5844 J. Agric. Food Chem., Vol. 57, No. 13, 2009
Regos et al.
concentration of condensed tannins. The EtOAc extracts were combined
and concentrated under reduced pressure, yielding a yellow-brown powder
after freeze-drying (1.5 g, 3% of total leaf and stem material). Extracts as
well as the various residues obtained during the extraction process were
tested by TLC and HPLC for their chemical composition. The EtOAc
extract, redissolved in 10% aqueous MeOH, was chromatographed on a
Sephadex LH-20 column (300 mm ꢀ 30 mm internal diameter) with
different volumes of aqueous MeOH (10-100% in increments of 10%).
The eluate was collected in 20 mL portions in 107 test tubes, which were
combined according to their phenolic composition on the basis of their
TLC behavior to afford 45 fractions (Table 1). Each fraction was
characterized by HPLC, TLC, and LC-MS/MS. Later, the fractions were
purified by HPLC and TLC.
HPLC-ESI-MS/MS. HPLC-ESI-MS/MS analysis was performed
with a Shimadzu LC-10A series liquid chromatograph (Shimadzu,
Hannover, Germany) followed by an API 3000 triple-quadrupole mass
spectrometer (Applied Biosystem, Darmstadt, Germany) fitted with an
electrospray ionization source. The HPLC system consisted of an SCL-
10Avp system controller, two LC-10ADvp pumps, an SIL-HTC refriger-
ated autosampler, a DGU-14A degasser, and a CTO-10ASvp column
oven. The autosampler temperature was set at 4 °C and the column oven
at 25 °C. The chromatographic separation was performed using a
Phenomenex Synergy Fusion-RP 18 column (50 ꢀ 2 mm, Phenomenex,
Aschaffenburg, Germany), and the flow rate was maintained at
0.2 mL/min. Solvent A was H2O and solvent B was MeOH, both
containing 0.1% HCO2H, and the gradient protocol was as follows: 0-
16 min, 6-17% B; 16-18 min, 17-20% B; 18-25 min, 20-40% B; 25-
35 min, 40-100% B. The MS/MS detector was operated in the following
conditions: polarity, negative; ion spray voltage, -4200 V; heater gas
temperature, 350 °C; entrance potential, -10 V; focusing potential,
-330 V; declustering potential, -50 V; and collision energy, -52 V.
Nitrogen served as nebulizer, curtain, drying, and collision gas. The
acquisition was performed in full-scan mode (m/z 50-2000 amu) using
Analyst 1.4.1 (Applied Biosystem, Darmstadt, Germany) as software.
UV-Vis Spectroscopy. UV-vis spectra of flavonol glycosides and
bathochromic shifts after addition of shift reagents were recorded with an
Uvikon 931 UV-vis double-beam spectrometer (Kontron, Germany)
according to the method of Mabry et al. (9 ).
Quantitative Extraction of Phenolic Compounds from Different
Plant Parts. Freeze-dried material was ground in a mortar or a ball mill,
depending on the available amount. The extraction was performed by
adding 500 μL of 80% aqueous methanol to 100 mg of powder for 30 min in
a cooled ultrasound water bath (7 °C). After centrifugation (10000 rcf,
10 min, 4 °C), the clear supernatant was transferred to an Eppendorf tube
and the residue was washed twice, each time with 250 μL of 80% aqueous
MeOH. After centrifugation, the corresponding supernatants were com-
bined, the solvent was evaporated, and the residue was redissolved in 100 μL
of MeOH. A 10 μL sample of the extract was injected for HPLC analysis.
Thin Layer Chromatography (TLC). Analytical TLC and prepara-
tive TLC were carried out on Merck cellulose plates and on polyamide
::
Acid and Enzymatic Hydrolysis. Acid hydrolysis was performed by
heating 100 μL samples with 100 μL of 0.1 M aqueous HCl in a boiling
water bath for 15 min. The aglycones were extracted from this solution
with EtOAc (3 ꢀ 300 μL). For the enzymatic hydrolysis a solution
containing the unknown compound (0.2-1 mg) was evaporated to dryness
under vacuum. The residue was further diluted with 300 μL of 0.1 M
NaOAc buffer (pH 4.6) and incubated with 2 mg of enzyme (sulfatase and
glucosidase from Sigma-Aldrich, Deisenhofen, Germany; tannase,
Braunschweiger Biotechnologie, Germany) in a water bath at 50 °C for
3 h. The aglycones were extracted with EtOAc (3 ꢀ 500 μL), and the sugars
were analyzed in the remaining aqueous phase.
plates from Macherey-Nagel (Duren, Germany) with n-BuOH/HOAc/
H2O (4:1:5) (BAW) and H2O/EtOH/ethyl methyl ketone/acetylacetone
(65:15:15:5) (WEEA), respectively. The chromatograms were evaluated
under UV light both with and without spraying with Naturstoffreagenz A
(diphenylboric acid 2-aminoethyl ester) and DMACA.
HPLC Analysis of Phenolic Compounds. The phenolic compounds
from sainfoin were analyzed and purified from the fractions F1-F45 with
an HPLC system consisting of two pumps (model 422, Kontron Instru-
ments, Germany), an automatic sample injector (model 231, Gilson
Abimed Systems, Germany), and a diode array detector (Kontron 540,
Kontron Instruments). For postcolumn derivatization a further Gynkotek
analytical HPLC pump (model 300 C, Germering, Germany) and a Vis
detector (640 nm, Kontron Detector 432, Kontron Instruments) were
used. Sainfoin compounds were separated on a Nucleosil column (250 ꢀ
4 mm, Macherey-Nagel) and eluted with a mixture of H2O containing 5%
HCO2H (solvent A) and MeOH (solvent B). The following gradient was
applied using a flow rate of 0.5 mL/min: 0-5 min, 5% B; 5-10 min, 5-
10% B; 10-15 min, 10% B; 15-35 min, 10-15% B; 35-55 min, 15% B;
55-70 min, 15-20% B; 70-80 min, 20% B; 80-95 min, 20-25% B; 95-
125 min, 25-30% B; 125-145 min, 30-40% B; 145-160 min, 40-50% B;
160-175 min, 50-90% B; 175-195 min, 90% B.
For their quantification, sainfoin compounds were grouped into 10
categories based on the maximum UV-vis absorption and were mon-
itored and analyzed at 280 nm (amino compounds, simple phenolic acids,
hydroxybenzoic acids, and dihydroflavonols), 320 nm (hydroxycinnamic
acids, flavones), 350 nm (flavonols), and 540 nm (anthocyanins). Addi-
tionally, postcolumn derivatization with DMACA was used for selective
detection of flavanols at 640 nm (8 ). Quantification was performed as
RESULTS AND DISCUSSION
Identification of Phenolic Compounds from Sainfoin (O. viciifo-
lia). The acetone extract of a mixed sample of sainfoin whole
plants was dominated by low molecular weight secondary plant
metabolites consisting of simple phenolic acids, hydroxybenzoic
and hydroxycinnamic acids, dihydroflavonols, flavones, flava-
nols, and flavonols (see Figure 1B-H for chemical structures).
Additionally, amino compounds were also identified (Figure 1 A).
Sephadex LH-20 gel chromatography using a stepwise water/
methanol gradient as shown in Table 1 proved to be very effective
to enrich and separate sainfoin compounds by different families
in 45 fractions. Fraction components were further purified for the
subsequent characterization of these structures through various
techniques. TLC on polyamide plates was found to be a useful
technique for the purification of flavonols with different degrees
of glycosylation. The structures of the isolated compounds were
established by means of acid and enzymatic hydrolysis, spectro-
metric, and chromatographic methods: UV with shift reagents
(NaOMe, NaOAc, H3BO3, AlCl3, and HCl), the LC-ESI-MS/
MS technique, TLC, and HPLC. The spectral data obtained were
confirmed by comparison with those previously reported. When
standards were available, HPLC co-injection was performed. The
63 compounds identified in sainfoin are listed in Table 2 together
with their acronyms (A1-P3) and the chromatographic, UV, and
mass spectral characteristics.
follows:
nidin B2, rutin, and cyanidin 3-glucoside were available as standards,
hypaphorine was calculated as -tryptophan, and 8-β-glucopyranosylox-
L-tryptophan, arbutin, ellagic acid, catechin, epicatechin, procya-
L
ycinnamic acid was calculated as cinnamic acid. Hydroxybenzoic acids
were calculated as p-hydroxybenzoic acid, hydroxycinnamic acids as
chlorogenic acid, dihydroflavonols as dihydroquercetin, and the flavones
as vitexin. From the flavanols, gallocatechin and epigallocatechin were
calculated as epicatechin and the oligomers as procyanidin B2. The
flavonols were calculated as rutin.
HPLC Analysis of Sugars. The HPLC apparatus consisted of a
gradient pump (GP50, Dionex, Idstein, Germany), an interface (Advanced
Computer Interface, Dionex), a manual injector, and an electrochemical
detector (ED40, Dionex). The sugars were separated on a CarboPac PA-
100 column (250 ꢀ 4 mm, Dionex) with a mixture of 100 mM NaOH (B)
and deionized water (C). The flow rate was 1 mL/min, the injected sample
volume 25 μL, and the gradient as follows: 0-5 min, 10% B; 5-10 min,
10-15% B; 10-20 min, 15% B.
Amino Compounds. In addition to the amino acid L-tryptophan
(A1), previously reported by Marais et al. (10), hypaphorine (A2),
an R-N,N,N-trimethyltryptophan betaine, was isolated for the
first time from sainfoin. The UV spectrum of A2 showed a
maximum at 279 nm with shoulders at 273 and 287 nm. The