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Department of Biochemistry and
*
Department of Physiology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, The People’s Republic of China and
Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, The People’s Republic of China
2To whom correspondence should be addressed. E-mail: zhenyuchen{at}cuhk.edu.hk.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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TF2B TF2A
> TF1 EC > EGC. Four TF derivatives
also demonstrated a dose-dependent antioxidant activity in
Cu+2-mediated LDL oxidation at concentrations of 5–40
µmol/L. These results demonstrate that the TF present
in black tea possess at least the same antioxidant potency as catechins
present in green tea, and that the conversion of catechins to TF during
fermentation in making black tea does not alter significantly their
free radical–scavenging activity.
KEY WORDS: • black tea • catechins • green tea • low density lipoprotein • oxidation • theaflavins.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Catechins account for 6–16% of the dry green tea leaves
(1)
. Theaflavins (TF) are another group of polyphenol
pigments found in both black and oolong teas. TF are formed from
polymerization of catechins at the fermentation or semifermentation
stage during the manufacture of black or oolong tea (2)
.
TF contribute to the characteristic bright orange-red color of
black tea, accounting for 
2 g/100 g of the dried water extract of
black tea. The major TF in black and oolong tea are theaflavin
(TF1), theaflavin-3-gallate
(TF2A), theaflavin-3'-gallate
(TF2B) and theaflavin-3,3'-digallate
(TF3) (Fig. 1
). Both catechins and TF
have recently received much attention as protective agents against
cardiovascular disease and cancer (3
4
5)
. They are also
believed to have a wide range of other pharmaceutical benefits,
including antihypertensive (6
,7)
, antioxidative
(8
,9)
and hypolipidemic (10
,11)
activities.
|
,13)
. This idea is supported by the
following observations: 1) oxidatively modified LDL (ox-LDL)
are present in atherosclerotic plaque but absent in the normal artery
wall (14
,15)
; 2) ox-LDL are taken up by
macrophage scavenger receptors, promoting cholesterol ester
accumulation and foam cell formation (16)
; and
3) ox-LDL are toxic to endothelial cells and stimulate
monocyte adhesion to the endothelium (13)
. Catechins are
effective inhibitors of LDL oxidation induced by copper and
peroxyl free radicals (17
,18)
. However,
information is very limited concerning the relative anti-LDL
oxidation activity of individual TF derived from black tea compared
with that of individual catechins found in green tea. The objective of
the present study was to isolate theaflavins from Chinese black tea and
examine the relative antioxidant potencies of
TF1, TF2A,
TF2B and TF3 in comparison
with those of EC, ECG, EGC and EGCG, using human LDL oxidation as a
model. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Total catechins from Chinese longjing green tea (Huangshan Forestry
Farm, Xiaoshan, Zhejiang, China) were extracted and isolated as
previously described (17)
. To purify TF, qimen black tea
(2.25 kg) was purchased locally and was first extracted three times
using 13.5 L of 70% ethanol. After the removal of ethanol in a rotary
evaporator, the remaining water solution was extracted subsequently
using chloroform (3 L), ethyl acetate (2 L) and butanol (2 L). The
ethyl acetate extract was then applied onto a silica gel column (80
x 6.5 cm i.d.; silica gel 60M, 230–240 mesh). The total TF
fraction was obtained when the column was eluted with a mixture of
chloroform and ethyl acetate (1:1, v/v) followed by increasing the
ratio of chloroform to ethyl acetate to 4:1 (v/v). The total TF
fraction was then subjected to a Sephadex LH-20 column (50 x6.0 cm
i.d.) and eluted with 15 L of 70% ethanol to obtain crude
TF1, TF2A, TF2B and TF3
fractions. TF1 was purified on a Sephadex LH-20 column B
(50 x 2.5 cm i.d.) and eluted using 4 L of 30% acetone in water
containing 2% acetic acid. TF2A, TF2B and TF3
were similarly isolated and purified. The chemical structures of the
four purified TF were verified using the melting point test, TLC, UV
spectrometry, liquid chromatography-mass spectrometry and
1H nuclear magnetic resonance spectrometry. The results
were in agreement with those previously reported (19)
.
LDL isolation.
Fresh blood was collected and pooled from healthy subjects
(n = 20) at the Prince of Wales Hospital, The
Chinese University of Hong Kong, Shatin, Hong Kong. To prevent the
lipoprotein from oxidative modification, EDTA (2.7 mmol/L) and
NaN3 (7.7 mmol/L) solutions were immediately added before
LDL were isolated from serum according to the method previously
described (17)
. The protein content of isolated LDL was
determined using the method of Lowry et al. (20)
. The
protocol was approved by the Committee of Human Ethics, The Chinese
University of Hong Kong.
LDL oxidation.
Oxidation of LDL was conducted as previously described by Puhl et al.
(21)
. In brief, the stock LDL fraction (5 g protein/L) was
dialyzed against 100 volumes of the degassed dialysis solution (pH
= 7.4) containing 0.01 mol/L sodium phosphate, 9 g/L NaCI, 10
µmol/L EDTA and 7.7 mmol/L NaN3 in the
dark for 24 h. The dialysis solution was changed at least four
times. Then, the dialyzed LDL were diluted to 250 mg protein/L with
0.01 mol/L sodium phosphate buffer (pH = 7.4). For the control
incubation tubes, 0.4 mL LDL (250 mg/L) was mixed with 50
µL of 50 µmol/L CuSO4
solution and 50 µL of 0.01 mol/L sodium phosphate
buffer (pH = 7.4), and incubated at 37°C for up to 24 h.
For the experimental tubes, 0.4 mL LDL (250 mg protein/L) was
preincubated with 50 µL of varying concentrations of
individual catechins and TF for 5 min. Then, 50 µL of
50 µmol/L CuSO4 solution was added to
initiate the oxidation, followed by incubation at 37°C for up to
24 h. The oxidation was then stopped by the addition of 25
µL of 27 mmol/L EDTA and cooled to 4°C. The degree
of LDL oxidation was monitored by measuring the production of
thiobarbituric acid–reactive substances (TBARS) as previously
described (17)
. The LDL-incubated tubes were
immediately combined with 2 mL of 0.67% thiobarbituric acid
and 15% trichloroacetic acid in 0.1 mol/L HCl solution. The incubation
mixture was then heated at 95°C for 1 h, cooled on ice and
centrifuged at 1000 x g for 20 min. TBARS were
then determined by measuring the absorbance at 532 nm. The calibration
was done using a malondialdehyde (MDA) standard solution prepared from
tetramethoxylpropane. The value of TBARS was expressed as nmol MDA/mg
LDL protein.
Conjugated dienes.
Oxidation of LDL was also monitored by measuring the production of
conjugated dienes. In brief, the same amount of human LDL was incubated
at 37°C and the oxidation was initiated by the same concentration of
Cu+2 as described above. The absorbance of conjugated
dienes at 234 nm was recorded at 0, 4, 8, 12, 16, 20 and 24 h. The
change in the absorbance (
234 nm) was used as an index of LDL
oxidation.
Statistics.
Data were expressed as means ± SD, n = 6–7. For the dose effect of individual TF on Cu+2-mediated LDL oxidation, the linear regression was conducted between the induction time and the dose of each TF. The trends were considered significant when P < 0.01. For the TBARS assay, differences were analyzed by a three-factor ANOVA with concentrations, types of catechins and TF, and the time of incubation as factors. Sigmastat (Jandel Scientific Software, San Rafael, CA) was used for all analyses. The differences were considered significant when P < 0.01.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. In
contrast, qimen black tea is one of the most popular Chinese black teas
exported. In this study, it was found by HPLC that the total
theaflavins in qimen black tea were 1.58 g/100 g dry leaves.
TF3 was the most abundant (1.05%) followed by
TF2A (0.34%), TF2B
(0.11%) and TF1 (0.08%).
TBARS were used as an index of LDL oxidation. When all catechins and TF
were compared at 5 µmol/L, the antioxidant activity was in
the order: TF3 > ECG > EGCG TF2B TF2A > TF1 EC > EGC (Fig. 2
). A similar order was seen when oxidation was measured by conjugated
diene production (data not shown) Under the same experimental
conditions, vitamin C (ascorbic acid) had little or no protective
activity for human LDL.
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, P for trend < 0.01). When 5 µmol/L
concentrations of TF1,
TF2A, TF2B and
TF3 were incubated with LDL, the lag times were
4, 7, 7.5 and 16 h, respectively. When 10 µmol/L
concentrations of the four TF derivatives were incubated, the lag times
were extended to 8, 12, 16 and 32 h, respectively. As the
concentrations of the four TF were increased to 20 and 40
µmol/L, LDL showed little or no oxidation during the test
period.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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EGCG TF2B TF2A > TF1 EC > EGC in decreasing order using
human LDL as the oxidation model. Together with the study by Yoshino et
al. (22)
, who showed that green tea and black tea
infusions had similar antioxidant activities in rat liver homogenates,
the present study clearly suggests that the TF present in black tea
possess at least the same antioxidant potency as the catechins present
in green tea.
TF are the products formed when catechins are oxidized and dimerized.
TF1 was formed by the oxidation and dimerization
of EC and EGC (23)
. As shown in Figure 2
, the antioxidant
activity of TF1 was similar to that of EC (lag
time = 4 h) but it was stronger than that of EGC
(P < 0.01). Similarly, TF2A had
an antioxidant activity similar to that of EGCG but it was more
effective than EC; the former was produced when the latter two
underwent oxidation and dimerization (23)
. Compared with
the two precursors, TF2B was weaker than ECG but
stronger than EGC in protecting LDL from oxidation (Fig. 2)
.
Interestingly, TF3 was more effective than its
two precursors, ECG and EGCG, in preventing LDL from oxidation (Fig. 2)
. The present comparisons indicate that TF are at least as effective
as their precursors in protecting human LDL from oxidation. The
conversion of catechins to theaflavins during the manufacture of black
tea does not affect their free radical–scavenging potency on the same
molar basis.
It is interesting that TF3 containing two gallate
groups inhibited LDL oxidation more than TF2A and
TF2B, which have only one gallate group.
Similarly, TF2A and TF2B
had stronger antioxidant activities than TF1
because the former two have one gallate group, whereas the latter
contain no gallate group. This observation is consistent with that of
Shiraki et al. (24)
, who showed that the galloyl moiety of
TF was essential for their potent antioxidant activities. Similarly, EC
and EGC were less active than their corresponding gallate derivatives,
ECG and EGCG, as antioxidants against LDL oxidation (17)
.
Perhaps an additional group increases the total number of phenyl
hydroxyl groups and makes the gallate-containing catechins and TF
more able to donate a proton due to the resonance delocalization.
The present study in vitro, although not directly applicable to humans,
may have some implications for individuals who often consume black or
green tea. It was previously demonstrated that the ingestion of green
and black teas significantly increased human plasma antioxidant
capacity in vivo (25)
. This was also in agreement with the
observation that total plasma lipid peroxides and oxidation of LDL were
significantly reduced in hamsters fed a high cholesterol diet and given
green and black tea (26)
. All data presented here suggest
that drinking black tea has benefits equal to those of drinking green
tea in terms of their antioxidant capacity.
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FOOTNOTES |
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3 Abbreviations used: EC, (-)-epicatechin; ECG,
(-)-epicatechin gallate; EGC, (-)-epigallocatechin; EGCG,
(-)-epigallocatechin gallate; MDA, malondialdehyde; ox-LDL,
oxidatively modified LDL; TBARS, thiobarbituric acid–reactive
substances; TF1, theaflavin; TF2A,
theaflavin-3-gallate; TF2B, theaflavin-3'-gallate;
TF3, theaflavin-3,3'-digallate.
Manuscript received March 2, 2001. Revision accepted May 31, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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