Characterization of phenolic compounds in rooibos tea

Article abstract of DOI:10.1021/jf703701n

Polyphenols present in rooibos, a popular herbal tea from Aspalathus linearis, were isolated in two steps. First, phenolic ingredients were separated by multilayer countercurrent chromatography (MLCCC). Preparative high-performance liquid chromatography (HPLC) was then applied to obtain pure flavonoids. The purity and identity of isolated compounds was confirmed by different NMR experiments, HPLC-diode array detector (DAD), or gas chromatography-mass spectrometry (GC-MS) analysis. This strategy proved to be valid to isolate material in up to gram quantities and to verify known and previously not published polyphenol structures. In addition the chemistry of dihydrochalcones and related intermediates was studied. The dihydrochalcone aspalathin was oxidized to the corresponding flavanone-C-glycosides ((R)/(S)-eriodictyol-6-C-β-D-glucopyranoside and (R)/(S)-eriodictyol-8-C- β-D-glucopyranoside). Flavanone-6-C-β-D-glucopyranosides were further degraded to flavones isoorientin and orientin.

Full text of DOI:10.1021/jf703701n

3368 J. Agric. Food Chem. 2008, 56, 3368–3376
Characterization of Phenolic Compounds in Rooibos
Tea
NICOLE KRAFCZYK AND MARCUS A. GLOMB*
Institute of Chemistry, Food Chemistry, Martin Luther University Halle-Wittenberg,
Kurt-Mothes-Strasse 2, 06120 Halle, Germany
Polyphenols present in rooibos, a popular herbal tea from Aspalathus linearis, were isolated in two
steps. First, phenolic ingredients were separated by multilayer countercurrent chromatography
(MLCCC). Preparative high-performance liquid chromatography (HPLC) was then applied to obtain
pure flavonoids. The purity and identity of isolated compounds was confirmed by different NMR
experiments, HPLC-diode array detector (DAD), or gas chromatography-mass spectrometry (GC-
MS) analysis. This strategy proved to be valid to isolate material in up to gram quantities and to
verify known and previously not published polyphenol structures. In addition the chemistry of
dihydrochalcones and related intermediates was studied. The dihydrochalcone aspalathin was oxidized
to the corresponding flavanone-C-glycosides ((R)/(S)-eriodictyol-6-C-ꢀ-D-glucopyranoside and (R)/
(S)-eriodictyol-8-C-ꢀ-D-glucopyranoside). Flavanone-6-C-ꢀ-D-glucopyranosides were further degraded
to flavones isoorientin and orientin.
KEYWORDS: Rooibos tea; multilayer countercurrent chromatography (MLCCC); reactions of dihydro-
chalcones; NMR; HPLC-DAD; HR-(GC)-MS
INTRODUCTION
alcones aspalathin and nothofagin, the major C-glycosylfla-
vonoids in unfermented rooibos, decreased significantly with
processing (10). Marais et al. reported on the degradation of
aspalathin during fermentation to the flavanones (S)- and (R)-
eriodictyol-6-C-ꢀ-D-glucopyranoside (9).
Rooibos is a shrubby legume that is indigenous to the
mountains of South African’s Western Cape. The genus
Aspalathus includes more than 200 species native to South
Africa, but only Aspalathus linearis (A. linearis) is edible (1).
Its leaves and stems are used for the production of rooibos tea,
a beverage with acclaimed beneficial health effects. Rooibos
tea boasts high yields of flavonoids with exceptional high
contents in C-glycosides. Furthermore, this tea is rich in
minerals, is caffeine-free, and has low tannin contents (2).
Usually, two types of rooibos are available, unfermented and
fermented rooibos tea. The fermentation process is an oxidation
process brought about for the formation of the characteristic
red-brown color and sweetish flavor of the infusion.
Separation and purification of rooibos using conventional
methods such as column chromatography and high-performance
liquid chromatography (HPLC) requires several steps resulting
in low amounts of products. Multilayer countercurrent chro-
matography (MLCCC) is a unique liquid–liquid partition
chromatography, which uses no solid matrix. Therefore, it
eliminates irreversible adsorptive loss of samples onto the solid
support used in conventional chromatographic setups. This
method has been successfully applied to analysis and separation
of various natural products (11). The aim of this work was to
comprehensively isolate phenolic compounds from rooibos by
MLCCC and preparative HPLC. In addition, the oxidative
degradation mechanism of dihydrochalcones was studied in-
depth.
First determination of phenolic ingredients of A. linearis was
established by Koeppen et al. (3–5). The presence of aspalathin,
orientin, isoorientin, isoquercitrin, and rutin was described after
separation by paper chromatography. Other flavonoids like
quercetin and luteolin have been isolated from processed rooibos
tea by special extraction methods (6). Furthermore, vitexin,
isovitexin, chrysoeriol, and some phenolic acids such as
p-hydroxybenzoic acid, protocatechuic acid, caffeic acid, vanillic
acid, p-coumaric acid, and ferulic acid were identified in
processed rooibos (7). The occurrence of nothofagin, (S)- and
(R)-eriodictyol-6-C-ꢀ-D-glucopyranoside, syringic acid, and (+)-
catechin was established (8, 9). The content of the dihydroch-
MATERIALS AND METHODS
Chemicals. Chemicals of the highest quality available were obtained
from Roth (Karlsruhe, Germany) unless otherwise indicated. Methanol
was HPLC grade from Merck (Darmstadt, Germany). Caffeic acid,
ferulic acid, heptafluorobutyric acid, p-hydroxybenzoic acid, (+)-
catechin, and pyridine were purchased from Fluka (Taufkirchen,
Germany). Dimethyl sulfoxide (DMSO-d6) was obtained from
Chemotrade (Leipzig, Germany). Gallic acid was ordered from Serva
(Heidelberg, Germany). Vanillic acid, sinapinic acid, p-coumaric acid,
* To whom correspondence should be addressed. Fax: +49-345-
5527341. E-mail: marcus.glomb@chemie.uni-halle.de.
10.1021/jf703701n CCC: $40.75  2008 American Chemical Society
Published on Web 04/10/2008

Phenolic Compounds in Rooibos
J. Agric. Food Chem., Vol. 56, No. 9, 2008 3369
(PTFE) tubing. The total capacity was 300 mL. The MLCCC was run
at a revolution speed of 800 rpm and a flow rate of 2 mL/min in head
to tail modus.
Samples of 1 g were dissolved in a 1:1 mixture (10 mL) of light
and heavy phase and injected into the system. The solvent system for
separation of ethyl acetate extract or n-butanol extract consisted of ethyl
acetate/n-butanol/water (3:1:4 (v/v)). Diethyl ether extracts were
separated by using water/ethyl acetate (2:1 (v/v)).
Analytical HPLC-DAD. A Jasco (Gross-Umstadt, Germany) qua-
ternary gradient unit PU 2080, with degasser DG 2080-54, autosampler
AS 2055, column oven (Jasco Jetstream II), and multiwavelength
detector MD 2015 was used. Chromatographic separations were
performed on stainless steel columns (VYDAC CRT, no. 218TP54,
250 × 4.0 mm, RP 18, 5 µm, Hesperia, CA) using a flow rate of 1.0
mL min-1. The mobile phase used was water (solvent A) and MeOH/
water (7:3 (v/v), solvent B). To both solvents (A and B), 0.6 mL/L
heptafluorobutyric acid (HFBA) was added. Samples were injected at
10% B; the gradient then changed linear to 30% B in 30 min, to 65%
B in 40 min, and to 100% B in 2 min and were held at 100% B for 8
min. The column temperature was 25 °C. The effluent was monitored
at 280 and 350 nm.
Preparative HPLC-UV. A Besta HD 2-200 pump (Wilhelmsfeld,
Germany) was used at a flow rate of 8 mL min-1. Elution of materials
was monitored by a UV detector (Jasco UV- 2075, Gross-Umstadt).
Chromatographic separations were performed on stainless steel columns
(VYDAC CRT, no. 218TP1022, 250 × 23 mm, RP18, 10 µm, Hesperia,
CA). The mobile phase used was solvents A and B, identical to the
analytical HPLC-DAD system. From the individual chromatographic
fractions, solvents were removed under reduced pressure. After addition
of water, solutions of polyphenols were freeze-dried.
Figure 1. HPLC-DAD chromatogram of ethyl acetate extract from
fermented and unfermented rooibos tea at λ ) 280 nm. Retention times
are given in parentheses: 1, (S)-eriodictyol-6-C-ꢀ-D-glucopyranoside (26.3
min); 2, (S)-eriodictyol-8-C-ꢀ-D-glucopyranoside (29.1 min); 3, (R)-
eriodictyol-6-C-ꢀ-D-glucopyranoside (29.5 min); 4, (R)-eriodictyol-8-C-ꢀ-
D-glucopyranoside (30.4 min); 5, aspalathin (39.4 min); 6, orientin (40.9
min); 7/8, isoorientin/vitexin (44.1 min); 9, nothofagin (47.8 min); 10,
isovitexin (49.7 min); 11, rutin (50.6 min); 12, hyperoside (51.6 min); 13,
isoquercitrin, (52.1 min) 14, chlorogenic acid (19.3 min).
Preparative Thin-Layer Chromatography (TLC). Separation was
performed on silica gel 60 F254 plates, 2 mm (Merck), with ethyl acetate/
hexane (1:1 (v/v)) as the mobile phase. For isolation of substances,
spots were scratched off. Then, target material was eluted from silica
gel with methanol.
Accurate Mass Determination. The high-resolution positive and
negative ion ESI mass spectra (HR-MS) were obtained from a Bruker
Apex III Fourier transform ion cyclotron resonance (FT-ICR) mass
spectrometer (Bruker Daltonics, Billerica, USA) equipped with an
Infinity cell, a 7.0 T superconducting magnet (Bruker, Karlsruhe,
Germany), a radio frequency (RF)-only hexapole ion guide and an
external electrospray ion source (APOLLO; Agilent, off-axis spray).
Nitrogen was used as the drying gas at 150 °C. The samples were
dissolved in methanol, and the solutions were introduced continuously
via a syringe pump at a flow rate of 120 µL h-1. The data were acquired
with 256k data points and zero filled to 1024k by averaging 32
scans.
Figure 2. Separation of ethyl acetate extract from unfermented rooibos
tea by MLCCC: A, mixture of flavonoids; B, (S)-eriodictyol-8-C-ꢀ-D-
glycopyranoside (2); C, chlorogenic acid (14), (S)-eriodictyol-6-C-ꢀ-D-
glucopyranoside (1), (R)-eriodictyol-6-C-ꢀ-D-glucopyranoside (3), (R)-
eriodictyol-8-C-ꢀ-D-glucopyranoside (4), and rutin (11); D, isoorientin (7);
E, aspalathin (5); F, orientin (6) and vitexin (8); G, nothofagin (9) and
isovitexin (10); H, hyperoside (12) and isoquercitrin (13).
Magnetic Resonance Spectroscopy. Nuclear magnetic resonance
(NMR) spectra were recorded on a Varian Unity Inova 500 instrument
(Darmstadt, Germany). Chemical shifts are given relative to external
Me4Si.
and N,O-bis(trimethylsilyl)acetamide were obtained from Sigma-Aldrich
(Taufkirchen, Germany). Salicylic acid was ordered from Kosmos
(Stuttgart, Germany).
NMR Data of Vitexin (8). 1H NMR (DMSO-d6): δ 6.76 (1 H, s,
H3), 6.26 (1 H, s, H6), 8.01 (2 H, d, J ) 8.7 Hz, H2′, H6′), 6.87 (2 H,
d, J ) 8.7 Hz, H3′, H5′), 4.67 (1 H, d, J ) 9.8 Hz, H1′′), 3.81 (1 H,
t, J ) 9.7 Hz, H2′′), 3.29 (1 H, m, H3′′), 3.37 (1 H, t, J ) 9.2 Hz,
H4′′), 3.23 (1 H, m, H5′′), 3.75 (1 H, dd, J ) 11.9 Hz, 2.2 Hz, H6a′′),
3.51 (1 H, dd, J ) 6.0 Hz, 11.7 Hz, H6b′′) ppm. 13C NMR (DMSO-
d6): δ 163.91 (C2), 102.41 (C3), 182.06 (C4), 155.96 (C5), 98.10 (C6),
162.51 (C7), 104.01 (C8), 160.35 (C9), 104.58 (C10), 121.57 (C1′),
128.92 (C2′, C6′), 115.77 (C3′, C5′), 161.10 (C4′), 73.34 (C1′′), 70.80
(C2′′), 78.63 (C3′′), 70.51 (C4′′), 81.81 (C5′′), 61.26 (C6′′) ppm.
NMR Data of IsoVitexin (10). 1H NMR (DMSO-d6): δ 6.77 (1 H, s,
H3), 6.51 (1 H, s, H 8), 7.91 (2 H, d, J ) 8.8 Hz, H2′, H6′), 6.91 (2
H, d, J ) 8.8 Hz, H3′, H5′), 4.57 (1 H, d, J ) 9.9 Hz, H1′′), 4.02 (1
H, t, J ) 8.9 Hz, H2′′), 3.19 (1 H, t, J ) 8.5 Hz, H3′′), 3.11 (1 H, t,
J ) 9.2 Hz, H4′′), 3.15 (1 H, m, H5′′), 3.68 (1 H, dd, J ) 10.4 Hz,
H6a′′), 3.40 (1 H, dd, J ) 5.9 Hz, 10.4 Hz, H6b′′) ppm. 13C NMR
(DMSO-d6): δ 163.96 (C2), 103.22 (C3), 182.39 (C4), 161.60 (C5),
109.31 (C6), 163.67 (C7), 94.20 (C8), 156.64 (C9), 103.85 (C10),
Extraction of Rooibos Tea. Rooibos tea from the Biedouw Valley
of South Africa was obtained from Ronnefeldt (Worpswede, Germany).
A 100 g amount of fermented (red-brownish) or unfermented (green)
rooibos was extracted with acetone/water (7:3 (v/v)) at 5 °C for 24 h
under argon atmosphere and decanted. Acetone was removed under
reduced pressure. The residual H2O phase was successively extracted
with diethyl ether (2 × 200 mL), ethyl acetate (2 × 200 mL), and
n-butanol (2 × 200 mL). From the individual extracts, solvents were
removed under reduced pressure.
Multilayer Countercurrent Chromatography. The MLCCC sys-
tem (Ito, Multilayer Separator-Extractor Model, P.C. Inc., Potomac)
was equipped with a Waters constant-flow pump (model 6000 A), a
Zeiss spectraphotometer PM2D operating at 280 nm, and a sample
injection valve with a 10 mL sample loop. Eluted liquids were collected
in fractions of 8 mL with a fraction collector (LKB Ultrorac 7000).
Chromatograms were recorded on a plotter (Servogor 200). The
multilayer coil was prepared from 1.6 mm i.d. poly(tetrafluoraethylene)

3370 J. Agric. Food Chem., Vol. 56, No. 9, 2008
Krafczyk and Glomb
Table 1. 1H (500 MHz) and 13C NMR (125 MHz) Spectroscopic Data of Dihydrochalcones Aspalathin (5) and Nothofagin (9) (in DMSO-d6)a
a δ, chemical shift; J, coupling constant. Hydrogen/carbon assignments were verified by HMBC, HMQC, and 13C-DEPT measurements.
121.53 (C1′), 128.90 (C2′, C6′), 116.41 (C3′, C5′), 161.08 (C4′), 73.48
(C1′′), 70.64 (C2′′), 79.37 (C3′′), 71.04 (C4′′), 82.00 (C5′′), 61.57 (C6′′)
ppm.
cases, only very rudimentary previous NMR data of rooibos
ingredients has been published . Therefore, it was necessary to
reinvestigate some structures, present in A. linearis.
Gas Chromatography. High-resolution GC-MS (HRGC-MS) was
performed on a Finnigan Trace GC Ultra (Thermo Finnigan, Bremen,
Germany): quartz capillary column (30 m, 0.32 I.D., HP-5, 0.25 µm;
J&W Scientific, Cologne, Germany); injection port, 220 °C; He, 27.5
cm/s; split ratio, 1:10; detector, 270 °C. After injection of samples at
100 °C, the oven temperature was raised at 5 °C min-1 to 200 °C,
then raised at 10 °C min-1 to 270 °C, and held for 25 min (temperature
program 1). For temperature program 2 samples were injected at 200
°C, the oven temperature was then raised at 2.3 °C min-1 to 270 °C
and held for 30 min. The gas chromatograph was connected to a Trace
DSQ (Thermo Finnigan, Bremen, Germany): transfer line, 220 °C; EI
at 70 eV; full scan, 50–650 m/z.
DeriVatization of Samples. Isolated material from MLCCC separation
of diethyl ether extracts was dissolved in 100 µL of pyridine, and 100
µL of N,O-bis(trimethylsilyl)acetamide was added. After a reaction time
of 1 h at room temperature, the solution was injected into the GC-MS
system.
Two dihydrochalcone structures, aspalathin (5) and nothofagin
(9), were isolated and identified from MLCCC peak E and G.
NMR data of both substances are demonstrated in Table 1. The
presence of the dihydrochalcone skeleton was evident from 13
C
NMR (δ 45.45 ppm (C-R), 29.67 ppm (C-ꢀ), the HMBC
correlation from H-ꢀ to C-1 (δ 132.35 ppm)) and from H-R to
C-1′. HR-MS verified a molecular mass of m/z 452.1 for 5 (m/z
451.1248 (found); m/z 451.1246 (calculated for C21H23O11) [M
- H]-) and a molecular mass of m/z 436.1 for 9 (m/z 435.1295
(found); m/z 435.1297 (calculated for C21H23O10) [M - H]-).
In both dihydrochalcones glucose is fixed in the ꢀ-glycoside
position, because the H-atom at position 1′′ had a chemical shift
3
1
of 4.50 ppm coupling to H 2– at J ) 9.8 Hz. H NMR data
from 5 were in line with Koeppen and Roux (5, 12) and Rabe
et al. (7). Results from 9 were corresponding to Bramati et al.
(13) and Hillis and Inoue (14).
Incubations. Isolated substances (4.5 mM) were dissolved in
phosphate buffer (0.2 M, pH ) 7.4) and incubated (37 °C) for 48 h in
a shaker incubator (New Brunswick Scientific, New Jersey). After
evaporation, the final residue was separated by HPLC-DAD.
Flavone-C-glycosides were isolated from MLCCC peak D
(isoorientin (7)), peak F (orientin (6), vitexin (8)), and peak G
(isovitexin (10)). NMR data of the flavone-8-C-ꢀ-D-glucopy-
ranoside orientin and flavone-6-C-ꢀ-D-glucopyranoside isoori-
entin are specified in Table 2. In both structures glucose is
bound in the ꢀ-glycoside position (δ 1H ) 4.66 ppm,3J ) 10.0
RESULTS AND DISCUSSION
Isolation and Elucidation of Polyphenolic Ingredients.
Ethyl Acetate and n-Butanol Extracts. Ethyl acetate and n-
butanol extract were screened for polyphenols by analytical
HPLC-DAD. Basically both extracts revealed the same ingre-
dients. The HPLC-DAD chromatogram of ethyl acetate extract
is shown in Figure 1. The first step of purification was
separation of these extracts by MLCCC (Figure 2) into eight
fractions (A-H). Stationary-phase retention was between 50
and 55%. Afterward, fractions A-H were separated by prepara-
tive HPLC to isolate pure substances. Final structural evidence
1
3
Hz and δ H ) 4.58 ppm, J ) 9.8 Hz, respectively). HR-MS
delivered a molecular mass of m/z 448.1 for 6 (m/z 447.0930
(found); m/z 447.0933 (calculated for C21H19O11) [M - H]-)
and a molecular mass of m/z 448.1 for 7 (m/z 447.0929 (found);
1
m/z 447.0933 (calculated for C21H19O11) [M - H]-). The H
NMR data of flavone-C-glycosides were in line with Koeppen
(4) and Rabe et al. (7).
The NMR data and HR-MS data verified the structure of 8and
10. These are isomers of 6 and 7 lacking the hydroxyl group at
position 3′ (Table 2, R ) H, orientin f vitexin, isoorientin f
isovitexin). Therefore, 3′ and 5′ like 2′ and 6′ have the same
1
was achieved by H and 13C NMR, as well as heteronuclear
multiple quantum coherence (HMQC) and heteronuclear mul-
tiple bond correlation (HMBC) NMR experiments. In most
1
chemical shift in H and 13C NMR experiments.

Phenolic Compounds in Rooibos
J. Agric. Food Chem., Vol. 56, No. 9, 2008 3371
Table 2. 1H (500 MHz) and 13C NMR (125 MHz) Spectroscopic Data of Flavone-C-glycosides Orientin (6) and Isoorientin (7) (in DMSO-d6)a
a δ, chemical shift; J, coupling constant. Hydrogen/carbon assignments were verified by HMBC, HMQC, and 13C-DEPT measurements. The asterisk (/) indicates
rotamers (21, 22).
In both flavones the glucose was fixed in the ꢀ-glycoside
position, too. HR-MS data from 8 delivered a molecular mass
of m/z 432.1 (m/z 431.0986 (found); m/z 431.0984 (calculated
for C21H19O10) [M - H]-) and a molecular mass of m/z 432.1
for 10 (m/z 431.0983 (found); m/z 431.0984 (calculated for
C21H19O10) [M - H]-). UV and MS data of vitexin and
isovitexin were corresponding to Rabe et al. (7) and Bramati et
al. (13).
449.10870 (found); m/z 449.1089 (calculated for C21H21O11) [M
- H]-) and a molecular mass of m/z 449.1092 for 3 (m/z
449.1092 (found); m/z 449.1089 (calculated for C21H21O11) [M
- H]-). Compound 2 gave a molecular mass of m/z 450.1 in
HR-MS (m/z 449.1083 (found); m/z 449.1089 (calculated for
C21H21O11) [M - H]-) and 4 gave a molecular mass of m/z
450.1 in HR-MS (m/z 449.1090 (found); m/z 449.1089 (calcu-
lated for C21H21O11) [M - H]-).
Four flavanone-C-glycosides (1–4) were isolated from ML-
CCC peaks B and C (Figure 2). 2 + 4 and 1 + 3 are
diastereoisomeres, respectively, and gave different retention
times in HPLC analysis. Although the absolute stereochemistry
was not determined, the peak eluting first was tentatively
assigned the S-isomer on the basis of Philbin and Schwartz (15).
However, NMR results for each group were basically identical
(Table 3). This must be due to the almost planar A and C ring
system of the flavanone isomers resulting in an almost identical
electronic environment for H-3 and H-2. Additionally, both pairs
of diastereoisomeres are in equilibrium via ring opening to a
quinine methide configuration. Incubations of isolated 1 in
phosphate buffer resulted in immediate conversion to a 1:1
mixture of 1 + 3 (Figure 3). The NMR results are in line with
the data from a diastereomeric mixture of (R)- and (S)-
eriodictyol-6-C-ꢀ-D-glucopyranoside by Marais et al. (9). The
data for the 8-C-glycosides are presented for the first time herein.
HR-MS delivered a molecular mass of m/z 450.1 for 1 (m/z
Flavonol-O-glycosides were isolated from MLCCC peak C
(rutin 11) and peak H (hyperoside (12) and isoquercitrin (13)).
NMR data of these substances are shown in Table 4. The sugar
of these three flavonol-O-glycosides is fixed in the ꢀ-glycoside
position. Compound 11 gave the molecular mass m/z 610.1
(m/z 609.1465 (found); m/z 609.1461 (calculated for C27H29O16)
[M - H]-), HR-MS delivered a molecular mass m/z 464.1
for 12 (m/z 503.0587 (found); m/z 503.0586 (calculated for
C21H20O12K) [M + K]+), and a molecular mass m/z 464.1
for 13 (m/z 487.0859 (found); m/z 487.0847 (calculated for
C21H20O12Na) [M + Na]+). All data for rutin were in line with
Sawabe et al. (16).
Hyperoside was isolated from A. linearis, and the NMR data
unequivocally were established for the first time in comparison
to 13 and 11. The detailed structure and selected HMBC
correlations are shown in Figure 4. The presence of the flavone
skeleton was evident from the UV (λmax ) 256 and 350 nm),
13C NMR (δ 177 (C-4), 156 (C-2), and 133 ppm (C-3)), and

3372 J. Agric. Food Chem., Vol. 56, No. 9, 2008
Krafczyk and Glomb
Table 3. 1H and 13C NMR Spectroscopic Data of Flavanone-C-glycosides (R)/(S)-Eriodictyol-6-C-ꢀ-D-glucopyranoside (1 + 3) and
(R)/(S)-Eriodictyol-8-C-ꢀ-D-glucopyranoside (2 + 4) (in DMSO-d6)a
a δ, chemical shift; J, coupling constant. Hydrogen/carbon assignments were verified by HMBC, HMQC, and 13C-DEPT measurements.
) 19.3 min) and absorption spectrum (λmax ) 320 nm) to
commercial reference material (Figure 5). Additionally, HR-
MS gave a pseudo-molecular mass m/z 353.1 for 14 (m/z
353.0873 (found); m/z 353.0878 (calculated for C16H17O9) [M
- H]-).
Diethyl Ether Extract. The diethyl ether extract was screened
for polyphenols by analytical HPLC-DAD (Figure 6). In
contrast to ethyl acetate and n-butanol extracts benzoic and
cinnamic acids were found in high yields. In addition diethyl
ether extract contained hydrophobic flavonoide aglycons. The
extract was separated into eight signals (I-P) by MLCCC
(Figure 7). Stationary-phase retention was between 60 and 65%.
After workup, material was derivatized and subjected to coupled
gas chromatography-mass spectrometry (temperature program
1). From the various MLCCC fractions the following polyphe-
nolic structures were unequivocally identified by comparison
with authentic material: syringic acid (23; m/z), 342 (M+•, 2×
silylated, 62%), 327 (70%), 312 (64%), 297 (27%), 283 (25%),
253 (28%), 223 (25%), 141 (19%), 89 (9%), and 73 (100%);
gallic acid (15; m/z), 458 (M+•, 4× silylated, 35%), 443 (16%),
281 (71%), 179 (11%), and 73 (100%); gentisic acid (18; m/z),
355 (M
Figure 3. HPLC-DAD chromatogram of the incubation of 1 in phosphate
buffer: (A) 24 and (B) 0 h.
4
the HMBC correlation from H-6 (δ 6.18 ppm, d, 1H, J ) 2
Hz) to C-5, C-7, C-8, and C-10. H-8 correlated to C-9, C-7,
C-10, and C-6. The 1H NMR spectrum displayed the anticipated
systems for the A and B ring, the protons for 7-OH, 3′-OH,
and 4′-OH (δ 9.1–12.6 ppm) and the chelated proton at 5-OH.
The ꢀ-glycoside configuration was verified by H-1′′ at δ 5.35
+ - 15, 3× silylated, 50%), 193 (10%), 147 (9%), and
3
73 (100%); protocatechuic acid (16; m/z), 370 (M+•, 2×
silylated, 32%), 355 (18%), 311 (9%), 223 (5%), 193 (100%),
165 (8%), 137 (5%), and 73 (53%); vanillic acid (21; m/z), 312
(M+•, 2× silylated, 67%), 297 (100%), 282 (26%), 267 (69%),
253 (55%), 223 (29%), 193 (24%), and 73 (90%); caffeic acid
ppm coupling with J ) 7.7 Hz, which correlated to C-3 at
133.46 ppm. NMR data of 12 and 13 were identical to work
published by Krasnov et al. (17).
From MLCCC peak B chlorogenic acid (5-O-caffeoyl-quinic
acid (14)) was identified by comparing the retention time (RT

Phenolic Compounds in Rooibos
J. Agric. Food Chem., Vol. 56, No. 9, 2008 3373
Table 4. 1H and 13C NMR Spectroscopic Data of Flavonol-O-glycosides Rutin (11), Hyperoside (12), and Isoquercitrin (13) (in DMSO-d6)a
a δ, chemical shift; J, coupling constant. Hydrogen/carbon assignments were verified by HMBC, HMQC, and 13C-DEPT measurements.
Figure 4. Structure of hyperoside 12. Selected HMBC correlations are
marked by arrows.
Figure 5. HPLC-DAD chromatograms of n-butanol extract (A) and
chlorogenic acid (B). Both gave a peak at 19.3 min with identical absorption
spectra, λ ) 320 nm.
(22; m/z), 396 (M+•, 3× silylated, 17%), 219 (16%), 207 (11%),
147 (21%), and 75 (100%). As an example the identification of
23 is shown in Figure 8. Results from protocatechuic acid,
caffeic acid, and vanillic acid were corresponding to Lin and
Harnly (18). Data from 23 were in line with Ferreira et al. (8).
After MLCCC separation the retarded stationary phase was
also silylated and separated by HRGC-MS (temperature program
1), leading to the identification of the following structures: 3,5-
dihydroxybenzoic acid (16; RT ) 18.3 min) m/z 370 (M+•, 3×
silylated, 35%), 355 (10%), 281 (6%), 193 (100%), 147 (16%),
and 73 (84%); p-hydroxybenzoic acid (19; RT ) 14.2 min),
m/z 282 (M+•, 2× silylated, 12%), 267 (86%), 223 (78%), 193
(41%), 147 (29%), and 75 (100%); p-coumaric acid (24; RT >

3374 J. Agric. Food Chem., Vol. 56, No. 9, 2008
Krafczyk and Glomb
Figure 6. HPLC-DAD chromatogram of diethyl ether extract from
unfermented rooibos tea at λ ) 280 nm. Retention times are given in
parentheses: 15, gallic acid (4.9 min); 16, protocatechuic acid (7.8 min);
17, 3,5-dihydroxybenzoic acid (7.8 min); 18, gentisic acid (12.3 min); 19,
p-hydroxybenzoic acid (12.3 min); 20, (+)-catechin (15.9 min); 21, vanillic
acid (17.0 min); 22, caffeic acid (19.0 min); 23, syringic acid (21.9 min);
24, p-coumaric acid (28.0 min); 25, salicylic acid (32.7 min); 26, ferulic
acid (33.4 min); 27, sinapinic acid (38.7 min); 28, quercetin (66.7 min);
29, luteolin (70.9 min); 30, chrysoeriol (75.7 min).
Figure 8. Signal O (Figure 4) of MLCCC separation of diethyl ether extract
was derivatized by silylation: (I) HRGC-MS chromatogram, syringic acid
(23) eluted at 19.8 min; (II) corresponding EI-mass spectrum.
Figure 7. Separation of diethyl ether extract from rooibos tea by MLCCC:
I-K, Mixture of flavonoids; L, gallic acid (15); M, gentisic acid (18); N,
protocatechuic acid (16); O, syringic acid (23); P, vanillic acid (21) and
caffeic acid (22).
Figure 9. Aglycons isolated from the stationary phase of diethyl ether
extract after MLCCC separation: 28, quercetin; 20, (+)-catechin; 29,
luteolin; 30, chrysoeriol.
) 20.5 min) m/z 308 (M+•, 2× silylated, 24%), 293 (53%),
249 (22%), 219 (35%), 179 (10%), 147 (15%), and 73 (100%);
salicylic acid (25; RT ) 11.8 min) m/z 267 (M+•, 2× silylated,
100%), 193 (7%), 149 (10%), 135 (12%), 91 (7%), and 73
(68%); ferulic acid (26; RT ) 23.2 min) m/z 338 (M+•, 2×
silylated, 38%), 323 (32%), 308 (27%), 293 (16%), 249 (19%),
219 (14%), 147 (26%), 117 (13%), and 73 (100%); sinapinic
acid (27; RT ) 25.2 min) m/z 368 (M+•, 2× silylated, 25%),
353 (23%), 338 (30%), 323 (11%), 129 (15%), 117 (26%), 73
(100%).
Data from p-coumaric acid (24) and ferulic acid (26)
correspond to Lin et al. (18).
The residual stationary phase contained nonpolar aglycons
such as (+)-catechin (20), quercetin (28), luteolin (29), and
chrysoeriol (30) (Figure 9). For isolation of 29 + 30 the
solventless residuum was separated by preparative thin-layer
chromatography. After elution, target material was derivatized
and injected into the HRGC-MS system (temperature program
2). The following polyphenolic structures were unequivocally
identified by comparison with authentic material: chrysoeriol
(30; RT ) 18.5 min) m/z 486 (M+ - 15, 5× silylated, 1%),
371 (69%), 313 (7%), 281 (10%), 239 (28%), 207 (29%), 203
(35%), 147 (85%), 129 (51%), 117 (37%), 103 (27%), 75
Figure 10. Hydroxybenzoic acids isolated from diethyl ether extract: 15,
gallic acid; 16, protocatechuic acid; 17, 3,5-dihydroxybenzoic acid; 18,
gentisic acid; 19, p-hydroxybenzoic acid; 21, vanillic acid; 23, syringic
acid; 25, salicylic acid.
(100%), and 73 (96); luteolin (29; RT ) 23.7 min) m/z 574
(M+•, 4× silylated, 1%), 488 (2%), 429 (2%), 399 (27%), 281
(10%), 207 (38%), 147 (77%), 129 (30%), 75 (100%), and 73
(62%). Quercetin (28) and (+)-catechin (20) could be unam-
biguously identified direct from the stationary phase by HRGC-
MS (temperature program 2) after silylation: quercetin (30; RT

Phenolic Compounds in Rooibos
J. Agric. Food Chem., Vol. 56, No. 9, 2008 3375
) 34.7 min) m/z 486 (M+ - 15, 5× silylated, 1%), 371 (69%),
313 (7%), 281 (10%), 239 (28%), 207 (29%), 203 (35%), 147
(85%), 129 (51%), 117 (37%), 103 (27%), 75 (100%), and 73
(96); (+)-catechin (20; RT ) 26.40 min) m/z 650 (M+•, 5×
silylated, 7%), 578 (8%), 488 (3%), 383 (16%), 368 (100%),
296 (70%), 280 (66%), 179 (44%), 73 (89%), and 75 (10%).
Data were in line with Ferreira et al. (8), Fischer et al. (19),
and Amani et al. (20).
Figure 11. Hydroxycinnamic acids isolated from diethyl ether extract: 22,
caffeic acid; 24, p-coumaric acid; 26, ferulic acid; 27, sinapinic acid.
In conclusion the following substances from A. linearis were
isolated and identified: (S)/(R)-eriodictyol-6-C-ꢀ-D-glucopyra-
noside (1 + 3), (S)/(R)-eriodictyol-8-C-ꢀ-D-glucopyranoside (2
+ 4), aspalathin (5), orientin (6), isoorientin (7), vitexin (8),
nothofagin (9), isovitexin (10), rutin (11), hyperoside (12),
isoquercitrin (13), chlorogenic acid (5-CQA, 14), gallic acid
(15), protocatechuic acid (16), 3,5-dihydroxybenzoic acid (17),
gentisic acid (18), p-hydroxybenzoic acid (19), (+)-catechin
(20), vanillic acid (21), caffeic acid (22), syringic acid (23),
p-coumaric acid (24), salicylic acid (25), ferulic acid (26),
sinapinic acid (27), quercetin (28), luteolin (29), and chrysoeriol
(30). Within this context 2 + 4, 12, 14, 15, 17, 18, 25, and 27
were isolated from rooibos for the first time. Identified benzoic
and cinnamic acids are shown in Figure 10 and Figure 11.
The established method is able to isolate multiple and minor
polyphenolic substances and applicable to further natural
products.
Figure 12. Degradation of aspalathin 5 to (R)/(S)-eriodictyol-6-C-ꢀ-D-
glucopyranoside (1 + 3), (R)/(S)-eriodictyol-8-C-ꢀ-D-glucopyranoside (2
+ 4), orientin (6), and isoorientin (7). Yields of intermediates are calculated
on the basis of the initial concentration of aspalathin.
Degradation of Dihydrochalcones. Prolonged incubation of
flavanone-C-glycosides resulted in several distinct structures.
Figure 13. Oxidative degradation of aspalathin (5) to orientin (6) and isoorientin (7). Eriodictyols 1–4 (1-(S)-eriodictyol-6-C-ꢀ-D-glucopyranoside, 2-(S)-
eriodictyol-8-C-ꢀ-D-glucopyranoside, 3-(R)-eriodictyol-6-C-ꢀ-D-glucopyranoside, 4-(R)-eriodictyol-6-C-ꢀ-D-glucopyranoside) are key intermediates. R )
glucose.

3376 J. Agric. Food Chem., Vol. 56, No. 9, 2008
Krafczyk and Glomb
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During the process eriodictyols 1 + 3 were the major products
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The degradation of 5 to eriodictyols 1–4 must be explained
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(S)-eriodictyol-6-C-ꢀ-D-glucopyranoside immediately changed
to a 1:1 mixture of 1 + 3 (Figure 3) and, more slowly and to
a much lower extent, to the eriodictyol-8-C-ꢀ-D-glucopyrano-
sides 2 + 4. In equilibrium the ratio of 1 + 3 to 2 + 4 was
about 10 to 1. Reactions of authentic 4 yielded 1 + 3, while 4
was readily decomposed. Reactions of 1 + 3 also produced 7
and, unexpectedly, even higher amounts of 6. In contrast, 2 +
4 never resulted in any 6. This excludes direct oxidation of 2
+ 4 to 6. Compounds 6 and 7 were also incubated separately.
Isoorientin was converted to orientin, although it was only a
minor product among uncharacterized brown material. This is
an irreversible reaction step, because 6 was not converted to 7.
Conversion must be initiated by opening of the vinyl ester
structure of 7 followed by bond rotation and recyclization, also
known as Wessely-Moser rearrangement. Conversion including
reductive steps could be excluded as no eriodictyols were
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considerably faster than degradation of 6 (half-life, 208 h). This
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ACKNOWLEDGMENT
We thank D. Ströhl from the Institute of Organic Chemistry,
Halle, Germany, for recording NMR spectra, and J. Schmidt
from the Leibnitz Institute of Plant Biochemistry, Halle,
Germany, for performing LC-MS/accurate mass analysis.
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