MuUgeneds vol.11 no.2 pp.189-194, 1996
Antimutagenicity and catechin content of soluble instant teas
Anne Constable1, Natacha Varga, Janique Richoz and
Richard H^tadler
Nestec Ltd Research Centre, Vers-Chez-les-Blanc, PO Box 44, CH-1000
Lausanne 26, Switzerland
'To whom correspondence should be addressed
The antimutagenic properties of soluble instant teas were
examined using the bacterial Ames assay. Inhibition of the
numbers of revertants induced from a number of known
mutagens indicates that aqueous extracts of instant teas
have antimutagenic activity and antioxidative properties,
and can inhibit nitrosation reactions. Despite a significant
reduction in the amounts of major green tea catechins,
quantified using reversed-phase HPLC with electrochemical detection, no differences in antimutagenicity were
observed between the instant teas, a black fermented tea
and a green tea. Oxidation of polyphenolic compounds
which occurs during the production of instant tea does not
therefore decrease the antioxidant, free radical scavenging
and antimutagenic properties. This suggests that catechins
are not the only compounds responsible for the protective
effects of teas.
Introduction
Tea (Camellia sinensis) is one of the most popular beverages
worldwide, and its consumption has been claimed to be
associated with beneficial health effects (Conney et al., 1992;
Yang and Wang, 1993; Imai and Nakachi, 1995). The inhibitory
effects against tumorigenesis shown by green tea and green
tea extracts, including also semi-fermented and fermented tea,
are apparently linked to the polyphenolic constituents of this
traditional beverage (Yang and Wang, 1993). These green tea
polyphenols, commonly referred to as tea catechins, can
account for up to 30% of dry wt materials in green tea
leaves (Graham, 1992). Structurally, catechins are monomeric
flavanols and are extremely base-labile due to their catecholic
moieties. The major catechins found in green tea are
(-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin-3-gallate
and (-)-epigallocatechin-3-gallate, and as a minor component
( + )-catechin. These natural chemicals exhibit excellent radical
scavenging and hence antioxidative properties (Zhao et al,
1989; Yuting et al, 1990), are inhibitory against nitrosation
reactions (Stich, 1982), modulate carcinogen metabolizing
enzymes (Khan et al, 1992; Sohn et al, 1994) and inhibit
cell proliferation (Mukhtar et al, 1992; reviewed in Yang and
Wang, 1993).
An estimated 2.5 million metric tons of dried tea are
manufactured annually. Of this, only 20% is green tea and is
consumed mainly in Asian countries (Yang and Wang, 1993).
Black tea accounts for 78% of total tea production, mainly
consumed in the West. In recent years instant teas have been
available on the market. It has been reported that the European
consumption of iced tea has grown by 31% from 1993, and
that in 1994, 285 000 gallons of iced tea were consumed, with
© UK Environmental Mutagen Society/Oxford University Press 1996
Switzerland leading the way with 9.85 gallons consumed per
capita (Food Institute Report, May, 1995). Instant teas are
manufactured from fermented black teas, and then made
soluble by a treatment in oxidative and/or alkaline conditions.
These processing conditions, including fermentation itself, can
lead to rapid dimerization and polymerization of catechin
monomers via oxidative phenolic coupling to furnish bisflavonols and other compounds. The fermentation and solubilization processes therefore decrease the amounts of catechins,
suggesting a decrease in the associated beneficial properties.
The detection and quantification of catechins is usually performed by reversed-phase HPLC with UV detection, a method
of adequate sensitivity considering the abundance of these
compounds in green tea. However, most of the analytical
HPLC data published to date cover green and fermented teas
(Hoefler and Coggon, 1976; Bailey et al, 1990; Liang et al,
1990; Kuhr and Engelhardt, 1991; Graham, 1992), with only
little information concerning the catechin content of commercial soluble instant tea powders. Similarly, no data are available
concerning the antimutagenic or antitumorigenic properties of
instant teas; much of the work has concentrated on green teas,
with less on black teas. Using the in vitro bacterial Ames test,
we have compared the antimutagenic properties of green, black
and two instant teas. In addition, a rapid and sensitive analytical
method using reversed-phase HPLC with electrochemical
detection (ECD) has been developed to measure the amounts
of the major green tea catechins in commercial instant teas.
Materials and methods
Chemicals
All solvents were analytical grade. The mutagens 2-amino-l-methyl-6-phenylimidazo[4,5fe]-pyridine (PhD?), 2-amino-3,4-dimethylimidazo[4,5/)quinoline
(MelQ) and 2-amino-3-methylimidazo[4,5/]quinoline (IQ) were purchased
from Toronto Research Chemicals, Ontario, Canada. 2-Nitroflourene (2-NF)
was purchased from Merck, Darmstadt, Germany. Stock solutions were
prepared in DMSO or methanol. Working concentrations of r-butylhydroperoxide (f-BOOH, Sigma Chemical Co., Basle, Switzerland) were prepared in
water and used immediately. The commercial tea catechin standards
(-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin-3-gallate, (-)-epigallocatechin-3-gallate and ( + )-catechin were purchased from Roth (Karlsruhe,
Germany). Chromabond Ci8 EC cartidges (500 mg) were from Macherey &
Nagel (DUren, Germany).
Preparation of aqueous extracts of tea
Aqueous extracts of green tea extract powder (Nikkon Foods Ltd, Japan), a
North American instant tea (1009fc) powder (Instant A), a European 100%
instant tea powder (Instant B) and fermented black tea leaves (bought on the
market in Russia) were prepared by the addition of boiled water to the tea
powder or leaves to a concentration of 15 mg tea/ml and left to brew for
10 min. The teas were then sterilized through a 0.45 u.m filter. As a control,
a herbal tea consisting of dried lime leaves was purchased from a local
supermarket and treated in the same manner.
Antimutagenicity assays
Antimutagenicity was assessed using the standard Ames plate reversion assay
(Maron and Ames, 1983). Salmonella typhimurium strains were co-treated
with the tea extracts and mutagen in the presence or absence of metabolic
activation mix (S-9 mix) where appropriate. The doses of mutagens chosen
for these experiments are within the linear portions of appropriate doseresponse curves, result in sufficient numbers of revertants to detect any
modulation of mutagenicity and are not toxic. Dilutions of each tea were
189
A.Constable et al
Table I. Effect of teas on the mutagenicity of heterocyclic aromatic amines
Mutagen
Tea (mg/plate)
Instant A
Instant B
Green
None
MelQ ( + S9)
7.5"
0
0.5
1.5
2.5
5.0
7.5
0
0.5
1.5
2.5
5.0
7.5
0
0.5
1.5
2.5
5.0
7.5
0
0.5
1.5
2.5
5.0
7.5
0
1175
559
308
25
0
0
1459
902
653
16
0
0
406
329
242
31
0
0
358
367
561
369
305
315
0
1038
657
228
0
0
0
1509
1005
514
0
0
0
399
579
274
0
0
0
361
386
338
338
293
279
0
1085
983
585
192
56
0
1277
1050
637
112
17
0
418
549
358
46
0
0
359
379
410
362
408
400
I Q ( + S9)
PhIP (+ S9)
2-NF (-S9)
±
±
±
±
18
70
80
80
±
±
±
±
40
157
26
8
±
±
±
±
45
30
109
14
±
±
±
±
±
±
4
2
54
17
22
25
± 91
± 86
± 34
± 8
± 236
± 154
± 25
± 82
± 54
±
±
±
±
±
±
25
1
30
22
52
29
Black
±
±
±
±
±
10
258
118
57
10
±
±
±
±
±
59
120
27
10
21
±
±
±
±
7
8
21
8
±
±
±
±
±
±
47
22
6
20
40
1
0
1133
1101
1071
191
21
0
1084
1139
833
115
0
0
779
749
583
295
37
0
366
451
338
443
383
369
Lime leaves
±
±
±
±
±
43
80
142
30
5
±
±
±
±
89
99
113
6
i
±
±
±
±
209
80
76
49
1
±
±
±
±
±
±
8
10
19
16
1
55
0
nt
nt
nt
nt
nt
nt
1812
2499
2263
1917
1944
2585
nt
nt
nt
nt
nt
nt
193
218
278
341
302
312
±
±
±
±
±
±
175
201
110
156
216
92
±
±
±
±
±
±
5
30
46
40
25
90
Values are the mean ± SD of the numbers of histidine revertants from two plates. Spontaneous reversion rates of TA98 (55 ± 7) have been subtracted. When
this corrected number was <10, the number of revertants is given as 0.
T h e values observed with the highest concentration of tea in the absence of mutagen.
made with sterile water to give concentrations from 0 to 7.5 mg/plate in a
0.5 ml volume. The S-9 was obtained from Moltox (Annapolis, MD, USA),
and was prepared from the livers of male Sprague-Dawley rats pretreated
with Aroclor 1254. The S-9 was stored at -20°C and immediately before use
a cofactor solution containing 10% S-9 was prepared (Maron and Ames,
1983). The tea extracts (0.5 ml) were each plated on three minimaJ agar plates
with 0.1 ml bacterial culture and, when required, 0.5 ml S-9 mix and 0.1 ml
of the appropriate mutagen. The HAAs (500 ng PhIP, 5 ng IQ and 3 ng
MelQ) were tested on strain TA98 with S-9 mix, 2-NF (3 |ig) on strain TA98
without S-9 and f-BOOH (1 umol) on strain TA102 without S-9. The Petri
plates were incubated for 3 days at 37°C. The colonies were counted on an
automatic colony counter. Fisher Count-All 880, linked to a desktop computer.
Nilrosation assays
The method of Stich et al. (1982) was used. A stock of 15 mg7ml tea was
prepared in Buffer A (0.068 M citric acid and 0.064 M disodium phosphate,
pH 3.6) and filter sterilized. The teas were diluted in buffer A to give a dose
range from 0 to 8 mg/plate. To each dilution 25 mM /V-methylurea (Fluka,
Buchs, Switzerland) and 100 mM sodium nitrite (Merck) was added. After
20 min the nitrosation reaction was terminated by the addition of 0.75%
sodium bicarbonate and the volume made up to 3 ml with 10X PBS. The
Ames test was performed as before using strain TA1535.
Evaluation of results
The number of spontaneous revertants were subtracted from the values
obtained in the presence of mutagens and/or tea extracts. At each dose of tea
the percentage inhibition was calculated by: (1 - A/B) XI00, where A =
number of revertants induced in the presence of mutagen and tea extract and
B = number of revertants induced by mutagen. The presence of mutagen
alone is considered as 0% inhibition.
Quantification of catechins in instant teas
Solid-phase extraction. Instant tea powders (20 mg) were dissolved in 6 ml
Millipore grade water. To aliquots of 1 ml, 50 u.1 of 1 M HC1 was added to
remove polymeric compounds. Individual aliquots were spiked with four
standard catechins, (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin-3gallate and (-)-epigailocatechin-3-gallate, to give final concentrations of 0, 2,
5 and 10 ng of added catechin/ml of tea solution. These were then centrifuged
(14 000 r.p.m., 1 min) and the supematants (1 ml) loaded onto Ci 8 EC
cartridges, and preconditioned with two bed volumes of methanol, followed
by two bed volumes of water and finally one bed volume of 10 mM HC1.
After gravity-induced penetration, the cartridges were rinsed with 1 ml of
water. The catechins were eluted (2 ml) with the solvent mixture A + B
(1 + 1, v/v), comprising A = 95% water, 5% methanol, 2 mM acetic acid
190
and B = 95% methanol, 5% water, 2 mM acetic acid, and the effluent
subsequently dried by vacuum centrifugation. The residues were redissolved
in 1 ml of the elution solvent mixture A + B (1 + 1, v/v). Samples were
stored at 4°C in the dark and aliquots (20 |il) analysed directly by HPLC.
HPLC with ECD. Analytical reversed-phase HPLC was carried out using a
Hewlett Packard Model 1050 Ti series chromatograph, consisting of a 1050
UV/vis detector and an Antec Decade electrochemical detector with an
integrated pulse dampener and thermostated electrode oven chamber. Data
acquisition and peak integration (UV and ECD) was done with a HPChem
workstation. HPLC separation was with a Suplecosil LC-18 DB column (25
cmX4.6 mm i.d.), 5 nm particle size, attached to a precolumn (2 cmX4.6
mm i.d.), operated at ambient temp, and at a flow rate 0.8 ml/min. HPLC
solvents were A = 5 mM citnc acid (Sigma) adjusted to pH 5.1 with solid
NaOH; B = 100% methanol, filtered through 0.22 u.m membrane filters
(Milliporc Corp., MA, USA) and constantly sparged with helium during
chromatographic operation. The citric acid solvent was prepared fresh prior
to use and not stored for a longer period than two consecutive days. Solvent
flow commenced lsocratically at 15% B for 20 min, increasing to 80% B
after 34 min and returning to initial conditions after 6 min Typical retention
times of the pertinent analytes (min) were: (-)-epigaIlocatechin, 18.5; (+)
catechin, 19.1; (-)-epigallocatechin-3-gallate, 33.9; (-)-epicatechin, 34.7; and
(-)-epicatechin-3-gallate, 37.4. ECD was with a glassy carbon electrode
operated at 32°C, with the electrode potential set at a constant potential of
+0.5 eV versus the AgCl/KCI reference electrode, flow cell volume 0.04 (jJ
(50 |im spacer), output range 0.1 nA and filter 0.1 s. UV detection was at
272 nm.
Results and discussion
Suppression of HAA mediated mutagenicity
Heterocyclic aromatic amines (HAAs) are common food mutagens, produced by the heating of meat to high temperatures
(Felton et al., 1981; Sugimura and Sato, 1983). They require
activation by the hepatic cytochrome P450 enzymes (Kato,
1986) to their reactive forms, which can interact with DNA
and are detected by the strain TA98 in the presence of metabolic
activation mix. Aqueous extracts of the four teas were tested
on strain TA98. None showed any mutagenic effects at the
highest dose tested. The HAAs PhIP (500 ng), IQ (5 ng) and
MelQ (3 ng) were added to the test system and all three were
Instant tea antlmutagenlclty and catechin content
b.IQ
100-r
.2
teamg/plate
teamg/plate
c PUP
12-NF
iooT
I""
•I 60..
20.
Fig. 1. Inhibition of mutagenic effects of HAAs by tea extracts. The number of revertants induced by each mutagen alone is regarded as 0% inhibition.
— • — (Instant A), —Q— (Instant B), —A— (Green), —X— (Black)
Table D. Effect of teas against the mutagenicity of /-BOOH
Tea (mg/plate)
7.5
0
0.5
1.5
25
5.0
7.5
1
Instant A
Instant B
Green
Black
148
702
639
497
499
3%
371
67
452
324
235
225
215
182
158 ± 92
750 ± 92
444+93
357 ± 70
320 ± 140
242 ± 71
264 ± 63
74
473
148
225
204
210
179
±
±
±
±
±
±
±
83
51
149
160
160
123
139
±
±
±
±
±
±
±
10
86
219
116
22
26
15
±
±
±
±
±
±
±
Lime leaves
9
107
42
23
48
37
23
31
332
272
391
491
537
498
±
±
±
±
±
±
±
7
37
57
27
50
57
53
Values are the mean ± SD number of histidine revertants from four plates from two independent experiments. The number of induced revertants have been
corrected for the spontaneous reversion rate of TA102 (306 ± 52).
The values observed with the highest concentration of tea in the absence of (-BOOH.
tested alone and in the presence of the tea extracts. When the
tea samples were added, the numbers of revertants induced by
each of the mutagens were reduced in a dose-dependent manner
(Table I and Figure 1). In all cases 5 mg/plate of tea was
sufficient for at least 90% inhibition. The mechanism of action
against heterocyclic amines has been proposed at the level of
the xenobiotic metabolizing enzymes. In vitro studies have
shown that the addition of tea extracts to microsomes from
the livers of rats can inhibit the activities of arylhydrocarbon
hydroxylase, 7-ethoxycoumarin-O-deethylase and 7-ethoxyresorufin deethylase (Wang et al., 1989) and cause a marked
decrease in the 0-dealkylation of methoxyresorufin, ethoxyresorufin and pentoxyresorufin (Bu-Abbas et al, 1994). A
similar inhibition of the reduction of cytochrome c has also
been observed, due at least partly to impairment of the electron
flow from NADPH to the cytochrome (Bu-Abbas et al, 1994).
When the mutagenic compound 2-NF (3 |ig) was tested, no
reduction of revertants was observed with any of the teas at
any dose. 2-NF is a direct-acting mutagen requiring no activation. This would support published results suggesting that the
mechanism of antimutagenicity against the heterocyclic amines
is at the level of interference of the xenobiotic metabolizing
g
1
if
Fig. 2. Inhibitory effects of tea extracts on the mutagenicity of /-BOOH.
The number of revertants induced by (-BOOH alone is regarded as 0%
inhibition.
— • — (Instant A), —D— (Instant B), —A— (Green), —X— (Black)
enzymes present in the S-9 mix. A herbal tea consisting of
lime leaves gave no protection against either 2-NF or IQ.
Antioxidant effects
Yen and Chen (1995) have demonstrated the antioxidant
and reducing power of green and black tea extracts by the
191
A.Constable et al
Table III. Inhibition of nitrosation reaction by teas
Tea (mg/plate)
Instant A
Instant B
Green
Black
8'
Nitnteb
0
2
4
6
8
0
563 ±
1413 ±
551 ±
487 ±
488 ±
513 +
0
3% it 38
1076 dt 69
475 dt 4
443 ;i 16
348 it 24
3 % ± 38
0
482 ± 12
1096 ± 268
468 ± 30
407 ± 35
395 ± 6
410 ± 1
0
376 ± 48
1194 ± 187
669 ± 69
414 ± 30
454 ± 47
358 ± 24
22
34
2
17
16
2
Values are the mean number ± SD of histidine revertants from two plates. Spontaneous reversion rates of TAI535 (29 ± 8) have been subtracted.
T h e values observed with the highest concentration of tea in the absence of mutagen.
•The values observed when TA1535 was treated with 100 mM sodium nitrite in the presence of the highest concentration of tea.
peroxidation of linoleic acid. In addition, they observed that
semi-fermented teas showed greater antioxidant activity, reducing power and scavenging effects on active oxygen and free
radicals. Tea extracts can therefore act as electron donors and
react with free radicals to convert them to more stable products,
and terminate radical chain reactions (Wang et al., 1989).
Reactive oxygen species may damage DNA, alter gene expression or affect growth and differentiation (Cerutti, 1989).
f-BOOH is a pro-oxidant which produces hydroxyl radicals
which can be directly detected by strain TA102. All five teas
when tested on strain TA102 induced a slight but nonsignificant increase in the number of revertants [using the
linear regression model of Moore and Felton (1983)]. The
number of revertants produced by 1 ^mol of r-BOOH was
reduced when any of the five teas were added (Table II and
Figure 2). Some dose dependency was observed, but the level
of inhibition was not as complete as that seen with the
heterocyclic amines. The highest inhibition was observed with
the black tea, while instant B and green tea showed the least.
However, all teas were effective at the highest dose tested,
and achieved a reduction in mutants by 46-62%. No decrease
in the number of revertants induced from /-BOOH was observed
when a herbal tea extract was tested.
Inhibition of nitrosation reactions
Nitrosation of secondary amines can form mutagenic /V-nitroso
compounds. Protective effects from this reaction by tea components have been demonstrated (Nakamura and Kawabata,
1981). To determine and compare the properties of the instant
teas with respect to inhibition of nitrosation reactions, the
method of Stich et al. (1982) was used. Methylureais nitrosated
in the presence of sodium nitrite at acid pH and induces
histidine reversions of the strains TA100 and TA1535. Methylurea not subjected to the nitrosation process is not mutagenic
to either strain (data not shown). Nitrous acid was generated
from nitrite at low pH, causing point mutations, and generated
485 ± 128 revertants of TA1535. Nitrosation of methylurea
increased this background number of revertants to 1036-1476.
When the reactions were performed in the presence of each
of the teas, the number of revertants of TA1535 (Table III and
Figure 3) was drastically reduced to the level observed when
only sodium nitrite alone was added to the system. This effect
was observed at the lowest dose of 2 mg/plate for all teas.
Experiments performed by Stich et al. (1982) indicated that
the effect of tea on the nitrosation reaction was probably due
to interference of the nitrosation reaction itself, rather than the
interaction of the nitrosated product, since addition of tea after
the formation of nitrosomethylurea had no effect on its
mutagenic activity.
192
Fig. 3. Modulation of nitrosation reactions in the presence of tea extracts.
0% inhibition is taken from the number of revertants produced from the
nitrosation of methylurea in the absence of tea extract
— • — (Instant A), — • — (Instant B), —A— (Green), —X— (Black)
Epigallocatechln-3-gallate
•200
nA
0
Epigallocatechin
Epicatachin
Eplcatechin-3-gallate
Brand B
Brand A
15
20
25
30
35
40
Time (min.)
Fig. 4. Excerpts of C-18 reversed-phase HPLC-ECD chromatograms of
solid-phase-treated instant tea powders. Equivalents of 0.067 mg of tea
powder were injected using HPLC conditions as described under Materials
and methods.
Quantification of catechins
The identification and quantification of the major catechins in
the instant tea powders was determined by spiking the tea
Instant tea antlmutagenicity and catechin content
Table IV. Quantitative analyses of the major catechins in instant teas (A-D) by reversed phase HPLC with ECD
Tea
A, batch
A, batch
A, batch
B, batch
B, batch
B, batch
C
D
Black"
Green"
1
2
3
1
2
3
EC
EGC
ECG
EGCG
Total catechins
0.18
0.12
0.07
0.62
0.46
0.32
0.92
1.16
1.21
1.98
0.14
0.15
0.07
1.47
1.1
2.1
3.93
2.81
1.09
8.42
0.15
0.16
0.12
0.7
0.62
0.45
0.91
0.83
3.86
5.20
0.2
0.21
0.1
1.95
1.74
1.38
2.94
3.12
4.63
20.29
0.67
0.64
0.36
4.72
3.92
4.25
8.7
7.92
10.79
35.09
Values arc expressed as Sb of tea powder solids Abbreviations are. (-)-epicatechin, EC; epigallocatechin, EGC; (-)-epicatechin-3-gallate, ECG; and (-)epigallocatechin-3-gallate, EGCG.
"Graham, 1984.
solutions with different known amounts of the four commercial
standards before extraction, and then comparing the retention
times and peak areas of the individual analytes after HPLC
analysis (Figure 4). Quantification of all four catechins was
performed by plotting peak areas against increasing known
amounts of the standard analytes and subsequent extrapolation
from a linear regression equation, which takes into account
recoveries of the individual flavanols. For all of the four
catechins the regression curves were linear, and the square of
correlation coefficients (r2) was >0.97. Repetitive analyses of
independent extracts of the same tea batch revealed deviations
not greater than 10%.
The catechin contents (% relative to dry tea powder) of the
same two different brands of instant teas used in the Ames
assays, and of two further instant tea brands (C and D) were
compared with the catechin contents of black and green tea
and are portrayed in Table IV. Comparison of three different
batches of the same tea brands (instant A and instant B)
showed consistency in catechin content, revealing for brand
A an average of ^echins = 0.56 ± 0.17% (RSD = 30) and
for brand B Xcatechins = 4.29 ± 0.40% (RSD = 9.3) of tea
powder solids. Instant B therefore contains 8-fold higher levels
of catechins in the tea powders versus brand A. The ratios of
the four major catechins in tea powders are comparable to
those found in black teas, in that in most cases epigallocatechin
and epigallocatechin-3-gallate were the most dominant flavanolic constituents, reaching up to 3.93 (brand C) and 3.12 g
(brand D) per 100 g dry wt respectively. To date, catechin
analyses of only two different instant tea powders have been
reported, reaching levels of 10.8 and 14.5% based on dry
powder and including the minor component catechin (Kuhr
and Engelhardt, 1991).
Certain tea powders with very low concentrations of
catechins cannot be quantified accurately by conventional
HPLC-UV detection techniques. The HPLC-ECD method
presented here enables rapid and sensitive analysis of tea
powders for their catechin content. Fluctuations that may be
encountered using different extraction methods (Kuhr and
Engelhardt, 1991) are overcome by spiking the powders prior
to solid-phase extraction with increasing amounts of standard
catechins, enabling direct compensation for loss in recovery
during the work-up procedure.
r-BOOH and the nitrosation of methylurea is not significantly
different to those of green and black fermented teas. There is
much data from in vitro and in vivo experimental systems
concerning the beneficial aspects of green tea, and most of
these properties are attributed to the presence of the catechins.
As shown in Table IV, the amounts of these compounds in
black fermented and instant soluble teas are reduced, yet their
antimutagenic effects are not significantly different to those
observed for green tea. This suggests that catechins are
not the only compounds responsible for the protective and
antioxidant effects of teas. The oxidation reactions which take
place during processing of green tea polymerizes the catechin
monomers, resulting in the formation of theaflavins, theaflavic
acid, bisflavanols and thearubigens. These components still
have many phenolic hydroxy groups, responsible in part for
antioxidative activities, and the thearubigens are known to be
a mixture of heterogeneous polyphenols. Strong antioxidative
effects from thearubigens and theaflavins have been observed
from experiments using in vitro peroxidation of rat liver
homogenate induced by f-BOOH (Shiraki et al, 1994).
Similarly, theaflavins inhibit the hydrogen peroxide-induced
cleavage of DNA (Yoshino et al., 1994). The gallic acid moiety
of theaflavins were essential for their potent antioxidative
abilities. As much as 2.62% dry wt theaflavins and 35.9% dry
wt thearubigens are present in black tea (Graham, 1984).
Theogallin is present as -0.58% in green tea, 0.2-0.95% in
black tea and ~2.5% dry wt in instant teas (Kuhr and Engelhardt,
1991). Gallic acid was also found to be slightly higher in
instant (0.29-1.15% dry wt) and black teas (~1% dry wt) than
in green teas (~0.1% dry wt). A reduction of catechin content
in fermented and instant teas does not correlate with the
antimutagenic effects observed in our bacterial assays, and so
may be compensated for by other compounds produced by
processing conditions. The composition of black and instant
teas with respect to beneficial health properties therefore seems
to warrant a closer examination.
Acknowledgements
We thank Drs P.-M.Leong-Morgenthaler and R.Turesky for helpful discussions
and cntica] reading of the manuscript, and Dr B.N.Ames for providing
S.typhimurium strains.
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Received on August 30, 1995; accepted on October 10, 1995
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