Quantitation of chafurosides A and B in tea leaves and isolation of prechafurosides A and B from oolong tea leaves

Article abstract of DOI:10.1021/jf900032z

A procedure was developed for the quantitative determination of chafuroside A, a flavone C-glycoside with potent anti-inflammatory activity, and its regioisomer chafuroside B, as well as isovitexin and vitexin, by selected reaction monitoring liquid chromatography-tandem mass spectrometry (SRM LC-MS/MS) analysis. This method was successfully applied to commercial leaves of green tea, houji tea, oolong tea, and black tea. High levels of chafurosides A and B were found in oolong tea leaves that had been heated at >140°C. Next, their precursors, prechafurosides A and B, were isolated from methanol extract of oolong tea leaves prepared from Shizu 7132, Camellia sinensis (L.) O. Kuntze, by partition with n-butanol and H₂O and chromatography on Diaion SP-825, Sephadex LH-20, and ODS C-18, guided by assay of chafuroside formation. Prechafurosides A and B gave chafurosides A and B, respectively, in good yields when heated at 160°C for 0.5 h. Solvolysis of prechafurosides A and B with pyridine and dioxane quantitatively afforded isovitexin and vitexin, respectively. On the basis of these results and physicochemical data (MS, UV, and NMR), prechafurosides A and B were concluded to be new flavone C-glycoside sulfates, isovitexin2"-sulfate and vitexin-2″-sulfate, respectively.

Full text of DOI:10.1021/jf900032z

J. Agric. Food Chem. 2009, 57, 6779–6786 6779
DOI:10.1021/jf900032z
Quantitation of Chafurosides A and B in Tea Leaves and
Isolation of Prechafurosides A and B from Oolong Tea Leaves
H
ITOSHI
I
SHIDA,* TOSHIYUKI
OSHIO
W
AKIMOTO, YUKIKO
K
ITAO, SHIMAKO
T
ANAKA
,
T
MIYASE AND ARUO
,
H
N
UKAYA
School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
A procedure was developed for the quantitative determination of chafuroside A, a flavone
C-glycoside with potent anti-inflammatory activity, and its regioisomer chafuroside B, as well as
isovitexin and vitexin, by selected reaction monitoring liquid chromatography-tandem mass spectro-
metry (SRM LC-MS/MS) analysis. This method was successfully applied to commercial leaves of
green tea, houji tea, oolong tea, and black tea. High levels of chafurosides A and B were found in
oolong tea leaves that had been heated at >140 °C. Next, their precursors, prechafurosides A and
B, were isolated from methanol extract of oolong tea leaves prepared from Shizu 7132, Camellia
sinensis (L.) O. Kuntze, by partition with n-butanol and H2O and chromatography on Diaion SP-825,
Sephadex LH-20, and ODS C-18, guided by assay of chafuroside formation. Prechafurosides A
and B gave chafurosides A and B, respectively, in good yields when heated at 160 °C for 0.5 h.
Solvolysis of prechafurosides A and B with pyridine and dioxane quantitatively afforded isovitexin
and vitexin, respectively. On the basis of these results and physicochemical data (MS, UV, and
NMR), prechafurosides A and B were concluded to be new flavone C-glycoside sulfates, isovitexin-
200-sulfate and vitexin-200-sulfate, respectively.
KEYWORDS: Oolong tea; tea leaf; chafuroside A; chafuroside B; isovitexin; vitexin; isovitexin-200-
sulfate; vitexin-200-sulfate
INTRODUCTION
leaves (7, 8). Interestingly, we found that oral administration of
chafuroside A prevents skin inflammation in an ICR mouse
atopic disease model at the dose of 10 μg/kg, which is much lower
than the effective doses of prednisolone and betamethasone
(10 and 0.8 mg/kg, respectively). Like indomethacin, chafuroside
A reduced AOM-induced development of colon aberrant crypt
foci in rats and APC-deficient mice at 10 ppm (9 ).
Furanoflavonoids show various biological activities, including
anti-inflammatory, antifungal, antiviral, and/or antibacterial
activities. A representative structure of this type of flavonoid is
flavone with a furan ring fused at C-7,8, such as lamceolatin (10).
In contrast, flavone C-glycosides with a furan ring between
sugar and aglycone are rare. Ketohexofuranosides have both
C- and O-glycosidic linkages at the same carbon (C2) of hexose,
forming a 5,5-spiroketalring system. Recently, pinnatifinosidesA
and B were reported to have unusual glycoside structures,
Tea is made from evergreen leaves of Camellia sinensis and is
the most widely consumed beverage after water in daily life
worldwide. The common commercial types are green tea, oolong
tea, pu-erh tea, and black tea. The processing methods of their
leaves are similar, except for the manner of fermentation and
firing. The fermentation level increases from unfermented green
tea to partially fermented oolong tea to well-fermented black tea
(1 ). Firing involves heating at high temperature by means of a hot
plate, hot air, or flame after drying, and some species of green tea
and oolong tea leaves are subjected to strong firing at >160 °C to
enhance stability, taste, and flavor.
There has been increasing interest in polyphenolic compounds
contained in foodstuffs, especially in tea leaves, because of their
beneficial effect on human health. Uehara et al. (2 ) showed that
daily drinking of oolong tea (1 L) for 4 weeks improved skin
condition in 62% of patients with atopic dermatitis. We recently
isolated new flavone C-glycosides with a condensed dihydrofuran
ring, chafurosides A and B (Figure 1), from oolong tea (unpub-
lished information) by means of fractionation guided by assay of
anti-inflammatory activity in an atopic dermatitis model (3 ).
Many biologically active polyphenolic compounds, such as
EGCG and theaflavins, have been isolated from green and black
tea leaves (4-6). There have been fewer studies on oolong tea
including β-D-allofuranose and R-D-allofuranose, respectively
(11). To our knowledge, however, the coexistence of C- and
O-glycoside linkages at separate positions in chafurosides A and
B is novel.
We considered that chafuroside A is a promising candidate as
an anti-inflammatory and chemopreventive agent. Only about
1 mg of chafuroside A was isolated from 3.2 kg of commercial
oolong tea leaves, so it is important to develop a method for
quantitative determination of chafurosides to identify teas with
high levels of this chemical, both for biosynthetic and metabolic
studies and for therapeutic use. In the present study, we developed
*Corresponding author (telphone +81.54.264.5620; fax
+81.54.264.5623; e-mail ishida@u-shizuoka-ken.ac.jp).
© 2009 American Chemical Society
Published on Web 07/02/2009
pubs.acs.org/JAFC

6780 J. Agric. Food Chem., Vol. 57, No. 15, 2009
Ishida et al.
pressure of approximately 1.3 ꢀ 10-4 mbar in the collision cell (Q2)
and scanning the second quadrupole mass spectrometer (Q3) in the range
of m/z 80-500.
A precursor ion for CID and diagnostic product ion for the target
were identified to carry out selective reaction monitoring (SRM) LC-
MS/MS analysis. The source and desolvation temperatures were main-
tained at 100 and 300 °C, respectively. The desolvation and cone gas
flows were set at 680 and 0 L/h. The electrospray capillary voltage and
cone voltage were optimized for each standard.
(2) Liquid Chromatography. Liquid chromatography was per-
formed on an Agilent 1100 LC system (Agilent Technologies, Wald-
bronn, Germany). Chromatographic separation of chafurosides A and
B, isovitexin, and vitexin was carried out on a 150 mm ꢀ 3 mm i.d., 3 μm,
Cadenza CD-C18 column (Intact, Kyoto, Japan). The column tempera-
ture was kept at 45 °C. The mobile phase reservoirs contained (A) H2O/
MeOH (40:60) for the analysis of authentic standards. HPLC conditions
were as follows: 100% A for 20 min to elute vitexin, isovitexin, and
chafurosides A and B and then a linear gradient to 100% MeOH at
22 min, hold for 8 min to wash the column, return to initial conditions in
31 min, and condition column for 9 min before the next injection. The
flow rate from 0 to 20 min was 0.3 mL/min and that from 20 to 40 min
was 0.5 mL/min.
Figure 1. Structures of chafurosides A and B from oolong tea.
a method for quantitative determination of chafurosides A and B,
together with their structural analogues, isovitexin and vitexin,
respectively, by using ion-spray LC-MS/MS and applied it to
analyze green tea, houji tea, oolong tea, and black tea from
C. sinensis. Here, we report on the concentrations of chafurosides
in various tea leaves, as well as the isolation, structural determi-
nation, and reactivity of chafuroside precursors in oolong tea
leaves.
MATERIALS AND METHODS
Quantitative Determination of Chafurosides A and B,
Isovitexin, and Vitexin by LC-MS/MS. Ten microliters of the
test sample from each type of tea leaves (0.1 g) in 0.5-10 mL of 50%
MeOH was used for LC-MS/MS analyses of chafurosides A and B,
isovitexin, and/or vitexin. Reference standard solution (5 μL) was
injected before and after each sample analysis. The peak area was used
for calibrating flavone C-glycoside amounts. The determination limit
was taken to be the amount giving a signal-to-noise ratio of 5.
General. 1H and 13C spectra were obtained on a JEOL ECA-500
spectrometer at 500 and 125 MHz, respectively, with chemical shifts
being reported as δ (ppm) from tetramethylsilane as an internal standard.
1H-1H COSY, 1H-13C COSY, and HMBC spectra were obtained with
the JEOL standard pulse sequences, and data processing was performed
with standard software Delta. IR and UV spectra were taken on a
JASCO WS/IR-8000 grating infrared spectrometer and on a Hitachi UV
absorption spectrometer U-2900. Optical rotation was measured with a
JASCO DIP-1000 digital polarimeter.
LC-MS/MS measurements were performed with triple-stage quadru-
pole instruments equipped with electrospray ionization (ESI) interfaces:
API 3000 system (Applied Biosystems, Foster City, CA). Analyst soft-
ware (version 1.4.1) was used for data acquisition and analysis. QTOF
MS and MS/MS measurements were performed with QSTAR Elite
(Applied Biosystems) equipped with electrospray ionization (ESI) inter-
faces.
Recovery Rates of Chafurosides A and B, Isovitexin, and
Vitexin. Recoveries of chafurosides A and B, isovitexin, and/or vitexin
were determined at concentrations of 5, 50, and 500 ng/g, respectively.
The H2O-soluble fraction from 100% MeOH extract of green tea leaves
from Shizu 7132 was used in this experiment. The H2O-soluble fraction
was spiked with the standards and extracted with n-BuOH (n = 3). The
n-BuOH-soluble fraction was evaporated to dryness, then the residue
was dissolved in 50% MeOH, and this solution was subjected to LC-MS/
MS analysis.
ESI-MS and MS/MS analyses for compound identification were
performed on a Q-Star XL Hybrid LC-MS/MS System (Applied
Biosystems, Foster City, CA) using an Agilent 1200 series binary HPLC
pump with a 50 mm ꢀ 2.1 mm i.d., 3 μm, Inertsil ODS-SP column (GL
Sciences, Tokyo, Japan). For system control and data acquisition we
used Analyst QS software.
Preparation of Tea Leaf Extract and Its Fractionation.
Leaves (0.5 g) of commercial green tea, houji tea (roasted green tea),
oolong tea, and black tea and those prepared from Shizu 7132 were
ground and extracted twice with H2O, 30% MeOH, or 100% MeOH
(5.0 mL) under reflux for 40 min. Each extract was partitioned between
n-BuOH (1 mL ꢀ 2) and H2O (1 mL). The extract, H2O-soluble fraction,
and n-BuOH-soluble fraction were subjected to LC-MS/MS analysis
for quantitation of chafurosides A and B, isovitexin, and vitexin.
Samples and Reagents. Commercial leaves of green tea, Yabukita
and Sayamakaori, houji tea of Yabukita, oolong tea, Shikisyu and
Suisen, and black tea, Darjeeling and Assam, were used. Green tea,
houji tea, oolong tea, and black tea leaves were prepared from fully
grown tea leaves of Shizu 7132, C. sinensis (L.) O. Kuntze, in summer
2007 according to the traditional manners described below. The tem-
peratures of green tea, oolong tea, and black tea leaves were strictly
controlled under 90 °C in the courses of their processing. Houji tea leaves
were prepared by roasting the green tea leaves, thus obtained, on a hot
plate at around 190-200 °C for 4 min.
All HPLC or analytical grade solvents were purchased from Wako
Pure Chemical Industries Ltd. (Osaka, Japan). Authentic isovitexin and
vitexin were isolated from Puriri (Vitex lucens) in New Zealand, using the
purification procedures described for flavone C-glycoside. Authentic
chafurosides A and B were synthesized from isovitexin and vitexin,
respectively, by means of modified Mitsunobu reaction with triphenyl-
phosphine and diethyl azodicarboxylate (12, 13). Sep-Pak plus C18
cartridges were purchased from Waters Corp. (Milford, MA), Diaion
SP 825 from Mitsubishi Kagaku (Tokyo, Japan) and Sephadex LH-20
from GE Healthcare Bio-Science AB (Uppsala, Sweden).
Heat Treatment of Tea Leaves, Extraction, and Fractiona-
tion. Leaves of commercial green tea, houji tea, oolong tea, and black
tea and those prepared from Shizu 7132, their extracts, and the fractions
derived from these extracts were placed in glass tubes and heated in an
oven (Nihon Buchi K.K., Tokyo, Japan).
Isolation of Chafuroside Precursors from Tea Leaves. Oolong tea
leaves prepared from Shizu 7132 (1 kg) were ground and extracted twice
with MeOH under reflux for 40 min. The extract (348 g) was partitioned
between n-BuOH and H2O. The aqueous fraction (142 g) was chroma-
tographed on Diaion SP 825 with H2O, 30% MeOH, and 100% MeOH
to obtain fractions 1-3, respectively. Fractions 1 and 2 afforded
substantial amounts of chafurosides on heating at 160 °C.
Fraction 2 gave an active fraction, fraction 4 (1.97 g), eluted between
Kd = 2.30 and 2.80 on Sephadex LH-20 using MeOH. Successive
chromatography of fraction 4 on ODS-C18 (Sep-Pak C18-Gel) afforded
active fractions 5 (87 mg) and 6 (63 mg). Finally, purification of both
fractions on ODS HPLC using Cadenza CD-C18 afforded the chafuro-
side A and B precursors, prechafuroside A (1) and its regioisomer,
prechafuroside B (2) (6 and 4 mg), respectively. Compound 1 gave
chafuroside A, and compound 2 gave chafuroside B in good yields,
respectively, when heated at 160 °C.
LC-ESI-MS/MS Analysis. Conditions. (1) ESI-MS/MS. In
both positive and negative modes, full-scan, single MS mass spectra were
acquired over the mass range of m/z 300-1200 by direct infusion of four
standards in H2O/MeOH eluent (flow rate = 0.2 mL/min).
Full-scan collision-induced dissociation (CID) spectra were acquired
by colliding the quadrupole 1 (Q1) selected precursor ion at an argon
During the isolation process, all eluates were monitored by means of
chafuroside formation assay (heating at 160 °C).

Article
J. Agric. Food Chem., Vol. 57, No. 15, 2009 6781
Figure 2. Negative product ion mass spectra of (A) chafuroside A and (B) chafuroside B with the [M - H]- ions at m/z 413 and 413 and of (C) isovitexin and
(D) vitexin at m/z 431 and 431 as precursors, respectively.
Chafuroside A (1) was obtained as a light yellow powder: [R]D26
=
Significant fragment ions were observed at m/z 293 and 117 in
the cases of chafurosides A and B and at m/z 341, 311, 283, and
117 in the cases of isovitexin and vitexin. The product ions at m/z
293 of the former compounds showed the highest intensity at a
collision energy of -36 eV and a capillary voltage of -4.0 kV,
and the product ions at m/z 311 of the latter compounds showed
the highest intensity at a collision energy of -32 eV and a
capillary voltage of -4.0 kV.
To achieve maximum selectivity, as well as quantitative
analysis, SRM was then implemented. On the basis of the
MS/MS fragmentations of chafurosides A and B, isovitexin,
and vitexin, combination precursor-product ions of m/z
413-293 for chafurosides A and B and of m/z 431-311 for
isovitexin and vitexin were considered to be suitable for con-
firmatory analysis. Typical chromatograms from the SRM LC-
MS/MS analyses of authentic standard chafuroside A, chafuro-
side B, isovitexin, and vitexin (100 ng/mL) are shown in Figure 3.
Good linearities (R2 > 0.99) of the peak areas with increasing
amounts of the four standards were confirmed over the range
from 0.005 to 1 ng per injection (0.5-100 ng/mL of H2O-soluble
fraction of the MeOH extract of oolong tea (0.1 g)). The
determination limits of chafurosides A and B, isovitexin,
and vitexin in the spiked H2O-soluble fraction part, based on
a signal-to-noise ratio of 5, were 0.01, 0.01, 0.02, and 0.02 ng/g of
the H2O-soluble fraction from MeOH extract of oolong tea,
respectively.
-45.0° (c 0.6, MeOH); UV λmax (MeOH) nm (log ε) 330 (4.34) and
273 (4.39); ESI-MS m/z 511 [M - H]-; HR-QTOFMS m/z 511.05612
[M - H]-; HR-FABMS m/z 511.0550 [M - H]- (calcd for C21H20O13
S - H, 511.05625); IR νmax (neat) 3600-3120, 1653, 1608, 1354, 1242,
1201,1178, and 812 cm-1; 1H and 13C NMR data in dimethyl sulfoxide
(DMSO)-d6 are shown in Table 6.
Chafuroside B (2) was obtained as a light yellow powder: [R]D26
=
-23.0° (c 0.6, MeOH); UV λmax (MeOH) nm (log ε) 326 (4.24) and 270
(4.31); ESI-MS m/z 511 [M-H]-. HR-QTOFMS m/z 511.05643 [M-H]-
(HR-FABMS m/z 511.0551 [M - H]- (calcd for C21H20O13S - H,
511.05625); IR νmax (neat) 3580-3100, 1655, 1610, 1356, 1244, 1200,
1180, and 814 cm-1; 1H and 13C NMR data in DMSO-d6 are shown in
Table 6.
Conditions for the Acquisition of TOFMS and MS/MS Spectra.
HPLC conditions were as follows. Acetonitrile (10%) was delivered
as the mobile phase at 0.2 mL/min. The column temperature was kept at
40 °C. ESI-TOFMS and ESI-Q-TOFMS were conducted in the negative
product ion scan mode; the ion spray voltage was set at -4500 V. Dry air
(GS1) was maintained at 75 psi. The declustering potentials 1 and 2 were
set at -50 and -15 V, respectively. On ion-dependent acquision (IDA)
for MS and MS/MS analyses of compounds 1 and 2, the collision energy
was set at -5 and -20 eV, respectively. Mass spectrometer calibration
was performed with a mixture of CsI ([M - H]-, m/z 126.90503),
taurocholic acid ([M - H]-, m/z 514.28440), and synthetic peptide
iPD1 (ALILTLVS) ([M - H]-, m/z 827.52477).
RESULTS AND DISCUSSION
Chafuroside Analysis with LC-MS/MS. Optimization of LC-
ESI-MS/MS Conditions. Negative ion MS of chafurosides A and
B, isovitexin, and vitexin standard solution was initially im-
plemented with H2O/MeOH or H2O/acetonitrile (1:1) as the
mobile phase, which promoted the selective and highly sensitive
formation of [M - H]- of chafurosides A and B at m/z 413
and of isovitexin and vitexin at m/z 431, as well as gave good
chromatographic performance during LC-MS analyses. In
further CID-MS/MS experiments, typical negative ion mass
spectra (range m/z 300-500) of chafurosides A and B, isovitex-
in, and vitexin standard solutions were obtained (Figure 2).
Recoveries of Chafurosides A and B, Isovitexin, and Vitex-
in. Recoveries of chafurosides A and B, isovitexin, and vitexin,
spiked at three dose levels, from the H2O-soluble fraction of
the MeOH extract of oolong tea leaves were 85-105, 80-112,
83-98, and 96-113%, respectively (Table 1).
Analyses of Green Tea, Oolong Tea, and Black Tea Leaves.
The MeOH extracts from tea leaf samples were examined using
the SRM LC-MS/MS method (Figure 3). The chromatographic
peaks eluted at the retention times of the authentic standards
showed that relatively large amounts of isovitexin and vitexin
(ng/g of tea leaves) were contained in the MeOH extracts

6782 J. Agric. Food Chem., Vol. 57, No. 15, 2009
Ishida et al.
Table 2. Contents of Chafurosides A and B, Isovitexin, and Vitexin in 100%
MeOH Extracts from Commercial Tea Leavesa
contents (ng/g of tea leaves) of
classification/variety chafuroside A chafuroside B
isovitexin
vitexin
green tea
Shiizu 7132
Yabukita
Sayamakaori
52 ( 4
38 ( 2
34 ( 6
42 ( 7
24 ( 3
24 ( 1
102110 ( 3040 88390 ( 2940
96440 ( 2840 89430 ( 2770
88800 ( 2250 74570 ( 2580
houji tea
Shiizu 7132
Yabukita-1
Yabukita-2
4980 ( 170
1980 ( 150
3140 ( 240
3650 ( 350
1770 ( 100
2590 ( 200
87060 ( 2370 184470 ( 2560
64520 ( 1910 60950 ( 1790
44540 ( 1610 51440 ( 1640
oolong tea
Shiizu 7132
Suisen
Shikisyu
50 ( 1
8580 ( 760
690 ( 70
39 ( 2
7960 ( 360
640 ( 60
76700 ( 2210 72620 ( 2290
32340 ( 970
41750 ( 890
28790 ( 710
40210 ( 790
black tea
Shiizu 7132
Assam
78 ( 2
18 ( 2
53 ( 2
23 ( 2
65580 ( 1490 59890 ( 1350
22580 ( 570
19880 ( 450
Darjeeling
97 ( 9
72 ( 9
20640 ( 440
19000 ( 420
a Results are mean ((SE) (n = 3).
Table 3. Contents of Chafurosides A and B in 100% MeOH Extracts from
Oolong Tea Leaves Prepared from Shizu 7132 after Heating at Various
Temperatures for 40 mina
contents (ng/g of tea leaves) of compound after heating at
compound
140 °C
160 °C
180 °C
200 °C
chafuroside A
chafuroside B
780 ( 16
556 ( 14
8650 ( 750
7260 ( 710
7760 ( 670
7140 ( 650
172 ( 10
169 ( 9
a Results are mean ((SE) (n = 3).
Table 4. Contents of Chafurosides A and B in 100% MeOH Extract of Oolong
Tea Leaves Prepared from Shizu 7132 after Heating at 160 °C
contents of compound (ng/g of tea leaves) after heating for
compound
20 min
40 min
60 min
80 min
chafuroside A 3480 ( 216
chafuroside B 2244 ( 224
7580 ( 750
6950 ( 610
8790 ( 770
7580 ( 640
8870 ( 610
7990 ( 590
Results are mean (SE (n = 3).
Figure 3. Selected reaction monitoring liquid chromatography-tandem
mass spectrometry chromatograms of authentic standards (100 ng/mL)
(A) and 100% MeOH extracts (mg/mL) from green tea and houji tea leaves
from Shizu 7132 (B, C), oolong tea Suisen (D) and Darjeeling black tea
(E) with negative ESI. Precursor-product ion combinations and polarity
used in SRM detection are shown.
(Table 2), as reported previously (14 ). The mean values were
highest in green tea and houji tea and lowest in oolong tea and
black tea. As noted above, the fermentation levels are different
among these teas, and this result indicates that isovitexin and
vitexin are gradually oxidized during oxidative fermentation in
the processing of oolong tea and black tea.
The leaves of green tea, houji tea, and black tea also contained
chafurosides A and B. Contents of these compounds in houji tea
and oolong tea (except oolong tea prepared from Shizu 7132)
were at similar levels, whereas the mean values of green tea and
black tea were also similar, but 100 times lower than those of
houiji tea and oolong tea. These results suggest that putative
precursors of chafurosides remain intact during the preparation
processes of green tea, houji tea, and oolong tea.
Traditionally green tea leaves are prepared as follows. Fresh
young tea leaves are steamed quickly after having been picked
and are then cooled and dried stepwise with rolling by hand on a
hot plate at around 40-80 °C. Black tea leaves are processed by
oxidative fermentation of young tea leaves by means of gradual
Table 1. Recovery of Chafurosides A and B, Isovitexin, and Vitexin from
Spiked H2O-Soluble Fraction of 100% MeOH Extract of Oolong Tea Leaves
from Shizu 7132a
recovery (%) of
concn (ng/g)
chafuroside A
85.3 ( 7.1
chafuroside B
80.4 ( 5.9
112.8 ( 8.5
87.5 ( 7.2
isovitexin
83.6 ( 9.3
98.3 ( 8.1
85.8 ( 3.1
vitexin
5
50
95.5 ( 7.9
113.5 ( 6.9
94.5 ( 9.9
105.2 ( 12.9
90.0 ( 9.9
500
a Results are mean ((SE) (n = 3).

Article
J. Agric. Food Chem., Vol. 57, No. 15, 2009 6783
drying at room temperature for several hours with thorough
rolling or after cutting, followed by rapid drying with hot air
(at around 100 °C). Houji tea leaves are prepared from the green
tea by roasting at 160-200 °C for several minutes. Oolong tea
leaves are produced from fully grown tea leaves by semifermen-
tation under sunlight for a few hours, followed by shaking on
a bamboo tray in a room, and then heated quickly at around
160-260 °C. They are finally dried at around 70-80 °C. Some
species of oolong tea are further subjected to additional strong
firing to intensify the taste and flavor. The common feature in
the preparation of houji and oolong tea leaves is strong firing.
Interestingly, oolong and houji tea leaves that contained large
amounts of chafurosides A and B showed signs of burning.
Thus, heating at high temperature is assumed to be important
for the production of chafurosides.
Table 5. Contents of Chafurosides A and B, Isovitexin, and/or Vitexin in 100%
MeOH, 50% MeOH, and H2O Extracts of Oolong Tea Leaves Prepared from
Shizu 7132 and Their H2O- and n-BuOH-Soluble Fractions Obtained by
Partition of These Extracts with n-BuOH and H2O before and after Heating at
160 °C for 40 mina
contents (ng/g of tea leaves) of compound
Extraction and Fractionation of Chafurosides and Their
Precursors. The contents of chafurosides A and B in MeOH
extract from oolong tea leaves prepared from Shizu 7132
were drastically increased after heating at 160-180 °C (Tables 2
and 3). The contents did not increase substantially when heating
was prolonged over about 40 min (Tables 2 and 4). Thus,
chafurosides are produced by heating oolong tea leaves at
around 160-180 °C for about 40 min.
extraction solvent compound
extract
n-BuOH fraction H2O fraction
Before Heating
100% MeOH
chafuroside A 41 ( 1
chafuroside B 33 ( 1
38 ( 1
30 ( 1
-
-
-
-
isovitexin
vitexin
92870 ( 2760 82760 ( 2550
79480 ( 2450 73450 ( 2640
Chafurosides A and B, isovitexin, and vitexin were detected in
MeOH extracts and their n-BuOH-soluble fractions, but not in
the H2O-soluble fractions (Table 5). The contents of chafuro-
sides A and B in all of the extracts and their H2O-soluble
fractions were drastically increased to similar levels after heating
at 160 °C for 40 min. Interestingly, the contents of chafurosides
in n-BuOH-soluble fraction were similar before and after heat-
ing. These results indicate that the precursors of chafurosides A
and B are not isovitexin and vitexin, but are more hydrophilic.
Isolation and Structure Determination of Chafuroside A and B
Precursors, Prechafurosides A and B, from Oolong Tea Leaves
from Shizu 7132. Isolation of Prechafurosides A and B. Isolation
of prechafurosides A (1) and B (2), in fraction 2 from MeOH
extract of oolong tea leaves prepared from Shizu 7132 was
achieved in four steps of column chromatography (Diaion SP
825, Sephadex LH-20, ODS-C18, and HPLC), as shown in
Chart 1. Fraction 1 contains other precursors of both chafuro-
sides. Isolation of these compounds is in progress.
50% MeOH
H2O
chafuroside A 50 ( 1
chafuroside B 39 ( 2
47 ( 1
35 ( 2
-
-
chafuroside A 52 ( 1
chafuroside B 45 ( 1
49 ( 1
41 ( 1
-
-
After Heating
100% MeOH
chafuroside A 7660 ( 440
chafuroside B 6950 ( 410
35 ( 1
29 ( 1
6580 ( 437
5850 ( 480
isovitexin
vitexin
85480 ( 2450 75880 ( 2390
77090 ( 2250 69660 ( 1940
-
-
50% MeOH
H2O
chafuroside A 12550 ( 770
chafuroside B 14290 ( 660
42 ( 1
33 ( 1
10180 ( 740
11580 ( 780
chafuroside A 13820 ( 780
chafuroside B 14850 ( 710
47 ( 1
41 ( 1
11270 ( 660
12540 ( 700
a Results are mean ( SE (n=3). -, not detected.
Chart 1. Isolation of Prechafuroside A (1) and Prechafuroside B (2) from Oolong Tea Leaves from Shizu 7132, Camellia sinensis (L.) O. Kuntze

6784 J. Agric. Food Chem., Vol. 57, No. 15, 2009
Ishida et al.
Figure 4. Preparation of (a) chafuroside A and isovitexin from isovitexin 200-sulfate (1) and (b) chafuroside B and vitexin from vitexin 200-sulfate (2).
Structure Determination of Prechafurosides A and B. The
precursors of chafurosides A and B, 1 and 2, have not yet been
crystallized. The IR spectra of compounds 1 and 2 suggested
the presence of hydroxyl, conjugated carboxyl, and sulfate
ester functions in both molecules. The UV characteristics of
1 and 2 are similar to those of isovitexin and vitexin, respecti-
vely (15 ).
As illustrated in Figure 4, compounds 1 and 2 quantitatively
afforded isovitexin and vitexin, respectively, on solvolysis
with pyridine-dioxane. Compounds 1 and 2 gave chafuroside
A and chafuroside B, respectively, in good yields when heated
at 160 °C. The 1H and 13C NMR data of 1, isovitexin, 2, and
vitexin are shown in Table 6. There were no significant
differences in the chemical shifts of the corresponding
carbons and protons, except for those of the C-200 carbons
and H-200 protons in the glucose moieties, between 1 and
isovitexin and between 2 and vitexin. Increases of the chemical
shifts of the C-200 carbons and protons of 1 and 2 by 5.1 and
0.86 ppm and by 5.7 and 0.80 ppm compared with those of
isovitexin and vitexin, respectively, indicated that the hydro-
xyl groups at C-200 of the two former compounds were
sulfated, on the basis of similar findings in heparin deriva-
tives (16 ).
On the basis of these data and other NMR (2D-COSY, C-H
shift COSY, and HMBC) spectra of prechafurosides A and B,
prechafuroside A was concluded to be isovitexin 200-O-sulfate
and prechafuroside B to be vitexin 200-O-sulfate.
The proposed structures were well supported by negative ion
Q-TOF (MS/MS) experiments (complete product scan) carried
out on the [M - H]- ion (m/z 511) of 1 and 2 (Figure 5). Bond
cleavage between C-200 and O-200-SO3H was evidenced by ion
formation of m/z 96.9548 from 1 and of m/z 96.9601 (calcd. for
HSO4, m/z 96.9596) from 2. Other prominent ions, m/z 431.1041
from 1 and m/z 431.0969 (calcd. for C21H29O10, m/z 431.0978)
from 2, were generated by characteristic bond cleavage between
O-200 and SO3H.

Article
J. Agric. Food Chem., Vol. 57, No. 15, 2009 6785
Table 6. 1H and 13C NMR Data for Prechafuroside A, Isovitexin, Prechafuroside B, and Vitexin in DMSO-d6
a
isovitexin
δH(mult, J in Hz)
prechafuroside A
δH(mult, J in Hz)
vitexin
δH(mult, J)
prechafuroside B
δH(mult, J in Hz)
no.
δC
δC
δC
δC
1
2
163.3
102.8
182.0
156.2
108.9
163.5
93.6
163.4
102.8
182.0
156.3
108.8
163.3
94.0
162.4
102.4
182.0
155.9
98.1
162.7
102.9
182.7
155.8
98.4
3
6.78 (s)
6.50 (s)
6.77 (s)
6.44 (s)
6.52 (s)
6.20 (s)
6.72 (s)
6.19 (s)
4
5
6
7
163.9
104.0
160.3
104.5
164.3
103.3
161.0
104.5
8
8a
4a
161.2
103.4
161.1
103.6
1’
2’
30
4’
50
6’
121.1
128.5
116.0
160.7
116.0
128.5
121.2
128.9
116.0
160.0
116.0
128.9
121.5
128.8
115.7
161.0
115.7
128.8
121.8
128.9
115.5
161.4
115.5
128.9
7.93 (d, 8.5)
6.91 (d, 8.5)
7.90 (d, 8.5)
6.90 (d, 8.5)
8.01 (d, 8.5)
6.89 (d, 8.8)
8.00 (d, 8.5)
6.86 (d, 8.5)
6.91 (d, 8.5)
7.93 (d, 8.5)
6.90 (d, 8.5)
7.90 (d, 8.5)
6.89 (d, 8.8)
8.01 (d, 8.5)
6.86 (d, 8.5)
8.00 (d, 8.5)
100
200
300
73.0
70.6
78.9
4.57 (d, 10.0)
70.6
75.7
78.3
4.64 (d, 9.5)
73.3
70.8
78.6
4.68 (d, 10.0)
71.1
76.5
78.4
4.74 (d, 10.0)
4.03 (dd, 10.0, 9.0)
3.14 (dd, 9.2, 9.0)
4.54 (dd, 9.5, 9.0)
3.39 (dd, 9.2, 9.0)
3.84 (dd, 10.0, 9.0)
3.28 (dd, 9.2, 9.0)
4.64 (dd, 10.0, 9.0)
3.49 (dd, 9.2, 9.0 )
400
70.2
81.6
61.5
3.12 (dd, 9.2, 9.0)
70.3
81.0
61.5
3.15 (dd, 9.2, 9.0)
70.5
81.7
61.3
3.39 (dd, 9.2, 9.0)
71.0
81.6
61.5
3.45 (dd, 9.2, 9.0)
500
3.10 (ddd, 9.0, 5.0, 2.5)
3.67 (dd, 12.0, 2.5)
3.38 (dd, 12.0, 5.0)
3.11 (ddd, 9.0, 5.0, 2.5)
3.70 (dd, 11.5, 2.5)
3.41 (dd, 11.5, 5.0)
3.24 (ddd, 9.0, 5.1, 2.5)
3.76 (dd, 12.2, 2.5)
3.54 (dd, 12.2, 5.1)
3.29 (ddd, 9.0, 5.1, 2.5)
3.74 (dd, 11.0, 2.5)
3.54 (dd, 11.0, 5.1)
600a
600b
a Assignments were based on 1H-1H-COSY, 1H-13C-COSY, and HMBC experiments.
Figure 5. Negative MS/MS spectra of (A, left) isovitexin 200-sulfate (1) and (B, right) vitexin 200-sulfate (2).
These data allowed us to assign the structures of prechafuro-
sides A and B as isovitexin 200-sulfate and vitexin 200-sulfate,
respectively.
1 and 2 may similarly involve an SN2 mechanism via a transition
state in which the anion formed by deprotonation of O-7 attacks
C-200 (Figure 6).
Formation Mechanism of Chafurosides A and B from Precha-
furosides A and B. It is reported that a sulfonyl group and an
adjacent free hydroxyl group in trans configuration on a sugar
unit in a polysaccharide undergo desulfation and subsequent
cyclization to give a relatively stable intermediate containing an
oxirane ring between C-2 and C-3, when a slightly alkaline
solution of heparin is simply freeze-dried (17, 18). This alkali-
catalyzed 2-O-desulfation and simultaneous etheric bond for-
mation between C-2 and C-3 are considered to involve an SN2
mechanism via a transition state in which an anion formed by
deprotonation of O-3 attacks C-2. Formation of chafurosides A
and B by heating under neutral conditions from their precursors
To our knowledge, this is the first report of the isolation of
flavone C-glycoside sulfate from oolong tea leaves.
In conclusion, procedures for quantitative determination of
chafurosides A and B, isovitexin, and/or vitexin by liquid
chromatography-tandem mass spectrometry were developed
and successfully applied to leaves of green tea, houji tea, oolong
tea, and black tea. Isovitexin and vitexin were detected in all tea
leaves, although only trace levels were found in one sample of
oolong tea leaves. We established an isolation procedure for
precursors of chafurosides A and B in oolong tea leaves from
Shizu 7132 and identified them as flavone C-glycoside sulfates,
isovitexin 200-sulfate (1) and vitexin 200-sulfate (2), respectively,

6786 J. Agric. Food Chem., Vol. 57, No. 15, 2009
Ishida et al.
Figure 6. Proposed formation mechanisms of etheric bond in isovitexin 200-sulfate (1) and vitexin 200-sulfate (2).
on the basis of chemical and physicochemical data. Study on the
formation mechanism of prechafurosides A and B in the oolong
tea leaves is ongoing.
Oolong tea rich in chafurosides may ameliorate atopic der-
matis, and our quantitation methods could be useful for
monitoring chafuroside content during the processing of tea
leaves, as well as for identifying tea species and other plants rich
in chafuroside precursors.
(9) Niho, N.; Mutoh, M.; Sakano, K.; Takahashi, M.; Hirano, S.;
Nukaya, H.; Sugimura, T.; Wakabayashi, K. Inhibition of intestinal
carcinogenesis by a new flavone derivative, chafuroside, in oolong
tea. Cancer Sci. 2006, 97, 248–251.
(10) Yadav, P. P.; Ahmed, G.; Maruya, R. Furanoflavonoids from
Pongamia pinnata fruits. Phytochemistry 2004, 65, 439–443.
(11) Maurya, R.; Yadav, P. P. Furanoflavonoids: an overview. Nat. Prod.
Rep. 2005, 22, 400–424.
(12) Furuta, T.; Kimura, T.; Kondo, S.; Mihara, H.; Wakimoto, T.;
Nukaya, H.; Tsuji, K.; Tanaka, K. Concise total synthesis of flavone
C-glycoside having potent anti-inflammatory activity. Tetrahedron
2004, 42, 9375.
(13) Nakatsuka, T.; Tomimori, Y.; Fukuda, Y.; Nukaya, H. First total
synthesis of structurally unique flavonoids and their strong anti-
inflammatory effect.. Bioorg. Med. Chem. Lett. 2004, 14, 3201.
(14) Engelhardt, U. H.; Finger, A.; Kuhr, S. Determination of flavone
C-glycosides in tea. Z. Lebensm. Unters. Forsch. 1993, 197,
239–244.
(15) Manki; Tomimori, T.; Ito, M. Constituents of Swertia japonica. I.
Structures of swertisin and isoswertisin. Chem. Pharm.Bull. 1967, 15,
263–269.
(16) Yates, E. A.; Santini, F.; Cristofano, D. B.; Payre, N.; Cosentino, C.;
Guerrini, M.; Naggi, A.; Torri, G.; Hricovini, M. Effect of substitu-
tion pattern on 1H, 13C NMR chemical shifts and 1JCH coupling
constants in heparin derivatives. Carbohydr. Res. 2000, 329,
239–247.
(17) Jaseja, M.; Rej, R. N.; Sauriol, F.; Perlin, A. S. Novel regio- and
stereoselective modifications of heparin in alkaline solution. Nuclear
magnetic resonance spectroscopic evidence. Can. J. Chem. 1989, 67,
1449–1456.
(18) Ishihara, M.; Kariya, Y.; Kikuchi, H.; Minamisawa, T.; Yoshida, K.
Importance of 2-O-sulfate groups of uronate residues in heparin
for activation of FGF-1 and FGF-2. J. Biochem. 1997, 121,
345–349.
LITERATURE CITED
(1) Kuroda, Y.; Hara, Y. Antimutagenic and anticarcinogenic activity
of tea polyphenols. Mutat. Res. 1999, 436, 69–97.
(2) Uehara, M.; Sugiura, H.; Sakurai, K. A trial of oolong tea in the
management of recalcitrant atopic dermatitis. Arch. Dermatol. 2001,
137, 143–148.
(3) Kuehl, F. A.; Humes, J. L.; Egan, R. W.; Ham, E. A.; Beveridge, G.
C.; Van Arman, C. G. Role of prostaglandin endoperoxide
PGG2 in inflammatory processes. Nature (London) 1977, 265,
170–173.
(4) Nagle, D. G.; Ferreira, D.; Zhou, Y.-D. Epigallocatechin-3-gallate
(EGCG): chemical and biomedical perspectives. Phytochemistry
2006, 67, 1849–1855.
(5) Higdon, J. V.; Frei, B. Tea catechins and polyphenols: health effects,
metabolism, and antioxidant functions. Crit. Rev. Food Sci. Nutr.
2003, 43, 89–143.
(6) Zaveri, N. T. Green tea and its polyphenolic catechins: medicinal
uses in cancer and noncancer applications. Life Sci. 2006, 78,
2073–2080.
(7) Nakai, M.; Fukui, Y.; Asami, S.; Toyoda-Ono, Y.; Iwashita, T.;
Shibata, H.; Mitsunaga, T.; Hashimoto, F.; Kiso, Y. Inhibitory
effects of oolong tea polyphenols on pancreatic lipase in vitro. J.
Agric. Food Chem. 2005, 53, 4593–4598.
(8) Hashimoto, F.; Nonaka, G.; Nishioka, I. Tannins and related
compounds. XC. 8-C-ascorbyl (-)-epigallocatechin 3-O-gallate
and novel dimeric flavan-3-ols, oolonghomobisflavans A and B,
from oolong tea. (3). Chem. Pharm. Bull. 1989, 37, 3255–3263.
Received January 5, 2009. Revised manuscript received March 19, 2009.
Accepted March 20, 2009.