minor constituents in grapevine extracts, the elucidation process
of stilbene oligomers from grapevine extracts can be carried out
more efficiently using the data reported here.
HPLC-ESI-MS/MS and High-Resolution ESI-MS. A HPLC
system (pump 1100 series, autosampler 1200 series) from Agilent Tech-
€
nologies (Boblingen, Germany) was connected with an Esquire LC-ESI-
MATERIALS AND METHODS
MS/MS from Bruker (Bremen, Germany). Mass spectra were recorded in
the negative mode, with the capillary set at 1500 V, the end plate at-500 V,
the capillary exit at -120.4 V, the dry gas at 310 °C, the gas flow at 9.0 L/
min, the nebulizer at 40 psi, the target mass at m/z 500, the scan range from
m/z 50 to 2200, the collision gas of helium, and a MS/MS fragmentation
amplitude at 1.0 V. HPLC conditions were the same as described in the
HPLC section above. High-resolution ESI-MS were recorded on a
Thermo Science LTQ Orbitrap mass spectrometer.
Materials. Chemicals and solvents were obtained from the suppliers
shown: acetone-d6, 99.96% (Deutero, Kastellaun, Germany); acetone, p.a.
(Roth, Karlsruhe, Germany); citric acid, anhydrous, p.a., 99.5% (Fluka,
Steinheim, Germany); disodium hydrogen phosphate dihydrate p.a.
(Merck, Darmstadt, Germany); horseradish peroxidase, 970 U/mg
(Fluka); aqueous hydrogen peroxide, p.a., 30% (Merck); methanol-d4,
99.96% (Deutero).
Preparative HPLC. Fractions were purified by preparative HPLC on
a Smartline system from Knauer (Smartline, pump 1000, manager 5000,
detector UV K-2600, Berlin, Germany). The preparative HPLC column
(Luna C18, 250 ꢀ 15.0 mm, 5 μm, Phenomenex, Aschaffenburg, Germany)
was operated with a binary solvent system of ultrapure water (A) and
methanol (B) at a flow rate of 4 mL/min. The gradient for the isolation of
trans-δ-viniferin 1 was composed of 0 min at 25% B, 35 min at 45% B, and
50 min at 60% B, and the gradient for the isolation of compound 2 and 3
was 0 min at 45% B, 20 min at 53% B, and 53 min at 63% B. The fractions
were monitored at λ = 280, 306, and 325 nm.
Solvents for HPLC analysis included acetic acid, HPLC quality (Baker,
Deventer, The Netherlands); acetonitrile, HPLC quality (Fisher Scientific,
Loughborough, U.K.); methanol, HPLC quality (Fisher); and ultrapure
water (NANOpure).
Solvents used for HSCCC were ethyl acetate, p.a. (Fisher); n-hexane
(distilled, industrial quality); methanol (distilled, industrial quality); and
ultrapure water (NANOpure).
trans-Resveratrol and (-)-ε-viniferin were isolated from the commer-
cial grapevine extract of Vitis vinifera, Vineatrol 30 (Breko GmbH,
Bremen, Germany).
Small-Scale Peroxidase Reaction of trans-Resveratrol and (-)-ε-
Viniferin. The peroxidase reaction was carried out according to the
method of Takaya et al. using optimized conditions (3). A suspension of
horseradish peroxidase (1.33 mg of enzyme in 1 mL of McIlvaine buffer)
was added to a mixture of trans-resveratrol (22.8 mg) dissolved in 4 mL of
acetone, 4 mL of water, and 1.6 mL of McIlvaine buffer (pH 6) and stirred
for 5 min at 22 °C. Aqueous hydrogen peroxide (10%, 40 μL) was added,
and the solution was stirred for 45 min at 22 °C, extracted with ethyl
acetate, evaporated, and lyophilized in the absence of daylight. The
biotransformation of (-)-ε-viniferin (5.0 mg) was carried out with aliquots
of chemicals as described above, as well as a mixture of both stilbenes
(5.0 mg). For the blank experiments, educts and chemicals without
application of peroxidase were used.
Nuclear Magnetic Resonance Spectroscopy. 1H, 13C, DEPT-135,
1H-1H -COSY, 1H-1H phase-sensitive ROESY, HSQC, and HMBC
experiments were carried out on Bruker Avance DMX 600 or DPX 300
NMR spectrometers. Chemical shifts were referenced to the solvent
signals.
trans-δ-Viniferin (resveratrol dehydrodimer), 1: amorphous powder; UV
(MeOH) λmax 311 nm; ESI-MS/MS m/z 453 [M - H]-; MS/MS fragments
m/z 435, 411, 369, 359, 347, 333, 307; 1H NMR (600 MHz, methanol-d4)
and 13C NMR (150 MHz, methanol-d4) see Table 2.
Resviniferin A, 2: amorphous powder; UV (MeOH) λmax 323 nm; ESI-
MS/MS m/z 679 [M - H]-; MS/MS fragments m/z 661, 643, 621, 585, 573,
451, 439, 345, 333; HR-ESI-MS m/z 681.2111 [M þ H]þ (calcd for
[C42H32O9 þ H]þ 681.2119); 1H NMR (600 MHz, acetone-d6) and 13C
NMR (150 MHz, acetone-d6) see Table 2.
Large-Scale Peroxidase Reaction of trans-Resveratrol and (-)-ε-
Viniferin. A suspension of horseradish peroxidase (6.3 mL; c 1 g/L) was
added at 22 °C to a mixture of trans-resveratrol (415.7 mg) and (-)-ε-
viniferin (214.3 mg) dissolved in 250 mL of an acetone/water mixture
(1:1, v/v) and 50 mL of McIlvaine buffer (pH 6). Aqueous hydrogen
peroxide (10%, 1.25 mL) was then added, and the solution was stirred for
45 min at 22 °C. The lyophilized product was obtained as described above.
HSCCC. A preparative high-speed countercurrent chromatograph
(model 1000, Pharma-TechResearch Corp., Baltimore, MD) wasequipped
with three coils connected in series (total capacity of 800 mL). The two-
phase solvent system was composed of n-hexane/ethyl acetate/methanol/
water (2:3:2:3, v/v/v/v), and a HPLC pump (Biotronic BT 3020) was used
to pump the solvents. The separation was carried out in the “head-to-tail”
elution mode in which the upper phase acts as the stationary phase. The
Resviniferin B, 3: amorphous powder; UV (MeOH) λmax 331 nm; ESI-
MS/MS m/z 679 [M - H]-; MS/MS fragments m/z 661, 585, 573, 451, 359,
345, 333; HR-ESI-MS m/z 681.2099 [M þ H]þ (calcd for [C42H32O9 þ H]þ
681.2119); 1H NMR (600 MHz, acetone-d6) and 13C NMR (150 MHz,
acetone-d6) see Table 2.
RESULTS AND DISCUSSION
Small-Scale Peroxidase Reaction of trans-Resveratrol and (-)-ε-
Viniferin. In this preliminary study trans-resveratrol and (-)-ε-
viniferin were used for the reactions with horseradish peroxidase
and hydrogen peroxide on a small scale. The conversion was
analyzed by HPLC-PDA and HPLC-ESI-MS/MS. All given