Stability of tea polyphenol (-)-epigallocatechin-3-gallate and formation of dimers and epimers under common experimental conditions

Article abstract of DOI:10.1021/jf0519055

(-)-Epigallocatechin-3-gallate (EGCG), the most abundant and biologically active compound in tea, has been extensively studied for its activities related to disease prevention in animal models and in vitro. However, its stability under different experimental conditions has not been well-characterized. In the present study, the stability of EGCG in animal drinking fluid and under cell culture conditions and the factors that affect its stability under these conditions were investigated. Our results demonstrated that auto-oxidation and epimerization are the two major reactions causing the instability of EGCG. The structures of the major oxidation products, EGCG dimers, were identified. The rates of these reactions were affected by the temperature, pH, the partial pressure of oxygen, the level of antioxidants, the concentration of EGCG, and other components of tea. In future studies with EGCG, its stability should be considered in order to avoid possible artifacts.

Full text of DOI:10.1021/jf0519055

9478 J. Agric. Food Chem. 2005, 53, 9478−9484
Stability of Tea Polyphenol (−)-Epigallocatechin-3-gallate and
Formation of Dimers and Epimers under Common Experimental
Conditions
†
†
†
‡
SHENGMIN SANG, MAO-JUNG LEE, ZHE HOU, CHI-TANG HO, AND
,
†
CHUNG S. YANG*
Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers University,
64 Frelinghuysen Road, Piscataway, New Jersey 08854-8020, and Department of Food Science,
Rutgers University, 65 Dudley Road, New Brunswick, New Jersey 08901-8520
1
(-)-Epigallocatechin-3-gallate (EGCG), the most abundant and biologically active compound in tea,
has been extensively studied for its activities related to disease prevention in animal models and in
vitro. However, its stability under different experimental conditions has not been well-characterized.
In the present study, the stability of EGCG in animal drinking fluid and under cell culture conditions
and the factors that affect its stability under these conditions were investigated. Our results
demonstrated that auto-oxidation and epimerization are the two major reactions causing the instability
of EGCG. The structures of the major oxidation products, EGCG dimers, were identified. The rates
of these reactions were affected by the temperature, pH, the partial pressure of oxygen, the level of
antioxidants, the concentration of EGCG, and other components of tea. In future studies with EGCG,
its stability should be considered in order to avoid possible artifacts.
KEYWORDS: Stability; EGCG; dimers; GCG; auto-oxidation; epimerization; cell culture; tea
INTRODUCTION
Tea, made from the leaves of Camellia sinensis, is one of
the most widely consumed beverages in the world. The three
major types of tea, green, oolong, and black, differ in their
manufacturing process and chemical compositions. Many studies
in humans, animal models, and cell lines suggest potential health
benefits from the consumption of tea, including the preven-
tion of cancer and heart disease (1-3). Many of the health
benefits have been attributed to the polyphenolic components
in tea. (-)-Epigallocatechin-3-gallate (EGCG) is the most
abundant and biologically active constituent in tea (Figure 1).
Green and oolong teas typically contain 30-130 mg of EGCG
per cup (237 mL), whereas black tea may contain up to 70 mg
of EGCG per cup (4).
Green tea, black tea, and EGCG, when administered to
animals in drinking fluid, have shown cancer preventive activity
against ultraviolet light-induced, chemically induced, and genetic
models of carcinogenesis (1-3). For example, we demonstrated
that orally administered green tea inhibited chemically induced
lung tumorigenesis in mice, esophageal carcinogenesis in rats,
and oral carcinogenesis in hamsters (5-7). Chung et al. (8)
showed that 2% green tea inhibited 4-(methylnitrosamino)-1-
(
∼
3-pyridyl)-1-butanone-induced lung tumor multiplicity by
45%. The corresponding amount of EGCG inhibited lung
Figure 1. Structure of EGCG and its epimerization and oxidation products.
*
To whom correspondence should be addressed. Tel: 732-445-3400
ext. 244. Fax: 732-445-0687. E-mail: csyang@rci.rutgers.edu.
tumor multiplicity by 28%. Intestinal tumorigenesis in the ApcMin
mice was shown by Orner et al. (9) to be inhibited by orally
†
Department of Chemical Biology.
Department of Food Science.
‡
1
0.1021/jf0519055 CCC: $30.25
© 2005 American Chemical Society
Published on Web 11/05/2005

Stability of (−)-Epigallocatechin-3-gallate
J. Agric. Food Chem., Vol. 53, No. 24, 2005 9479
administered green tea, and recently, such an activity was also
demonstrated in our laboratory with EGCG administered in
drinking fluid (10). The stability of EGCG and other tea
polyphenols in the drinking fluid under some of these experi-
mental conditions was not well-characterized.
maintained at 100% B for 10 min and then was reequilibrated to 4%
at 53 min for another run.
Liquid Chromatography/Mass Spectrometry (LC/MS) and NMR
Analyses. LC/MS analysis was carried out with a Finnigan Spectra
System, which consisted of a Finnigan model P4000 pump, a model
AS3000 refrigerated autosampler, and a Finnigan LCQ Deca mass
detector (ThermoFinigan, San Jose, CA) incorporated with an electro-
spray ionization (ESI) interface. A 75 mm × 2.1 mm i.d., 3 µm
Discovery HS C18 column (Supelco) was used for separation with a
flow rate of 0.2 mL/min. The column elution started with a 6 min
isocratic phase of 100% solvent A (5% aqueous methanol with 0.2%
acetic acid), followed by progressive, linear increases in B (95%
aqueous methanol with 0.2% acetic acid) to 20% at 15 min, 50% at 25
min, 55% at 30 min, and 100% at 36 min. The mobile phase was
maintained at 100% B for 8 min and then was reequilibrated to 100%
A at 45 min. The LC elute was introduced into the ESI interface. The
negative ion polarity mode was set for ESI ion source with the voltage
on the ESI interface maintained at approximately -4 kV. Nitrogen gas
was used as the sheath gas at a flow rate of 80 arb, and the auxiliary
gas was set at 2 arb, respectively. The heated capillary temperature
and voltage were maintained at 260 °C and -24 V, respectively. The
tube lens offset voltage was -55 V. The structural information of the
standard (-)-gallocatechin-3-gallate (GCG) and theasinensin A were
obtained by tandem mass spectrometry (MS/MS) through collision-
induced dissociation with a relative collision energy setting of 25%.
The mechanisms of the chemopreventive activity have been
studied extensively; however, they are not clearly understood.
Most of the mechanisms have been proposed based on the
studies in vitro. In cell lines, tea polyphenols, especially EGCG,
have been shown to cause growth inhibition and apoptosis in
many human tumor cell lines (11-14). Some of these activities
are probably due to the inhibition of specific protein kinases
leading to cell cycle arrest or blocking of the activation of
transcription factors, such as activator protein-1 and nuclear
factor-κB (15-20). The mechanisms of the action of EGCG in
cell culture systems, however, are complicated by the fact that
it is not stable under most cell culture conditions. We have
shown previously that EGCG was unstable in several cell culture
media, such as McCoy’s 5A, HBSS, and Ham’s H-12 (21). In
McCoy’s 5A culture media, the half-life of EGCG was less than
3
0 min, and the half-life increased to 130 min in the presence
of HT-29 human colon adenocarcinoma cells (21).
To fully understand the biological effects of EGCG and other
tea polyphenols, a careful study on their stability under common
experimental conditions is of importance. This information,
however, is lacking, even though the stability of tea catechins
in tea drinks under either direct brewing or industrial canning
processes as well as in bread production and storage after adding
tea catechins to bread has been studied (22-27). In the present
study, the stability of EGCG in drinking fluids used in most
animal experiments and under cell culture conditions as well
as the factors that affect its stability were investigated.
1
13
H (600 MHz), C (150 MHz), and all 2D NMR spectra were acquired
on a Varian Unity INOVA 600 NMR spectrometer (Palo Alto, CA)
equipped with a z-gradient inverse-detection triple resonance probe.
Isolation of GCG as a Product of EGCG in Phosphate Buffer.
EGCG (0.2 g) was incubated in 100 mL of phosphate buffer (50 mM
Na HPO , pH 7.4) at 37 °C for 2 days. After extraction with ethyl
2 4
acetate and evaporating the solvent in vacuo, the residue was applied
to a Sephadex LH-20 column and eluted with 35% aqueous acetone to
obtain GCG (30 mg) and EGCG (80 mg). GCG: 1H NMR (CD
OD,
600 MHz): δ 5.05 (1H, brd, J ) 4.8 Hz, H-2), 5.37 (1H, m, H-3),
.76 (1H, dd, J ) 4.8, 16.4 Hz, H-4a), 2.71 (1H, dd, J ) 5.4, 16.4 Hz,
H-4b), 5.96 (2H, s, H-6 and H-8), 6.40 (2H, s, H-2′ and H-6′), 6.97
2H, s, H-2′′ and H-6′′). 13C NMR (CD
OD, 150 MHz): δ 79.2 (d,
3
2
MATERIALS AND METHODS
(
3
Materials. Decaffeinated green tea extracts (GTE) (dehydrated water
extract of green tea; 1 g of powder derived from 6 g of dry leaves)
were obtained from Unilever Best Foods Inc. (Englewood Cliffs, NJ).
EGCG (100% pure) was provided by Mitsui Norin Co. Ltd. (Shizuoka,
Japan). Theasinensin A was synthesized previously in our laboratory
C-2), 71.0 (d, C-3), 23.7 (t, C-4), 157.6 (s, C-5), 95.6 (d, C-6), 158.0
(s, C-7), 96.4 (d, C-8), 156.3 (s, C-9), 99.5 (s, C-10), 131.0 (s, C-1′),
106.3 (d, C-2′ and C-6′), 146.9 (s, C-3′ and C-5′), 133.9 (s, C-4′), 121.4
(s, C-1′′), 110.2 (d, C-2′′ and C-6′′), 146.3 (s, C-3′′ and C-5′′), 139.8
(s, C-4′′), 167.7 (s, C-7′′).
1
13
(
(
28). The structure was confirmed by H, C, and two-dimensional
2D) NMR. Superoxide dismutase (SOD), ascorbic acid, CD OD, RP
Stability of EGCG or GTE under Different Conditions. Different
concentrations of EGCG (or GTE) were prepared and incubated under
different conditions {in Nanopure water at room temperature or 37 °C
or in 50 mM phosphate buffer (pH 7.4) in the absence or presence of
nitrogen (the buffer solution was flushed with nitrogen for 24 h in a
37 °C water bath before adding EGCG) or EDTA or in cell culture
medium [Ham’s and RPMI-1640 (50:50), pH 7.4] in the absence or
presence of SOD (5 units/mL) or as powder for long-term storage}.
Then at different time points, 20 or 100 µL samples were taken and
transferred to vials containing 380 µL or 9.9 mL of a solution containing
0.2% ascorbic acid and 0.05% EDTA to stabilize EGCG. These samples
were immediately analyzed or stored at -80 °C before analyzing by
HPLC. The quantification of EGCG and theasinensin A was based on
the standard curve of each compound.
3
C-18 silica gel, and Sephadex LH-20 gel were purchased from Sigma
St. Louis, MO). Thin-layer chromatography (TLC) was performed on
50 µm thickness, 2-25 µm particle size TLC plates (Sigma-Aldrich),
with compounds visualized by spraying with 5% (v/v) H SO in ethanol
(
2
2
4
solution. High-performance liquid chromatography (HPLC) grade
solvents and other agents were obtained from VWR Scientific (South
Plainfield, NJ). HPLC grade water (Nanopure water (18 MΩ) was
prepared using a Millipore Milli-Q purification system (Bedford, MA).
RPMI 1640 and Ham’s F12 medium were purchased from Mediatech
Inc. (Herndon, VA).
HPLC Analysis. EGCG levels were analyzed using an HPLC
system, consisting of a Waters 717 refrigerated autosampler, a Hitachi
L-6200A intelligent pump, and an ESA 5600 coulochem electrode array
system (CEAS). The potentials of the CEAS were set at -100, 200,
RESULTS AND DISCUSSION
400, and 600 mV. Separation was achieved using previously described
methods (29). In brief, a 150 mm × 4.6 mm i.d. Supelcosil C18
reversed-phase column (Supelco Co., Bellefonte, PA) was used. The
column and CEAS detector were housed in a temperature-regulated
compartment maintained at 25 °C. The autosampler was maintained at
Stability of EGCG and Other Tea Polyphenols in Drink-
ing Fluid. To study the stability of EGCG and other tea
polyphenols, we used three different concentrations of EGCG
(0.025, 0.1, and 0.32%) and GTE (0.05, 0.2, and 0.6%), which
6
°C. For binary gradient elution, mobile phases A (1.75% acetonitrile
and 0.12% tetrahydrofuran in 30 mM NaH PO , pH 3.35) and B (58.5%
acetonitrile and 12.5% tetrahydrofuran in 15 mM NaH PO , pH 3.45)
covered the range of concentrations used in most animal studies.
EGCG was rather stable in the 0.32% (6.99 mM) solution; no
significant change in EGCG concentration was observed in the
2
4
2
4
were used. The flow rate was maintained at 1 mL/min, and the mobile
phase began with a 5 min isocratic phase of 96% A and 4% B. It was
followed by progressive, linear increases in B to 15% at 15 min, 22%
at 25 min, 80% at 32 min, and 100% at 42 min. The mobile phase was
7
day experiment (Figure 2). The 0.1 (2.06 mM) and 0.025%
(0.51 mM) solutions were less stable, showing a gradual
decrease of EGCG concentration in the first 2 or 3 days and a

9480 J. Agric. Food Chem., Vol. 53, No. 24, 2005
Sang et al.
Figure 2. Stability of EGCG and GTE at room temperature. Three different concentrations of EGCG (0.025, 0.1, and 0.32%) and GTE (0.05, 0.2, and
.6%) were prepared in Nanopure water and kept in animal feeding bottles at room temperature.
0
Figure 3. Factors affecting the stability of 20
M EGCG after addition to 50 mM Na HPO (pH 7.4) buffer flushed with
nitrogen for 24 h at 37 C ( ). Stability of 20 M EGCG in HAM’s
F12:RPMI 1640 (1:1) medium (pH 7.4) at 37 C in the presence of SOD
5 U/mL) ( ). Stability of 20 M EGCG in water at 23 C in the presence
of 14 mM Na EDTA ( ). Stability of 20 M EGCG in water at 23 C in
the absence of 14 mM Na EDTA ( ). Stability of 20 M EGCG in 50
mM Na HPO (pH 7.4) buffer without saturated nitrogen at 37 C ( ).
Stability of 20 M EGCG in HAM’s F12:RPMI 1640 (1:1) medium (pH
.4) at 37 C in the absence of SOD ( ).
µM EGCG. Stability of 20
µ
2
4
°
b
µ
°
(
O
µ
°
2
9
µ
°
2
0
µ
2
4
°
2
µ
Figure 4. Time-dependent conversion of EGCG to dimers. HPLC spectra
of EGCG (20 M) samples after they were incubated in Ham’s F12 and
RPMI-1640 (50:50) culture medium (pH 7.4) at 37 C in the absence of
7
°
4
µ
°
maximal decrease of about 20% during the 7 day studies. In
.6% GTE solution [containing 1.57 mM EGCG, 1.33 mM
nitrogen at different time points (0, 30, 60, and 90 min).
0
(
(
-)-epigallocatechin (EGC), 0.77 mM (-)-epicatechin-3-gallate
ECG), and 0.56 mM (-)-epicatechin (EC)], EGCG was rather
appeared in the HPLC spectra (Figure 4). Two of them were
confirmed by LC/MS/MS to be the dimers of EGCG. Figure 5
shows the respective LC/MS/MS chromatograms and frag-
ment ion mass spectra of the standard theasinensin A (EGCG
dimer) and the corresponding dimer peak formed in cell culture
medium from 20 µM EGCG at 30 min. Theasinensin A and
the major dimer peak found in the cell culture medium exhibited
the same chromatographic retention times and molecular masses
and also had the same fragment ion mass spectra (Figure 5),
confirming the formation of theasinensin A. Figure 6 shows
the LC/MS/MS chromatograms and fragment ion mass spectra
of the other major dimer peak formed at 30 min. This dimer
peak showed a molecular ion mass of 883 (ESI negative,
stable with no change or a few percent decrease in concentration
in 7 days, and other catechins were also rather stable. In 0.2
and 0.05% GTE solutions, up to a 20% decrease of EGCG
concentration was observed after 3 or 4 days. In general, EGC
had a similar stability to EGCG at all three concentrations of
GTE, whereas ECG and EC were more stable (Figure 2). It is
interesting to note that in 0.05% GTE, the concentration of
EGCG and EGC dropped rapidly during days 4-7, and the
reason for this phenomenon is unknown.
Stability of EGCG in Cell Culture Medium. EGCG was
unstable in cell culture medium. When added to cell culture
medium [Ham’s F12 and RPMI-1640 (50:50), which was the
medium that we used to culture KYSE 150 and 510 human
esophageal squamous cell carcinoma cells], the EGCG concen-
tration decreased rapidly, with a half-life of about 30 min
-
[M - H] ), which is the same as that of P2, one of the major
auto-oxidation products of EGCG in mild alkaline fluids (30).
The major mass fragment ions of 883 were the ions that lost
fragments corresponding to one gallic acid group (m/z 713, [M
-
(Figure 3). After 6 h of incubation, EGCG was not detectable
- H - 170] ) and two gallic acid groups (m/z 543, [M - H -
-
in the medium. During the incubation, three major new peaks
170 × 2] ) (Figure 6), respectively. On the basis of the

Stability of (−)-Epigallocatechin-3-gallate
J. Agric. Food Chem., Vol. 53, No. 24, 2005 9481
Figure 5. Identification of theasinensin A by LC/MS/MS. ESI negative LC/MS/MS spectra of standard theasinensin A and related peaks in Ham’s F12
and RPMI-1640 (50:50) culture medium (20 M EGCG at 30 min). (A) LC and MS/MS spectra of standard theasinensin A (MW 914). (B) LC and MS/MS
µ
spectra of the peak found in buffer solution after EGCG was added at 30 min.
Figure 6. Identification of P2 by LC/MS/MS. ESI negative LC/MS/MS spectra of the major EGCG dimer peak corresponding to MW 884 in Ham’s F12
and RPMI-1640 (50:50) culture medium (20
µM EGCG at 30 min).
molecular ion mass and the major mass fragments of this peak,
we tentatively identified this peak as P2. The presence of minor
peaks (one in each of Figures 5 and 6), which had the same
molecular ion and mass fragments as that of theasinensin A or
P2, but with different retention times, suggests that they are
epimers of theasinensin A or P2. All of the dimers were unstable
in the cell culture medium. Their peak height reached a
maximum at 30-60 min and then decreased to an undetectable
level at 6 h.
LC/MS/MS (Figure 7). Corresponding to the ESI negative
molecular ion 457 [M - H] , two peaks showed up in the LC
-
spectrum of EGCG samples in the presence of SOD at 24 h.
The major peak was identified as EGCG by comparison with
the standard EGCG. The other peak showed almost the same
retention time and molecular masses and also had the same
fragment ion mass spectra as those of standard GCG. With the
inhibition of auto-oxidation by SOD, epimerization of EGCG
apparently became the predominant reaction.
Factors Affecting the Stability of EGCG. As shown in
Figure 3, the addition of SOD dramatically increased the
stability of EGCG in cell culture medium. The effect of SOD
suggests that the instability of EGCG is due to superoxide-
mediated auto-oxidation of EGCG. The dimer peaks (Figure
We found that the pH of Ham’s F12 and RPMI-1640
(50:50) culture medium changed from 7.40 to 8.34 during the
incubation with EGCG for 6 h. To maintain a constant pH, we
used a phosphate buffer (50 mM Na2HPO4, pH 7.4). EGCG
was slightly more stable in this phosphate buffer than in cell
culture medium (Figure 3). The major oxidation products were
the same as those formed in cell culture medium. To study the
effect of O2 on the stability of EGCG, the phosphate buffer (50
4
) were not detectable in the presence of SOD; instead, a new
peak appeared and increased with time. This peak was identified
as GCG (an epimer of EGCG) by using selected ion monitoring

9482 J. Agric. Food Chem., Vol. 53, No. 24, 2005
Sang et al.
Figure 7. Identification of GCG by LC/MS/MS. ESI negative LC/MS/MS spectra of standard GCG and related peak in cell culture medium (20
EGCG, with SOD at 24 h). (A) LC and MS/MS spectra of standard GCG (MW 458). (B) LC and MS/MS spectra of the peak found in cell culture medium
20 M EGCG with SOD at 24 h).
µM
(
µ
mM Na2HPO4, pH 7.4) was flushed extensively with N2 gas.
Under low O2 partial pressure, EGCG was greatly stabilized
with 95% remaining after 6 h. No dimers were detected, and
GCG was formed (Figure 3). An aqueous solution of EGCG
(20 µM) showing a pH of 5.4 was not very stable at room
temperature (23 °C). The stability was significantly increased
by the presence of EDTA (14 mM), suggesting the involvement
of metal in catalyzing the auto-oxidation of EGCG (Figure 3).
Concentration-Dependent Stability of Low Concentration
of EGCG and Theasinensin A. The stability of EGCG in water
at 37 °C was concentration-dependent, and the t1/2 increased
from 30 min in a 20 µM solution to 150 min in a 96 µM solution
(Figure 8). The EGCG in GTE was even more stable than the
pure 96 µM EGCG solution, even though the GTE solution
contained only 20 µM EGCG (48 µM in terms of total
catechins). The formation of theasinensin A was also concentra-
tion-dependent. The higher amounts of theasinensin A formed
from higher concentrations of EGCG also apparently had longer
half-lives (Figure 8).
EGCG was unstable in cell culture medium or in pH 7.4
buffer at low concentrations (20-100 µM), and EGCG dimers
were the major products under these conditions (Figures 3 and
Figure 8. Concentration-dependent stability of a lower concentration of
EGCG and theasinensin A. Stability of EGCG (A) and formation of
theasinensin A (B) at different concentrations of EGCG (20, 48, 72, and
4
). Incubating a higher concentration EGCG (0.2%, 4.12 mM)
in phosphate buffer (pH 7.4), however, produced GCG but not
the dimers. Corresponding to the decrease of EGCG, the amount
of GCG increased, reaching a maximum at day 2 (at 37 °C) or
day 5 (at 23 °C). At day 7 (at 37 °C) or day 10 (at 23 °C), both
EGCG and GCG were not detectable. During the incubation,
the color of the solutions became brown, with increasing
absorbance in the 400-800 nm range during days 4-7 (at 37
96
µ
M) and in GTEs (GTE contains 20
C.
µM EGCG and around 48 µM
total tea catechins) in water at 37
°
formed, and the factors that affect the stability. Our results
(Figure 2) indicated that in animal studies, the catechins were
rather stable at higher concentrations (0.32% EGCG or 0.6%
GTE). At lower concentrations (0.1 and 0.025% EGCG or 0.2
and 0.05% GTE), up to a 20% decrease in EGCG concentration
could occur in 2-3 days. In general, the stability of EGC was
about the same as EGCG, whereas ECG and EC were more
stable, possibly due to the oxidation of the trihydroxy structure
on the B-ring of EGC and EGCG. We also observed that EGCG
and other tea polyphenols were less stable if the solution was
made in tap water, possibly due to the presence of metal ions.
°
C) or 4-10 (at 23 °C). Apparently, larger molecular weight
compounds were formed. These colored compounds appeared
to be similar to the pinkish-brown material formed upon storage
of EGCG. The white-colored pure EGCG usually turns to
pinkish-brown upon long-term storage. The colored materials
had an absorbance between 400 and 800 nm, but HPLC and
1
H NMR analysis did not reveal any new compounds besides
the predominant EGCG and a trace amount GCG.
The purpose of this study was to determine the stability of
EGCG under common experimental conditions, the products
Our previous study in cell culture systems indicated that
EGCG underwent similar oxidation in the presence or absence

Stability of (−)-Epigallocatechin-3-gallate
J. Agric. Food Chem., Vol. 53, No. 24, 2005 9483
of cells, and cells could only slightly increase the stability of
EGCG (21). To simplify the experimental system, we conducted
the present study in the absence of cells. Under these conditions
(5) Liao, J.; Yang, G. Y.; Park, E. S.; Meng, X.; Sun, Y.; Jia, D.;
Seril, D. N.; Yang, C. S. Inhibition of lung carcinogenesis and
effects on angiogenesis and apoptosis in A/J mice by oral
administration of green tea. Nutr. Cancer 2004, 48, 44-53.
(pH 7.4, 37 °C), EGCG (20 µM) underwent auto-oxidation, with
(
6) Wang, Z. Y.; Wang, L. D.; Lee, M. J.; Ho, C. T.; Huang, M. T.;
Conney, A. H.; Yang, C. S. Inhibition of N-nitrosomethylben-
zylamine-induced esophageal tumorigenesis in rats by green and
black tea. Carcinogenesis 1995, 16, 2143-2148.
a half-life of less than 30 min; EGCG dimers and other products
were formed. Several factors, including pH, temperature, oxygen
levels, antioxidant levels, metal ions, concentration of EGCG,
and other ingredients in tea, could affect the stability of EGCG
(7) Li, N.; Chen, X.; Liao, J.; Yang, G.; Wang, S.; Josephson, Y.;
(Figures 3 and 8). The presence of SOD or nitrogen stabilized
Han, C.; Chen, J.; Huang, M. T.; Yang, C. S. Inhibition of
EGCG and increased its half-life to longer than 6 h. On the
basis of these observations, metal-catalyzed auto-oxidation of
EGCG is possible.
7
,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcino-
genesis in hamsters by tea and curcumin. Carcinogenesis 2002,
23, 1307-1313.
We observed that once the auto-oxidation of EGCG was
prevented by SOD or nitrogen, epimerization of EGCG to GCG
became appreciable. GCG was also the major product formed
from EGCG at high concentrations [0.2% (4.12 mM) solution
or higher concentration], whereas EGCG dimers were formed
mainly with low concentrations of EGCG (such as 20-100 µM).
In aged EGCG powder, GCG was also found. All of these results
suggest that there are two major reactions involved in the
instability of EGCG; one is auto-oxidation, and the other is
epimerization. The rates of these two reactions are affected by
the level of oxygen, the concentration of EGCG, and the level
of antioxidants.
(8) Chung, F. L.; Schwartz, J.; Herzog, C. R.; Yang, Y. M. Tea and
cancer prevention: studies in animals and humans. J. Nutr. 2003,
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(9) Orner, G. A.; Dashwood, W. M.; Blum, C. A.; Diaz, G. D.; Li,
Q.; Dashwood, R. H. Suppression of tumorigenesis in the Apc-
(
min) mouse: Down-regulation of beta-catenin signaling by a
combination of tea plus sulindac. Carcinogenesis 2003, 24, 263-
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2
(
10) Ju, J.; Hong, J.; Zhou, J.; Pan, Z.; Bose, M.; Liao, J.; Yang, G.;
Liu, Y. Y.; Hou, Z.; Lin, Y.; Ma, J.; Shih, W. J.; Carothers, A.
M.; Yang, C. S. Inhibition of intestinal tumorigenesis in
Apcmin/+ mice by (-)-epigallocatechin-3-gallate, the major
catechin in green tea. Cancer Res. 2005, in press.
As a pro-oxidant, EGCG can be oxidized to form phenolic
radicals, superoxide radical, and hydrogen peroxide. These
species may trigger a variety of biochemical reactions and
biological responses. For example, hydrogen peroxide may
contribute to cell apoptosis (11, 12), and the radical species may
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