Journal of Agricultural and Food Chemistry
Article
N2-t-Boc-N6-(diacetyldihydrocaffeoyl) Lysine t-Butyl Ester.
Diacetyldihydrocaffeic acid (632 mg, 2.38 mmol) and hydroxybenzo-
triazole (HOBt, 337.8 mg, 2.5 mmol) were dissolved in 12.5 mL of
dry tetrahydrofuran (THF) under an argon atmosphere at 0 °C.
Then, 466 mg (3 mmol) of 1-ethyl-3-(3-(dimethylamino)propyl)-
carbodiimide (EDC) was added. After 20 min, a solution of 756 mg
(2.5 mmol) of N2-t-Boc-lysine t-butyl ester in 12.5 mL of dry THF
was added dropwise. The mixture was stirred for 16 h on ice. The
solvent was evaporated under reduced pressure, and the residue was
dissolved in 10 mL of ethyl acetate and washed with 10 mL of a
saturated NaHCO3 solution and 1 M HCl, separately. The solvent
was evaporated, and the crude product was purified by column
chromatography (silica gel 60) using hexane/acetone (2:1). Fractions
containing the product [thin-layer chromatography (TLC): Rf = 0.29
in hexane/acetone (2:1), with ultraviolet (UV) and ninhydrin
detection] were collected, concentrated under reduced pressure,
and dried under high vacuum to afford a yellowish brown oil (256 mg,
20%). 1H NMR (400 MHz, CDCl3): δ 1.33 (m, 22H), 1.53 (m, 2H),
2.15 (s, 6H), 2.31 (t, 3J = 7.7 Hz, 2H), 2.80 (t, 3J = 7.7 Hz, 2H), 3.04
buffer. The solutions were mixed with hydrogen peroxide solutions of
different concentrations, resulting in a molar excess of hydrogen
peroxide of 1000×, 100×, 50×, and 10×, respectively, in relation to
phenol. The solutions were incubated for 24 h in a shaker at room
temperature. THOB was incubated in the same way.
Aspalathin Protein Incubation and Enzymatic Hydrolysis.
Mixtures containing aspalathin (100 mM) and bovine serum albumin
(BSA, 0.2 mM) were incubated under the above-mentioned aerated
conditions. The modified protein was precipitated with trichloroacetic
acid, washed with acetone, and dissolved in phosphate-buffered saline
(PBS) buffer. Enzymatic hydrolysis of the protein solution was carried
out according to Smuda et al.19
Analytical High-Performance Liquid Chromatography−
Diode Array Detection (HPLC−DAD). For polyphenol analyses,
a Jasco PU-2080 Plus quaternary gradient pump with a degasser (DG-
2080-54), quaternary gradient mixer (LG 2080-02), multiwavelength
detector (MD-2015 Plus) (Jasco, Gross-Umstadt, Germany), Waters
717 plus autosampler, and column oven (Techlab Jet Stream np K-3)
was used. Chromatographic separations were performed on stainless-
steel columns (Vydac CRT, 201TP54, 250 × 4.6 mm, RP-18, 5 μm,
Hesperia, CA, U.S.A.) using a flow rate of 1.0 mL/min. The column
temperature was always 25 °C. The mobile phase consisted of water
(solvent A) and MeOH/water (7:3, v/v, solvent B), and to both
solvents (A and B), 0.8 mL/L formic acid was added. Samples were
analyzed using a gradient system: samples were injected at 10% B.
The gradient was changed linear to 30% B in 30 min, to 65% B after
40 min, to 100% B after 2 min, and held at 100% B for 8 min. The
effluent was monitored at 220, 280, and 400 nm (DHC, tR = 14 min;
aspalathin, tR = 39 min). For quantitation, an external calibration
based on standard solutions of authentic isolated references dissolved
in water was used.
Analytical HPLC−FLD. For determination of DHC−amide, a
Jasco PU-980 gradient pump with a degasser (DG-2080-53), ternary
gradient mixer (LG 980-02), fluorescence detector (FP-920),
autosampler (851-AS) and column oven (Jasco, Gross-Umstadt,
Germany) set at 25 °C was used. Chromatographic separations were
performed on stainless-steel columns (Vydac CRT, 218TP54, 250 ×
4.6 mm, RP-18, 5 μm, Hesperia, CA, U.S.A.) using a flow rate of 1.0
mL/min. The mobile phase consisted of water (solvent A) and
MeOH/water (7:3, v/v, solvent B). A 1.2 mL/L aliquot of
heptafluorobutyric acid was added to both solvents (A and B). The
samples were injected at 2% B and held for 5 min, and the gradient
was changed to 90% B in 30 min and 100% B in 1 min and held for 5
min. The fluorescence detector was attuned to 340 nm for excitation
and 455 nm for emission (DHC−amide, tR = 19 min). Prior, a post-
column derivatization reagent was added at 0.5 mL/min. This reagent
consisted of 0.8 g of o-phthaldialdehyde, 24.73 g of boric acid, 2 mL of
2-mercaptoethanol, and 1 g of Brij 35 in 1 L of water adjusted to pH
9.75 with KOH. For quantitation with HPLC−FLD, an external
calibration based on standard solutions of authentic synthesized
references dissolved in water was used.
3
3
(dt, J = 6.6 Hz, J = 6.5 Hz, 2H), 3.98 (m, 1H), 5.15 (s, 1H), 6.22
(m, 1H) 6.89 (s, 1H), 6.94 (s, 2H). 13C NMR (100 MHz, CDCl3): δ
20.5, 22.5, 27.9, 28.2, 28.8, 30.9, 32.2, 37.7, 39.0, 53.8, 79.4, 81.5,
123.1, 123.2, 126.4, 139.8, 140.2, 140.8, 155.5, 168.2, 168.3, 171.8,
172.0.
DHC−Amide. N2-t-Boc-N6-(diacetyldihydrocaffeoyl) lysine t-butyl
ester (256 mg, 0.465 mmol) was dissolved in acetone and 6 M HCl (3
mL each). After stirring for 30 min, the mixture was diluted with 50
mL of water and evaporated to dryness. The crude product was
purified by column chromatography (silica gel 60) using acetonitrile/
water (15:4). Fractions containing the product [TLC: Rf = 0.23 in
acetonitrile/water (15:4), with UV and ninhydrin detection] were
collected, evaporated to dryness, and dried under high vacuum to
afford DHC−amide (130 mg, 90%). Verification was carried out by
NMR spectroscopy and HR-MS (Table 1).
Extraction of Rooibos Tea. Unfermented and fermented rooibos
teas from Biedouw Valley of South Africa were obtained from Mount
Everest Tea Company GmbH (Elmshorn, Germany) and from local
tea shops. For aspalathin isolation, 100 g of the unfermented rooibos
was extracted with 1 L of acetone/water (7:3, v/v) for 24 h under an
argon atmosphere at 4 °C. Acetone was evaporated under reduced
pressure at 30 °C, and the aqueous phase was extracted successively
with 2 × 200 mL each of diethyl ether and ethyl acetate, separately.
The solvent of the ethyl acetate extract was removed under reduced
pressure and freeze-dried. The dried extract was used for separation
via HPCCC.
For quantitation of DHC and DHC−amide, 1 g of the
unfermented and fermented rooibos was extracted with 20 mL of
the acetone/water (7:3, v/v) mixture for 1 h under an argon
atmosphere at room temperature in an ultrasonic bath. DHC−amide
was quantitatively enriched by repeated collection with analytical
HPLC (below).
Aerated Polyphenol Incubations. Mixtures containing either
aspalathin and N2-t-Boc-lysine (10 mM), aspalathin, phloridzin, or
phloretin (0.5 mM) in phosphate buffer (0.1 M, pH 7.0) were
incubated in screw-cap vials. Incubations were kept in a shaker for 7
days at 37 °C. The formation of DHC and dihydrocoumaric acid was
analyzed by gas chromatography−mass spectrometry (GC−MS) and
gas chromatography−flame ionization detection (GC−FID) after
silylation. DHC−amide was analyzed by high-performance liquid
chromatography−fluorescence detection (HPLC−FLD) after remov-
ing the N2-t-Boc group by adding 6 M HCl to the samples to a final
HCl concentration of 3 M and keeping them for 30 min at room
temperature.
Deaerated Polyphenol Incubations. The incubations were
modified by adding 1 mM diethylenetriaminepentaacetic acid to the
phosphate buffer. The buffer was degassed with helium before the
samples were placed in 2 mL screw cap vials without air and
incubated under an argon atmosphere.
Analytical High-Performance Liquid Chromatography−
Tandem Mass Spectrometry (HPLC−MS2). For quantitation of
DHC−amide as a protein modification, a Jasco PU-2080 Plus
quaternary gradient pump with a degasser (DG-2080-54), quaternary
gradient mixer (LG 2080-04) (Jasco, Gross-Umstadt, Germany), AS-
2057 Plus autosampler, and column oven (Jasco Jetstream II) set at
25 °C was used. Chromatographic separations were performed on
stainless-steel columns (Vydac CRT, 218TP54, 250 × 4.6 mm, RP-18,
5 μm, Hesperia, CA, U.S.A.) using a flow rate of 1.0 mL/min. The
eluents and gradient program were identical to that used for HPLC−
FLD analysis. The mass analyses were performed using an Applied
Biosystems API 4000 quadrupole instrument (Applied Biosystems,
Foster City, CA, U.S.A.). MS ionization was achieved using the
turbospray ionization source operated in positive-ion mode. The
settings were as follows: curtain gas (N2) at 40 psi, ion source gas 1 at
70 psi, ion source gas 2 at 80 psi, source temperature at 650 °C, and
ion spray voltage at 2500 V. Optimized mass spectrometric
parameters for DHC−amide were as follows: tR = 19 min, m/z
311.3/84.2 [declustering potential (DP), 65 V; collision energy (CE),
Singlet-Oxygen Polyphenol Incubations. The incubations
were modified by adding 10 mM sodium molybdate to the phosphate
C
J. Agric. Food Chem. XXXX, XXX, XXX−XXX