Determination of theaflavins including methylated theaflavins in black tea leaves by solid-phase extraction and HPLC analysis

Article abstract of DOI:10.1021/jf070312m

A quantitative method for four theaflavins and two methylated theaflavin derivatives in black tea leaves was developed by solid-phase extraction and a high-performance liquid chromatographic method with photodiode array detection. The theaflavins in black tea leaves were extracted three times with 40 vol 50% aqueous ethanol (mg dry tea powder/mL) containing 2% ascorbic acid. The ethanol extracts were diluted 4-fold with distilled water. All diluted extracts were directly applied to the solid-phase C₁₈ cartridge column without concentration. The fraction of theaflavins was obtained by 40% ethanol extraction after rinsing with water followed with 15% ethanol extraction. An aliquot of theaflavins after concentration was injected onto an ODS C ₁₈ reversed-phase column, and four theaflavins and two methylated theaflavins were sufficiently separated by a linear gradient system using distilled water and acetonitrile with 0.5% acetic acid. This analytical method is sensitive for the determination of a small amount of methylated theaflavins, since various interfering substances produced during the fermentation process were eliminated in advance by solid-phase extraction. Using this analytical method, we also demonstrated that methylated theaflavins were easily produced during the manufacture of black tea.

Full text of DOI:10.1021/jf070312m

7252 J. Agric. Food Chem. 2007, 55, 7252−7257
Determination of Theaflavins Including Methylated Theaflavins
in Black Tea Leaves by Solid-Phase Extraction and HPLC
Analysis
MASAMI NISHIMURA,† KINUKO ISHIYAMA,† AKIKO WATANABE,†
SETSUKO KAWANO,† TOSHIO MIYASE,‡ AND MITSUAKI SANO*,†
Division of Food Science & Nutrition, Nagoya Women’s University, 3-40 Shioji-Cho Mizuho-ku,
Nagoya 467-8610, Japan, and School of Pharmaceutical Sciences, University of Shizuoka,
52-1 Yada, Shizuoka 422-8526, Japan
A quantitative method for four theaflavins and two methylated theaflavin derivatives in black tea leaves
was developed by solid-phase extraction and a high-performance liquid chromatographic method
with photodiode array detection. The theaflavins in black tea leaves were extracted three times with
40 vol 50% aqueous ethanol (mg dry tea powder/mL) containing 2% ascorbic acid. The ethanol extracts
were diluted 4-fold with distilled water. All diluted extracts were directly applied to the solid-phase
C18 cartridge column without concentration. The fraction of theaflavins was obtained by 40% ethanol
extraction after rinsing with water followed with 15% ethanol extraction. An aliquot of theaflavins
after concentration was injected onto an ODS C18 reversed-phase column, and four theaflavins and
two methylated theaflavins were sufficiently separated by a linear gradient system using distilled
water and acetonitrile with 0.5% acetic acid. This analytical method is sensitive for the determination
of a small amount of methylated theaflavins, since various interfering substances produced during
the fermentation process were eliminated in advance by solid-phase extraction. Using this analytical
method, we also demonstrated that methylated theaflavins were easily produced during the
manufacture of black tea.
KEYWORDS: Black tea; methylated theaflavin; solid-phase extraction; HPLC analysis
INTRODUCTION
generated by fermentation. The biological activities of TFs,
which are dimerization products of catechins, have been reported
concerning antioxidant (11-14), antibacterial (15), antimu-
tagenic (16), and hypocholesterolemic functions (17). The
existence of methylated TFs in tea leaves and their biological
activity remain unknown because an appropriate analytical
method for methylated TFs has not been established. Although
analytical methods for TFs using high-performance liquid
chromatography (HPLC) techniques combined with UV detec-
tion (18) or electrospray mass spectrometry (19, 20) have
been reported, many of these methods are to determine TFs
in an aqueous tea infusion. An amount of TFs and various
oxidized breakdown products of catechins are still present in
the used tea leaves after extraction with boiling water several
times. The presence of oxidized products interferes with the
determination of a small amount of methylated TFs in black
tea leaves.
(-)-Epigallocatechin gallate (EGCG), which is the major
catechin in tea leaves (Camellia sinensis L.), has been reported
to exert various biological effects such as antioxidative (1, 2),
antimutagenic/anticarcinogenic (3, 4) and antibacterial (5). We
have reported that 3-O- or 4-O-methylated derivatives of the
galloyl moiety in EGCG exhibit strong anti-allergic properties
(6-8) and antioxidant activities (9). One of the methylated
catechins, eipgallocatehin-3-O-(3-O-methyl)gallate (EGCG3′′Me),
is concentrated in limited tea cultivars such as “Benihomare”
and “Benifuuki”, which are classified as “Assam” hybrids, but
the concentrations are much lower than that of EGCG (10).
Therefore, EGCG3′′Me was also detected in fresh leaves of
“Assam” cultivars used for the manufacture of black tea. Tea
catechins are degraded markedly during the black tea manu-
facturing process (fermentation), and theaflavins (TFs) and
high molecular weight products such as thearubigins are
In this study, we report a specific determination method for
TFs and methylated TFs in black tea leaves by solid-phase
extraction, followed by HPLC analysis. We also investigated
the conversion of EGCG or EGCG3′′Me to the corresponding
TFs or methylated TFs by using green tea, oolong tea, and black
tea manufactured from the same lot of leaves.
* Address for correspondence: Prof. Mitsuaki Sano, Division of Food
Science & Nutrition, Nagoya Women’s University, 3-40 Shioji-Cho Mizuho-
ku, Nagoya, 467-8610, Japan. Phone, +81-528529412; fax, +81-528527470;
e-mail, sano@nagoya-wu.ac.jp.
† Nagoya Women’s University.
‡ University of Shizuoka.
10.1021/jf070312m CCC: $37.00
© 2007 American Chemical Society
Published on Web 08/15/2007

Determination Method of Methylated Theaflavins in Black Tea
J. Agric. Food Chem., Vol. 55, No. 18, 2007 7253
Figure 1. Chemical structures of theaflavins and methylated theaflavins.
MATERIALS AND METHODS
phase were purchased from Wako Pure Chemical Co., Ltd. (Osaka,
Japan). Water was purified in a Milli-Q system (Millipore, Bedford,
MA). Other chemicals were of reagent grade.
Reagents and Samples. Commercial black tea leaves (Assam,
Ceylon, Darjeeling, and Earl Grey), manufactured by Lipton or
Twinings, were purchased from markets in Japan. Green tea, oolong
tea, and black tea manufactured from the same lot leaves of “Beni-
homare”, which contains a relatively high level of EGCG3′′Me, were
obtained from the National Research Institute of Vegetable and Tea
Science (Kanaya, Shizuoka, Japan). Authentic samples of theaflavin
(TF), theaflavin 3-O-gallate (TF3G), theaflavin 3′-O-gallate (TF3′G),
and theaflavin 3,3′-di-O-gallate (TF3,3′diG) at 94∼99% purity were
provided by Lipton Unilever Japan (Tokyo, Japan).
EGCG3′′Me, EGCG, and the other tea catechins were prepared
according to the method previously reported (6). Two methylated TFs,
theaflavin-3-O-(3-O-methyl)gallate (TF3MeG) and theaflavin-3-O-(3-
O-methyl)gallate-3′gallate (TF3MediG), were synthesized by the reac-
tion of EGCG3′′Me and catechol-type catechins such as EC and ECG
in the presence of cocklebur polyphenol oxidase according to the
method described by Tanaka et al. (21). Their purities (>91%) were
confirmed by 1H NMR. The two methylated TFs were used as reference
standard for calibration and identification purposes. The chemical
structures of catechins and TFs used in this study are shown in Figure
1. Ethanol, acetonitrile, and acetic acid for extraction or HPLC mobile
Apparatus. A C18 Bond Elut solid-phase cartridge column (Varian
Inc. Co., Harbor City, CA) set in vacuum manifold apparatus (GL
Science, Tokyo, Japan) and high-speed shaker (CM-1000, Eyela, Tokyo,
Japan) were used for TFs extraction and fractionation. The solid-phase
cartridge column was previously rinsed with 4 mL of ethanol, then 5
mL of water for activation just before use. TFs were analyzed using a
Shimazu LC-MS system (Shimazu, Kyoto, Japan) consisting of two
pumps (LC-20AD), an automated sample injector (SIL-20AC), a
column oven (Shimadzu, CTO-20AC), a system controller (CBM-20A),
a photodiode array detection (SPD-M20A), and mass detection
(MS2010EV) with a computer (LC-MS solution software). The HPLC
separation column was a Shimazu ODS 80Ts column (2.0 × 250 mm
i.d., Shimazu, Kyoto, Japan).
Analytical HPLC Method. The optimal mobile phase for the
separation of four TFs (TF, TF3G, TF3′G, and TF3,3′diG) and two
methylated TFs (TF3MeG and TF3MediG) was a gradient system
consisting of solvent A (15% acetonitrile/water containing 0.5% acetic
acid) and solvent B (80% acetonitrile/water containing 0.5% acetic
acid). These TFs were eluted from the column at 25 °C with a linear

7254 J. Agric. Food Chem., Vol. 55, No. 18, 2007
Nishimura et al.
-
Figure 2. HPLC chromatogram of authentic theaflavins and methylated theaflavins. A mixture of authentic theaflavins (0.5 nmol
µ
L
1) was used for
HPLC-UV analysis. Peaks are 1, TF (theaflavin); 2, TF3G; 3, TF3,3′diG; 4, TF3′G; 5, TF3MeG; 6, TF3MediG.
Figure 3. Chromatogram of Ceylon black tea extracts and mass spectra of peak a and peak b in ESI-negative mode. Peaks are 1, TF; 2, TF3G; 3,
TF3,3 diG; 4, TF3 G; a, TF3MeG; b, TF3MediG.
′
′
gradient of solvent B starting from 0% to 60% in 80 min at a flow rate
of 0.2 mL/min. Each peak of TFs and methylated TFs was monitored
by a photodiode array UV detector at 280 nm. The TF peak was
confirmed by the retention times of authentic samples and mass
spectrometric analysis with an electrospray interface (ESI). The
molecular weight of TFs was analyzed in negative and positive
ionization modes.
RESULTS AND DISCUSSION
Preparation of Tea Extracts for HPLC Analysis. Black
tea leaves were pulverized into powder for good extraction. TFs
were extracted from 50 mg of tea leaf powder three times with
2 mL of 50% ethanol solution containing 2% ascorbic acid (w/
v) at room temperature by shaking at 1500 rpm for 20 min.
Almost all theaflavins in tea powder were extracted under the
extraction conditions shown in Table 1. The three extractions
with 50% aqueous ethanol showed good efficiency, ranging from
92% to 105%. The three extracts were combined and centrifuged
at 5000 rpm at 4 °C for 10 min. The supernatant was diluted
4-fold with distilled water, and then, the diluted extracts were
applied to the solid-phase C18 cartridge column set in a vacuum
manifold apparatus. The column was rinsed with 6 mL of water,
followed with 6 mL of 15% ethanol solution. These two rinses
eliminated polar compounds and ascorbic acid added as an
antioxidant to the ethanol extraction. Almost all theaflavins were
LC-MS conditions are as follows: ODS80Ts column, 2.0 × 250
mm Shimadzu; flow rate, 0.2 mL/min; injection volume, 10 µL; column
temperature, 25 °C; probe voltage, +4.5 V (ESI-negative mode);
nebulizing gas flow, 1.5 L/min; drying gas pressure, 0.1 MPa; CDL
temperature, 250 °C; CDL voltage, -1.5 V; Q-array voltage, DC +
5.0 V, RF + 150 V; SIM, 563 (TF), 715 (TF3G, TF3′G), 729
(TF3MeG), 867 (TF3,3′diG), 881 (TF3MediG) (negative).
The analysis of EGCG3′′Me and major catechins (EGCG, EGC, EC,
and ECG) was carried out by the method of HPLC with electrochemical
detection as previously reported (11).
Statistical analysis was performed by the Student’s t test, with
significance at P < 0.05.

Determination Method of Methylated Theaflavins in Black Tea
J. Agric. Food Chem., Vol. 55, No. 18, 2007 7255
Table 1. Extraction of Theaflavins and Methylated Theaflavins from
Black Tea Leavesa
theaflavins (mg/g dry tea leaves)
extraction
TF
TF3G
TF3
′
G
TF3,3
′
diG
TF3MeG
TF3MediG
1st
2nd
3rd
4th
2.17
0.40
0.05
0.002
3.04
0.58
0.07
0.003
3.80
0.69
0.08
0.00
1.74
0.36
0.05
0.00
0.19
0.03
0.01
0.003
0.54
0.09
0.01
0.00
a Theaflavins in black tea powder (50 mg) were extracted with 2 mL of 50%
aqueous ethanol containing 2% ascorbic acid at room temperature for 20 min
using a shaker at 1500 rpm.
Table 2. Separation of Theaflavins from Solid-Phase C18 Cartridge
Column by Ethanol Eluatea
separation (mg/g dry tea leaves)
15%
EtOH
40%
EtOH
99.5%
EtOH
theaflavins
H2O
TF
TF3G
TF3,3
TF3
TF3MeG
nd
nd
nd
nd
nd
nd
0.03
0.02
0.02
0.01
0.01
0.01
2.06
3.01
3.89
1.79
0.20
0.60
nd
nd
0.01
nd
nd
nd
′
G
diG
′
TF3MediG
a nd, not detected.
eluted with 6 mL of 40% ethanol solution (Table 2). The eluate
was evaporated to dryness under vacuum. An aliquot (10 µL)
of the residue, redissolved with 500 µL of 50% ethanol, was
used for HPLC analysis.
HPLC Separation and Detection of TFs and Methylated
TFs. The authentic theaflavin mixtures consisting of four TFs
(TF, TF3G, TF3′G, and TF3,3′diG) and two methylated TFs
(TF3MeG and TF3MediG) were sufficiently separated under
the conditions described in the analytical HPLC method section
(Figure 2). The detection limits (S/N ) 3) of these theaflavins
were 20-25 pmol µL-1. Figure 3 shows typical HPLC
chromatograms of extracts from Ceylon black tea. The peak
assignments of the two methylated TFs were based on retention
times relative to authentic samples and were confirmed by using
the sensitive technique of selective ion monitoring of mass
spectrometry. The results suggested that TF3MeG and TF3MediG
were also produced by the enzymatic oxidative reaction between
EGCG3′′Me and catechol-type catechins (EC or ECG) during
the fermentation process. Linear calibration curves for standard
TFs and methylated TFs were obtained in the 2.5-10 nmol
range for the determination of theaflavins in tea leaves (Figure
4). This method could determine a small amount of TFs and
methylated TFs present in tea leaves by ethanol extraction and
solid-phase extraction treatment.
Figure 4. Calibration curves of standard TFs and methylated TFs Ten
microliters of standard TFs (a) and methylated TFs (b) was injected into
HPLC with photdiode array detection.
The levels of all TFs in Darjeeling teas were lower, whereas
TF3,3′diG in Assam teas was much higher than in the other tea
cultivars used. These results suggest that in Darjeeling tea the
degree of oxidation in the fermentation process is lower or the
contents of catechins is lower, and in Assam green tea leaves,
the contents of EGCG and ECG, which are materials of
TF3,3′diG, are higher than those of other tea cultivars (22).
Methylated TFs were recognized in all black tea leaves used.
The levels in Ceylon and Assam teas were higher than those of
Darjeeling and Earl Grey tea. The contents of TF3MeG and
TF3MediG were 130∼ 200 µg/g dry tea leaf in Ceylon and
Assam black tea and 440∼540 µg/g in Ceylon tea, respectively.
Theaflavin Concentrations of Black Tea Leaves. Theafla-
vins in various black tea leaves obtained from markets were
examined by the proposed HPLC method (Table 3).
Table 3. Theaflavin Concentrations in Various Black Tea Leaves Obtained from Markets
(mg/g dry tea leaves)
diG TF3
TF
TF3G
TF3,3
′
′
G
TF3MeG
TF3MediG
Assam
Earl Grey
Ceylon
1.90
1.86
2.26
1.24
±
0.30a
0.70a,b
0.20a
0.05b
3.59
±
±
±
±
0.40a
11.31
±
±
±
±
0.39a
0.43b
1.70b,c
0.17c
2.27
1.25
1.43
0.63
±
±
±
±
0.16a
0.48b
0.26b
0.05c
0.15
±
±
±
±
0.02a
0.37
0.38
0.35
0.13
±
±
±
±
0.03a
±
±
±
2.78
2.54
0.97
0.84a
1.13a
0.07b
3.79
3.02
1.54
0.17
0.16
0.05
0.09a
0.06a
0.01b
0.26a,b
0.20a,b
0.01b
Darjeelin
a-
c Values are mean
± SD (n ) 3−5 samples). Values with the same superscript notation are not significantly different (P > 0.05).

7256 J. Agric. Food Chem., Vol. 55, No. 18, 2007
Nishimura et al.
Figure 5. Quantitative changes of catechins during the fermentation process. Tea leaves used were prepared from the same lot of fresh leaves of
“Benihomare” cultivars. (a) The tea (”Sen-cha”) was prepared by steaming treatment which inactivates polyphenol oxidase in first step of green tea
production. (b) The tea (“Kamairi-cha”, panning green tea) was prepared by parching treatment instead of steaming. (c and d) These teas were prepared
by the fermentation for 20 min (c) and 60 min (d) during the process of black tea production, respectively.
Figure 6. Quantitative changes of theaflavins during the fermentation process. Tea leaves used were prepared from the same lot of fresh leaves of
“Benihomare” cultivars. (a) The tea (”Sen-cha”) was prepared by steaming treatment which inactivates polyphenol oxidase in first step of green tea
production. (b) The tea (“Kamairi-cha”, panning green tea) was prepared by parching treatment instead of steaming. (c and d) These tea were prepared
by the fermentation for 20 min (c) and 60 min (d) during the process of black tea production, respectively.
Quantitative Changes of Catechins and Theaflavins dur-
ing the Fermentation Process. TF3G, TF3,3′diG, TF, and
TF3′G are produced by oxidative reaction between EC and
EGCG, between ECG and EGCG, between EC and EGC, and
between ECG and EGC, respectively (23, 24). In addition, this
study suggests that TF3MeG and TF3MediG are also produced
from the reaction between EGCG3′′Me and catechins of the
catechol type on the B ring. The content of catechins and TFs
in tea leaves varies considerably among tea species and
cultivation areas; therefore, tea leaves (green tea, oolong tea,
and black tea) from the same lot of leaves of “Benihomare”
cultivars were used in this examination to assess quantitative
changes of tea catechins during the fermentation process.
All TFs, including methylated TFs in tea leaves, increased
with the decrease of catechins following the development of
fermentation (Figures 5 and 6). A marked change in catechin
and theaflavin levels was observed in the manufacturing process
of oolong tea to black tea. The quantity of TF3MediG produced
from EGCG3′′Me and ECG was higher than that of TF3MeG
from EGCG3′′Me and EC. The production ratio of TF3MediG
to TF3,3′diG in black tea leaves was about 25%, but the ratio
of EGCG3′′Me to EGCG in green tea was about 14%. This

Determination Method of Methylated Theaflavins in Black Tea
J. Agric. Food Chem., Vol. 55, No. 18, 2007 7257
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ACKNOWLEDGMENT
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