Stability of black tea polyphenol, theaflavin, and identification of theanaphthoquinone as its major radical reaction product

Article abstract of DOI:10.1021/jf050662d

In the current study, we have focused on isolation and detection of major radical oxidation products from theaflavin in order to better understand antioxidation mechanisms of this compound. Theanaphthoquinone was identified as a major oxidation product of theaflavin from two different oxidant model systems: DPPH and peroxidase/hydrogen peroxide. This result indicated that the benzotropolone moiety in theaflavin may play an important role in its antioxidant properties. The stability of theaflavin was studied in varying pH solutions: simulated gastric juice and buffer solutions of pH 5.5, pH 7.4, and pH 8.5. The results indicated that theaflavin is unstable in alkaline conditions, while it was stable in acidic conditions. Theanaphthoquinone was identified as an autoxidation product of theaflavin during its stability study in alkaline conditions.

Full text of DOI:10.1021/jf050662d

6146 J. Agric. Food Chem. 2005, 53, 6146−6150
Stability of Black Tea Polyphenol, Theaflavin, and Identification
of Theanaphthoquinone as Its Major Radical Reaction Product
JIN-WOO JHOO,† CHIH-YU LO,† SHIMING LI,† SHENGMIN SANG,†
CATHARINA Y. W. ANG,‡ THOMAS M. HEINZE,‡ AND CHI-TANG HO*,†
Department of Food Science, Rutgers University, 65 Dudley Road,
New Brunswick, New Jersey, 08901, and Division of Chemistry, National Center for Toxicological
Research, U.S. Food and Drug Administration, HFT-230, 3900 NCTR Road,
Jefferson, Arkansas 72079
In the current study, we have focused on isolation and detection of major radical oxidation products
from theaflavin in order to better understand antioxidation mechanisms of this compound. Theanaph-
thoquinone was identified as a major oxidation product of theaflavin from two different oxidant model
systems: DPPH and peroxidase/hydrogen peroxide. This result indicated that the benzotropolone
moiety in theaflavin may play an important role in its antioxidant properties. The stability of theaflavin
was studied in varying pH solutions: simulated gastric juice and buffer solutions of pH 5.5, pH 7.4,
and pH 8.5. The results indicated that theaflavin is unstable in alkaline conditions, while it was stable
in acidic conditions. Theanaphthoquinone was identified as an autoxidation product of theaflavin during
its stability study in alkaline conditions.
KEYWORDS: Theaflavin; theanaphthoquinone; oxidation; radical oxidation; pH stability
INTRODUCTION
that black tea has a similar peroxyl radical scavenging capacity
to green tea (3). Trolox equivalent antioxidant capacity values
of green tea and black tea are comparable (4). Black tea extracts
have been shown to exhibit protection of human red blood cells
against oxidative damage from lipid peroxidation (5). Sarkar
and Bhaduri (6) reported that black tea extracts are more
effective than green tea catechins in scavenging superoxide
anions.
As a popular beverage, tea (Camellia sinensis) has attracted
public attention because of accumulating scientific evidence
linking tea consumption with health benefits (1). Fresh tea leaves
contain four major tea catechins with unique biological activities;
however, black tea is manufactured through fermentation of the
tea leaves. During the fermentation process, important chemical
changes occur due to the action of polyphenol oxidase (PPO).
PPO is responsible for oxidizing the dihydroxylated B ring
(catechol) and trihydroxylated B ring (pyrogallol) of tea
catechins to their o-quinones. Subsequent chemical reactions
generate the various characteristic black tea polyphenolic
pigments that are not found in unprocessed tea leaves. Four
major theaflavins have been identified from black tea, namely,
theaflavin, theaflavin-3-gallate, theaflavin-3′-gallate, and theafla-
vin-3,3′-digallate (2).
Tea consumption may provide health benefits, and these
biological activities are believed to arise from the antioxidant
activity of polyphenolic compounds in tea, specifically their
ability to effectively scavenge reactive oxygen species (1). A
number of studies have demonstrated the antioxidant capacity
of black tea and black tea polyphenols using in vitro and in
vivo methods. Many in vitro studies have shown that black tea
has a comparable antioxidant capacity to green tea. Researchers
using the oxygen-radical absorbance capacity assay have shown
Several studies have reported a proposed mechanism for the
reaction of tea catechins with radicals and have proposed
mechanisms for the antioxidant properties of these compounds
(7, 8). When tea catechins were reacted with peroxyl radical
generated by thermolysis of the radical initiator 2,2′-azobis-
(2,4-dimethylvaleronitrile), the resulting seven-membered B ring
anhydride and symmetrical dimer were identified as the oxida-
tion products of EGCG and (-)-epigallocatechin (EGC) (8).
These results indicated that the principal sites for radical reaction
are on the B ring rather than the galloyl moiety. Although
different oxidation model systems, such as 2,2-diphenyl-1-
picrylhydrazyl (DPPH), gave different oxidation products, Sang
et al. have demonstrated that the catechin B ring is the preferred
site for oxidation (9). Theaflavins have been regarded as
biologically important active components in black tea. Little
information is available concerning a mechanism for the
antioxidative action of black tea polyphenols. Because the
antioxidant mechanisms of theaflavins are still unclear, we
sought to identify the oxidation products from theaflavin in order
to better understand these mechanisms. Identification of the
oxidation products of theaflavins can provide useful information
to understand the mechanism giving rise to the antioxidant
* To whom correspondence should be addressed. Tel: 732-932-9611
ext. 235. Fax: 732-932-6776. E-mail: ho@aesop.rutgers.edu.
† Rutgers University.
‡ U.S. Food and Drug Administration.
10.1021/jf050662d CCC: $30.25
© 2005 American Chemical Society
Published on Web 07/01/2005

Radical Reaction of Theaflavin
J. Agric. Food Chem., Vol. 53, No. 15, 2005 6147
PPO was prepared from commercial banana fruit. Fresh banana flesh
(400 g) was homogenized with 800 mL of cold 100 mM potassium
phosphate buffer (pH 7.0, 4 °C). The homogeneous solution was
centrifuged at 4 °C for 20 min (10000g), and clear supernatant was
collected. The same volume of cold acetone (-40 °C) was slowly
poured into the collected solution with stirring. Then, precipitated
proteins were collected by centrifugation (10000g, 20 min, 4 °C). The
resulting pellets were washed with the same buffer three times and
dissolved in the same buffer. The protein solution was lyophilized; the
resulting powder was used as a crude PPO.
EC (1 g) and EGC (1 g) were dissolved in 200 mL of phosphate-
citrate buffer (50 mM, pH 5.0) along with 2 g of crude PPO enzyme.
The enzymatic oxidation was carried out at room temperature for 6 h
with stirring. The reaction solution was then subjected to partition three
times with the same volume of ethyl acetate. The organic layer was
concentrated under reduced pressure. The resulting residues were
subjected to Sephadex LH-20 and RP-18 column chromatography
successively to afford a reddish theaflavin (yield, 15.2%).
Theaflavin Oxidation Model Using DPPH Oxidant System.
Theaflavin (720 mg) was dissolved in 30 mL of acetone, and the radical
oxidation was initiated by adding DPPH (1000 mg) into this solution.
The reaction was carried out for 20 h with stirring in the dark at room
temperature. The reaction solution was dried under reduced pressure.
The resulting residue was subjected directly to Sephadex LH-20 column
chromatography eluting with 95% ethanol. Six fractions were collected
(each 200 mL), and fraction 3 was subjected to LH-20 (95% ethanol)
and RP-18 (50% aqueous methanol) successively for further purification
and yielded a deep reddish amorphous solid, theanaphthoquinone (24
mg). Further purification of fraction 5 afforded unreacted theaflavin
(155 mg).
Theaflavin Oxidation Model Using Peroxidase/Hydrogen Per-
oxide Oxidant System. Theaflavin (500 mg) was dissolved in 30 mL
of 20% aqueous acetone along with 1 mg of peroxidase. One milliliter
of 3.11% hydrogen peroxide solution was added to the reaction solution
every 40 min. The reaction was carried out at room temperature for
3.5 h with stirring. Then, the reaction solution was dried under reduced
pressure. The resulting residues were subjected to Sephadex LH-20
column chromatography eluting with 95% ethanol. Among the collected
seven fractions (each 180 mL), fraction 3 was subjected to LH-20 (95%
Figure 1. Chemical structure of compound 1.
properties of these molecules. Also, limited information is
available about the stability of theaflavins in various pH
solutions. In this study, we focused on examination of the
stability of theaflavin in different pH conditions.
MATERIALS AND METHODS
Materials and General Procedures. Epicatechin (EC), DPPH, and
horseradish peroxidase were purchased from Sigma (St. Louis, MO).
All high-performance liquid chromatography (HPLC) grade solvents
were from Fisher Scientific (Fair Lawn, NJ). Tea catechin, EGC, was
isolated from commercial green tea polyphenol extract using an LH-
20 column chromatography. Thin-layer chromatography (TLC) was
performed on Sigma-Aldrich silica gel TLC plates (250 µm thickness,
2-25 µm particle size), and the spots were detected by UV illumination
1
and spraying with 5% (v/v) H2SO4 in an ethanol solution. H NMR
spectra were obtained on a Varian 600 instrument (Varian Inc., Palo
Alto, CA). The compound was analyzed in acetone-d6 with tetrameth-
ylsilane as an internal standard. Atmospheric pressure chemical
ionization (APCI)-MS were recorded on a Micromass Platform II
system (Micromass Co., Beverly, MA) equipped with a Digital DECPC
XL 560 computer for data analysis.
Theaflavin Preparation. Theaflavin was prepared by a modified
enzymatic oxidation method from Tanaka et al. (10). In brief, crude
Figure 2. Proposed mechanism of theanaphthoquinone formation in radical reaction.

6148 J. Agric. Food Chem., Vol. 53, No. 15, 2005
Jhoo et al.
compound formed from the catechin B ring, it was necessary
to identify the principal site of oxidation that gives rise to the
antioxidant properties of these compounds. Therefore, we carried
out a theaflavin oxidation experiment using two different
oxidation model systems. The DPPH is a stable radical, which
is widely used for determination of the antioxidant activity of
test compounds. The DPPH radical forms a reduced DPPH
paired with a hydrogen from the test compounds. Heme-
containing peroxidases reduce hydrogen peroxide to water, while
oxidizing various substrates. The horseradish peroxidase/
hydrogen peroxide system has been used to generate substrate-
derived radicals that can undergo further reactions.
Theaflavin was subjected to oxidation using the above two
model systems, and successive purification steps using LH-20
and RP-18 column chromatography afforded a compound (1)
Figure 3. Theaflavin degradation in different pH solutions. Data were
expressed as means
± SD (n ) 3).
1
from both oxidation systems. After comparison with the H
NMR spectrum of theaflavin and those of isolated compound 1
along with published spectral data (11), it was found that the
chemical shift of the characteristic three proton signals in the
benzotropolone moiety of compound 1 was different from those
of theaflavin. Specifically, the chemical shifts of three aromatic
protons in the benzotropolone moiety of theaflavin were δH 8.04
(1H, s), 8.01 (1H, s), and 7.58 (1H, s); however, those of
compound 1 were δH 7.40 (1H, s), 7.28 (1H, s), and 6.74 (1H,
ethanol) and RP-18 (50% aqueous methanol) column chromatography
to afford theanaphthoquinone (35 mg). Fractions 5 and 6 were combined
and subjected to RP-18 column chromatography eluting with a gradient
of 40-50% of aqueous methanol to yield unreacted theaflavin
(123 mg).
Theanaphthoquinone. Deep red amorphous powder. APCI-MS [M
1
+ H]+ at m/z 535. H NMR (600 MHz in acetone-d6): δH 7.40 (1H,
s), 7.28 (1H, s), 6.74 (1H, s), 6.01 (1H, d, J ) 2.4 Hz), 6.01 (1H, d, J
) 2.4 Hz), 5.96 (1H, d, J ) 2.4 Hz), 5.96 (1H, d, J ) 2.4 Hz), 5.35
(1H, br s), 5.11 (1H, br s), 4.40 (1H, d, J ) 3.6), 4.37 (1H, br s),
2.68-2.93 (4H, m).
1
s). When we compared the H NMR spectrum of compound 1
with those of theanaphthoquinone, which was reported by
Tanaka et al. (12), we concluded that the spectral characters of
this compound were consistent with those of theanaphtho-
quinone (Figure 1). Moreover, positive ion APCI-MS ([M +
H]+ at m/z 535) data of compound 1 strongly supported this
conclusion. This compound has been reported as an oxidation
product of theaflavin by Tanaka et al. (10). They reported that
this compound is both an enzymatically and a nonenzymatically
generated from theaflavin. As shown in Figure 2, we propose
a radical oxidation mechanism of theaflavin and theanaphtho-
quinone formation through one-electron oxidation. From these
results, it is probable that the benzotropolone moiety of
theaflavin plays an important role in affording antioxidant
protection for the preferred oxidation site. Jovanovic et al. (13)
reported that although theaflavin radicals have a higher reduction
potential than the tea catechin EGCG, theaflavins have signifi-
cantly higher reaction rates with superoxide radicals than EGCG.
These authors proposed that the benzotropolone moiety might
be responsible for electron donation because of the existence
of resonance forms. As part of our continuing chemical study
of the antioxidant mechanisms of theaflavins in our laboratory,
A ring fission products of theaflavin 3,3′-digallate have been
reported using the hydrogen peroxide oxidant system (14). This
result indicated that the preferred oxidation site of theaflavin
3,3′-digallate is placed in the A ring of flavan-3-ol rather than
the benzotropolone moiety. Although the experiment was carried
out with a different oxidant system, this observation suggests
that gallated theaflavins may have a different antioxidation
mechanism as compared to nongallated theaflavins. Interestingly,
Jovanovic et al. (13) reported that gallated theaflavins have a
lower superoxide scavenging activity than nongallated theafla-
vin. They proposed that the gallate moiety may prevent reaction
between the radicals and the benzotropolone moiety in theafla-
vins. It might be that further chemical studies are needed to
understand the antioxidation mechanism of monogallated theafla-
vin.
Theaflavin Stability in Various pH Solutions. The stability of
theaflavin (4 mg in 16 mL) was examined in different pH solutions,
such as simulated gastric juice (0.2% sodium chloride, 0.24% hydro-
chloric acid), pH 5.5 sodium acetate buffer (60 mM), pH 7.4 phosphate
buffer (60 mM), and pH 8.5 phosphate buffer (60 mM). Aliquots (1
mL) of the sample were collected and analyzed at various time intervals
using HPLC to examine the degradation rate of theaflavin at ambient
temperature. The HPLC system was fitted with a Zorbax ODS HPLC
column (5 µm, 4.6 mm × 250 mm, RP-18) and equipped with an
autosampler (Waters, 717) and UV detector (Varian, 2050) at a
wavelength of 375 nm. Theaflavin analysis was performed with a linear
increasing gradient from 10 to 60% acetonitrile in water with constant
0.1% acetic acid in 15 min. The flow rate was 1.0 mL/min. The
degradation rate of theaflavin was measured based on decreasing
theaflavin peak area.
To identify oxidation products of theaflavin in alkaline conditions,
an aliquot of sample in pH 8.5 was partitioned with ethyl acetate, and
the organic part was dried under reduced pressure. The sample was
dissolved in water and analyzed with LC/electrospray ionization (ESI).
The analysis was performed on the Finnigan TSG 7000 mass
spectrometer equipped with an HP 1100 HPLC system (Hewlett-
Packard, Palo Alto, CA). The mass spectrometer was operated in the
negative ESI mode. Full scans were acquired from m/z 100 to 650/s.
The information for MS condition is as follows: voltage, 3.5 kV;
capillary temperature, 300 °C; sweep gas, 70 psi; and auxillary gas, 5
units. HPLC was performed with a Prodigy ODS(3) HPLC column (5
µm, 100 Å, 2.0 mm × 250 mm, Phenomenex, Torrance, CA). The
mobile phase delivered at 0.2 mL/min was a linear gradient from 5 to
95% acetonitrile in water with constant 0.1% formic acid in 40 min.
RESULTS AND DISCUSSION
Identification of Theaflavin Oxidation Product from
DPPH and Peroxidase/Hydrogen Peroxide Oxidant Model
Systems. A major oxidation product of theaflavin was isolated
using column chromatography methods from two different
oxidant model systems, DPPH and peroxidase/hydrogen per-
1
oxide, and its structure was identified by interpretation of H
NMR and MS spectra. It has been reported that the dihydroxy
B ring and the trihydroxy B ring are the major sites for
antioxidant action of tea catechins (7). As theaflavin is dimeric
Stability of Theaflavin in Different pH and Its Oxidation
Product. In the present study, solutions with various pH values,
including simulated gastric juice and buffer solutions of pH 5.5,

Radical Reaction of Theaflavin
J. Agric. Food Chem., Vol. 53, No. 15, 2005 6149
Figure 4. Theaflavin degradation product at alkaline condition.
Figure 5. ESI spectrum of theaflavin.
Figure 6. ESI spectrum of DEG-1.
pH 7.4, and pH 8.5, were chosen to examine the stability of
theaflavin in acidic and alkaline conditions. It has been reported
that plant flavonoids are vulnerable in alkaline condition, and
several studies have demonstrated that tea catechins are unstable
in neutral and alkaline pH (15-17). For example, the tea
catechins, EGC and EGCG, are completely degraded in pH 7.4
phosphate buffer solution within 3 h (17). Interestingly, EGC
and EGCG, which contain pyrogallol in the B ring, were much
less stable than tea catechins EC and ECG, which have a
catechol B ring. One reason for the difference is that pyrogallol-
containing catechins form semiquinones more readily than those
containing the catechol moiety (17).
When theaflavin was incubated in the acidic solutions, pH
5.5 buffer solution, and simulated gastric juice, as shown in
Figure 3, this compound exhibited a high degree of stability
during a 24 h incubation period. In contrast, theaflavin was
unstable in alkaline conditions. At pH 7.4 buffer solution, 34.8%
of theaflavin was degraded after 8 h of incubation. Moreover,
when theaflavin was added into pH 8.5 buffer solution, it rapidly
changed to a dark brown color, and 78.4% of theaflavin was
degraded after 2 h of incubation.
experiment revealed that the DEG-1 has a molecular ion at m/z
533 [M - H]- (Figure 6). In Figure 6, the pseudo-molecular
ion peak at m/z 535 [M - H]- was also identified as a result of
the reduction product. This result indicated that the spectral
character of DEG-1 was the same as theanaphthoquinone
reported by Tanaka et al. (12). After comparison of HPLC
analysis of an isolated theanaphthoquinone standard and DEG-
1, we concluded that DEG-1 is theanaphthoquinone. This
observation might be important to the black tea beverage
industry. It is possible that theaflavin in black tea beverage
undergoes a change to theanaphthoquinone in aqueous solution.
To date, little scientific information is available about the
biological activities of theanaphthoquinone. It might be interest-
ing to understand its biological activity and to examine its
presence in commercial black tea beverage products.
In conclusion, theanaphthoquinone was identified as an
oxidation product of theaflavin using DPPH and peroxidase/
hydrogen peroxide oxidant model systems. This indicated that
the benzotropolone moiety in theaflavin may play an important
role in its antioxidant activity. The stability of theaflavin was
examined in solutions of varying pH. The results confirmed that
theaflavin is unstable in alkaline conditions in agreement with
a recent report of Su et al. (18). In contrast, theaflavin was stable
in acidic condition. Theanaphthoquinone was identified as an
autoxidation product of theaflavin during its stability study in
alkaline conditions. As shown in Figure 2, the formation of
theanaphthoquinone from theaflavin can be facilitated in alkaline
conditions.
During the course of this stability study, it was noteworthy
that a major degradation product of theaflavin was identified
during HPLC analysis in alkaline conditions: pH 7.4 and pH
8.5 (Figure 4). To identify the degradation product, the solution
was partitioned with ethyl acetate and the organic part was
analyzed with LC/ESI. Figure 5 represents the negative ion ESI
spectrum of theaflavin (m/z 563 [M - H]-). The LC/ESI

6150 J. Agric. Food Chem., Vol. 53, No. 15, 2005
Jhoo et al.
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Received for review March 23, 2005. Revised manuscript received May
25, 2005. Accepted May 27, 2005.
JF050662D