Antioxidant capacity of different types of tea products

African Journal of Biotechnology Vol. 6 (19), pp. 2287-2296, 4 October 2007
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2007 Academic Journals




Ful Length Research Paper
Antioxidant capacity of different types of tea products

Karori, S. M.1, Wachira, F. N. 1*, Wanyoko, J. K.2 and Ngure, R. M.1

1Department of Biochemistry and Molecular Biology, Egerton University, P.O Box 536, Njoro, Kenya.
2Department of Chemistry, Tea Research Foundation of Kenya (TRFK), P.O Box 820 Kericho, Kenya.

Accepted 14 June, 2007

In the present study, twelve different types of commercial tea samples were assayed to determine their
phenolic composition and antioxidant activity. Reverse phase high performance liquid chromatography
using a binary gradient system was used for the identification and quantification of individual
catechins. Subsequently, total phenolic content was determined spectrophotometrically according to
the Folin-ciocalteus method. Total theaflavins and thearubigins were also determined. The radical
scavenging behavior of the polyphenols on 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) was also
studied spectrophotometrically. The results showed that total polyphenols, total catechins and antioxi-
dant activity were significantly (P<0.05) different in the commercial tea samples. Green tea had the
highest levels of catechins, total polyphenols and total antioxidant activity. White tea (silvery tip) a rare
specialty type of tea was not significantly different from green tea. Statistical analysis showed an
essential catechin content influence of the tea extracts on antioxidant activity. Epigallocatechin gallate
(EGCG) was the most potent catechin and the most potent in antioxidant activity (r = 0.989***). Epigallo-
catechin (EGC) (r = 0.787, P<0.001), epicatechin (EC) + catechin (+C) and epicatechigallate (ECG) also
showed significant (P<0.05) antioxidant activity. Black tea contained high levels of theaflavins and
thearubigins, which accounted for most of the antioxidant potential in this type of tea product (r =
0.930*** and r = 0.930*** respectively). These results suggest that conversion of catechins during black
tea processing did not affect the free-radical potency of black tea. Gallic acid (GA) also showed
significant(r = 0.530*) contribution to the antioxidant activity in black tea. Green, black and white tea
products processed from Kenyan tea cultivars originally selected for black tea had significantly
(P<0.05) higher antioxidant activity than green tea processed from tea cultivars from Japan and China.
These results seem to suggest that the cultivar type is critical in determining the antioxidant potency of
tea product and that black teas processed from suitable cultivars could be potent in antioxidant activity
when compared to green teas.

Key words: Antioxidant capacity, DPPH, catechins, polyphenols, EGCG, theaflavins.


INTRODUCTION

Processed tea, which is one of the most popular bevera-
world can be classified as non-fermented/aerated green
ges, is manufactured from the young tender leaves of the
tea, semi-fermented (oolong) tea and fermented black tea
plant Camel ia sinensi (Cabrera et al., 2003). Two types
(Reeves et al., 1987), processing has diversified to the
of tea products are most widely consumed; green and
production of specialty teas e.g. white tea, flavored teas,
black tea. In both cases, it is the chemical composition of
organic teas, decaffeinated teas, herbal teas, scented
the tea shoots and the reactions that occur during pro-
teas and various other blends. The manufacturing techni-
cessing that determine the nature of the finished product
ques of the above types of tea products, which may
and its quality. Though most of the tea produced in the
either be orthodox or non-orthodox, vary considerably

and have a pronounced impact on the formative and

degradative patterns of various cel ular components. The

conventional orthodox method which consists of rol ing
*Corresponding author. E-mail: fwachira@yahoo.com. Tel:
the leaf on a rol ing bed, stretching and tearing the leaf
+254-722644279.
has in some cases been replaced with non-orthodox me-

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2288 Afr. J. Biotechnol.







thods or curl, tear and crush (CTC) which have a quicker
(GC) and (-)-gal ocatechin gal ate (GCG). Catechins are
and more severe leaf disruption leading to production of
present in large amounts in green tea (Peterson et al.,
smal er fragments and consequently more oxidation
2005). Based on their chemical structure, catechins that
(Mahanta and Hemanta, 1992).
contain three hydroxyl groups in the B ring (positions 3’,
In the preparation of green tea, the withered leaves are
4’ and 5’) are cal ed gal ocatechins while gal ic acid
steamed and then dried relatively rapidly after plucking to
substitution in position 3 of the ring is characteristic of
minimize chemical and enzymatic reactions. This stops
catechin gal ate (Pel ilo et al., 2002). Catechins account
the polyphenol oxidase [PPO] enzyme [EC 1.10.31]
for 6 - 16% of the dry green tea leaves with EGCG
catalyzed oxidation of tea leaf catechins (Wilson and
constituting 10 - 50% of catechins and being the most
Clifford, 1992). In contrast, during black tea processing,
potent due to its degree of gal ation and hydroxylation
tea shoots are macerated to initiate oxidation by PPO
(Stewert et al., 2004). TFs and TRs are another group of
before firing. This reaction enables the catechins to con-
polyphenolic compounds found in both black and oolong
dense with the orthoquinones arising from the oxidation
teas (Obanda et al., 2001).
of the B ring di- and trihydroxylated catechins to form
The tea beverage has continued to be considered a
thea-flavins (TFs). TFs are homogenous substances,
medicine since the ancient times because of its polyph-
which give a yel ow red coloration in fermented black tea
enols. Research on the effects of tea on human health
and contribute to the briskness and brightness of tea
has been fuel ed by the growing need to provide natural y
liquor. They act as oxidizing agents for substrates like
healthy diets that include plant-derived polyphenols. In
gal ic acid to form epitheaflavic acids. These acids
line with this, there is need to elucidate how known
combine with TFs to produce the chemical y heteroge-
functional components in foods could expand the role of
neous substances cal ed thearubigins (TRs) that are
diet in disease prevention and treatment (Mandel et al.,
responsible for the color, body and taste of tea. Molecular
2006). There is already growing evidence that tea poly-
oxygen is essential in the formation of these compounds
phenols reduce the risk of heart diseases and cancer in
that are characterized by a benzotropolone ring structure
humans (Vanessa and Wil iamson, 2004). In some
(Obanda et al., 2001).
studies, tea has been associated with antial ergic action
The manufacturing process for semi-fermented oolong
(Yamamoto et al., 2004) and antimicrobial properties
tea consists of solar withering, panning, rol ing and
(Paola et al., 2005). Further studies have demonstrated
drying. During this process, the characteristic floral aroma
that the co-administration of drugs with catechins (EC
of oolong tea is produced. White tea is a rare specialty
and EGCG) inhibits glucoronidation and sulfonation of
tea that gets its name from a specific tea plant variety, as
oral y administered drugs thereby increasing the bioavai-
wel as a particular post-harvest processing method that
lability of such drugs (Hang et al., 2003). Moreover some
raises smal silvery hairs on the dried buds. White tea
epidemiological studies have associated consumption of
contains a higher proportion of the buds that are covered
tea with a lower risk of several types of cancer including
with fine “silvery” hairs that impart a light white colour to
those of the stomach, oral cavity, oesophagus and lungs
the tea. The brew from white tea is pale yel ow in colour
(Cabrera et al., 2003; Hakim and Chow, 2004).
with no “grassy” undertones sometimes associated with
Therefore, tea appears to be an effective chemo-
green tea. True white tea is lightly fermented, rapidly
preventive agent for toxic chemicals and carcinogens.
steamed and dried leaving the leaves “fresh”. Unlike
The ability to scavenge for free radicals by tea polyphe-
black, green and oolong teas, white tea is not rol ed or
nols due to possession of a phenolic hydroxyl group
crushed but it is steamed rapidly and air-dried to preserve
attached to the flavan-3-ol structure has been associated
most of the polyphenols. This unique processing prod-
with teas’ therapeutic action against free radical mediated
uces a rare and expensive but highly refreshing drink.
diseases thereby attracting tremendous research interest
The differences between the various processes of
(Amie et al., 2003). Free radicals are known to contribute
manufacture result in differences in the polyphenol profile
to numerous disorders in humans including cancer, arthe-
between black, green, oolong and white tea. Figure 1
roscerolosis, arthritis, ischemia, Central Nervous System
outlines the processing of tea in more details.
(CNS) injury, gastritis, dementia, renal disorders and
The chemical composition of tea is complex and
Acquired Immune Deficiency Syndrome (AIDS) (Pourmo-
includes polyphenols, alkaloids (caffeine, theophyl ine
rad et al., 2006; Rao et al., 2006). Free radicals are
and theobromine), amino acids, carbohydrates, proteins,
constantly generated due to environmental pol utants,
chlorophyl , volatile compounds, minerals, trace elements
radiation, chemicals, toxins, physical stress and the
and other unidentified compounds. Among these, polyph-
oxidation process of drugs and food. Many plant phenol-
enols constitutes the most interesting group and are the
lics have been reputed to have antioxidant properties that
main bioactive molecules in tea (Cabrera et al., 2003).
are even much stronger than vitamins E and C. In addi-
The major polyphenolic compounds in tea are the flavan-
tion, currently available synthetic antioxidant like
3-ols cal ed catechins which include: (-)-epicatechin (EC),
butylated hydroxyl anisole (BHA), butylated hydroxyto-
(-)-Epigal ocatechin (EGC), (-)-epicatechin gal ate (ECG),
luene (BHT) and gal ic acid esters have been suspected
(-)-epigal ocatechingal ate (EGCG), (-)-Gal ocatechins
to cause or prompt negative health effects and hence the

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Karori et al. 2289



Tea Processing
White Tea
Green Tea
Oolong Tea
Black Tea
Partialy Steamed
Steaming
Outdoor withering for 0.5-1hr
Indoor withering without roling
Air dried in natural sunlight
Parching (inactivation of endogenous enzymes)
Indoor withering with gentle roling for 6-8hrs
Roling
White tea
Roling & drying
Parching (inactivation of endogenous enzymes)
Fermentation
Final firing
Roling & drying
Final firing (inactivation of endogenous enzymes)
Green unfermented tea
Final firing
Black Fermented Tea
Oolong semi fermented tea


Figure 1. Schematic diagram of the conventional manufacture process of green, black, oolong
and white tea.



need to substitute them with natural y occurring antioxi-
indoor withering at room temperature with turned over treatment for
dants (Amie et al., 2003; Aqil et al., 2006; Pourmorad et
6 - 8 h and then rol ing and final firing at 100oC for about 30 min.
al., 2006).
The green teas had been manufactured using standard green tea
There is therefore an increased quest to obtain natural
manufacturing procedures of steaming for 1 h and then final firing in
a fluid bed drier at 120oC for 20 - 25 min. White tea had been proc-
antioxidants with broad-spectrum action. Despite the
essed from the hairy tip buds only by partial steaming and air-drying
upsurge of interest in the therapeutic potential of plants
in natural sunlight. Preliminary assay were carried out to establish
as sources of natural antioxidants few studies have been
the appropriate amount of sample for analysis and to ensure that
carried-out using black tea. In addition information on the
the samples had not been damaged or destroyed during transport-
antioxidant properties of white tea, which is a rare
tation. Al biochemical analysis was carried out in duplicate.

specialty tea is grossly lacking.

In the present study, a set of twelve tea samples; eight
Sample treatment for polyphenol and catechin analysis
commercial Kenyan tea samples that included black,

green, oolong and white tea and two of each Japanese
Tea samples of a coarse granular structure were minced and
and Chinese green teas were analyzed for total polyphe-
ground to a fine powder. 2 g of the sample was placed on a pre-
weighed moisture dish and left for 16 h at 103oC in the oven to dry
nols, total catechins, total TFs, total TRs and their antioxi-
for the determination of dry matter. Of these, 0.2 ± 0.001 g was
dant activity. Additional y, two popularly consumed fresh
weighed into an extraction tube. Five mil iliter of hot 70% v/v
unprocessed vegetables namely onion and spinach were
methanol/water was dispensed into the sample as an extraction
analyzed for antioxidant activity and compared to the tea
mixture and vortexed. Heating of the extraction tube continued in
samples. The objective of the study was to investigate
the water bath for 10 min with mixing in the vortex mixer after every
5 min. The extraction tubes were then removed from the water bath
the relationship between the phenolic content with the
and al owed to cool. The tubes were then placed in a centrifuge at
antioxidant activity of the tea samples.
3500 rpm for 10 min. The supernatant was decanted into a gradua-

ted tube and the extraction procedure repeated. The extracts were

combined and made up to 10 ml with cold methanol/water mixture.
MATERIALS AND METHODS
One mil iliter of the sample extract was transferred into a graduated

Samples
tube and diluted to 5 ml with a stabilizing solution (10% v/v acetone-

trile with 500 µg/ml EDTA and ascorbic acid). The solution was
A set of twelve tea samples; eight commercial Kenyan teas that
further filtered through a 0.45 µm nylon membrane filter. A 20 µl
included fermented (black), semi fermented (oolong), non- fermen-
aliquot of this solution was injected into HPLC for analysis.
ted (green) and white tea from different tea factories in Kenya and

two of each Japanese and Chinese green teas were analyzed. The

samples had been manufactured in commercial factories using
Catechin analysis using HPLC
standard manufacturing conditions. Black teas had been manufac-

tured using physical withering up to 50 - 65% moisture content for
A modified method of Zuo et al. (2002) was used. A Shimadzu LC
18 hours; fermentation at 24oC for 1 - 2 h and a final firing in a fluid
20 AT HPLC fitted with a SIL 20A auto sampler and a SPD-20 UV-
bed drier at 120oC for 20 - 25 min. The oolong teas had been
Visible detector with a class LC 10 chromatography workstation
processed using outdoor withering under sunlight for 30 - 60 min;
was used for the analysis of the prepared samples. A Luna TM 5

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2290 Afr. J. Biotechnol.







µM C18, 25 cm x 4.6 i.d (Phenomenex, Torrance, CA, USA) column
% Inhibition against DPPH = [(AB -AA)/AB] x100
with a Reodyne precolumn filter 7335 model was used. Al solvents

were filtered through a 0.45 µm mil ipore membrane filter disk and
Where AB is the absorbance of the blank sample (50 µl double
degassed before injection into a HPLC system. A gradient elution
distil ed water and 2 ml DPPH) and AA is the absorbance of the
was carried out using the fol owing solvent systems: Mobile phase
tested sample after 15 min.
A (acetonitrile/acetic acid/double distil ed water- 9/2/89 v/v/v),

Mobile phase B (acetonitrile/acetic acid/double distil ed water -

80/2/18 v/v/v). The mobile phase composition for a binary gradient
Statistical analysis
condition started at 100% solvent A for 10 min then over 15 minutes

a linear gradient to 60% mobile phase A, 32% mobile phase B and
Al determinations were carried out in duplicates and data were
held at this composition for 10 min. The condition was reset to 100
subjected to analysis of variance using SPPS version 11.5 soft-
% mobile phase A and al owed to equilibrate for 10 min before the
ware. The Duncan’s Multiple Range Test (DMRT) was used to
next injection. The flow rate of the mobile phase was 1 ml/ min and
separate the means.
the temperature at the column was performed at 35 ± 0.5oC. The

identification of individual catechins was carried-out by comparing

the retention times and UV- absorbance of unknown peaks with
RESULTS AND DISCUSSION
peaks obtained from the mixed known standards under the same

conditions. The quantification of catechins was performed at 278
Separation chromatograms of the major catechins by
nm and was achieved using a caffeine external standard with a
calibration curve R2 = 0.9984 in conjunction with the consensus
reverse phase (RP) HPLC is as shown in Figure 2. To
individual catechin relative response (RRF) values with respect to
obtain an adequate resolution of the peaks within a
caffeine calculated on dry matter basis. Total catechin as percent-
reasonable time of analysis, a gradient elution program
tage by mass on a sample dry matter basis was given on the
was developed. The best results were obtained with
summation of individual catechins.
double distil ed water–acetonitrile-acetic acid at a flow

%Total catechin = [%ECG + (%+C) + (%EC) + %EGCG + %ECG]
rate of 1 ml /min which al owed the separation of catec-
content.
hins within 18 min. To avoid interaction of the free hydro-

xyl groups of catechins and the stationery phase, al

solutions were prepared in an acidic media because
Total polyphenols determination
catechins are stable in acidic media (Pel ilo et al., 2002).

The Luna TM Phenomenex column was chosen because
The Folin-ciocalteu reagent method was used to determine total
polyphenols as described by Pourmorad et al. (2006).
of its high stationery phase surface and a constant

support dimension that permitted a complete separation

of catechins within a short time. The column technique
Total theaflavins (TF) content analysis/flavognost
employed was exclusively RP because of its high reso-

lution for separation and quantification of phenolic
Total TF were determined by the flavognost method of Hilton
substances. In addition, 70% aqueous methanol solution
(1973).

was used for extraction because of its protective role on
Specrophotometric measurement of total Thearubigins (TR)
phenolic substances from being oxidized (Proestos et al.,

2006). The major catechins were identified by a compare-
Total thearubigins were determined using the method of Roberts
son of their retention times with those of authentic
and Smith (1961).
standards at UV absorption spectra of 278 nm. Under


these operating conditions, the retention times in minutes
Free radical scavenging activity determination
for the studied compounds were as fol ows: EGC (8.1),

+C (10.2), caffeine (13.5), EC (14.8), EGCG (16.4), ECG
The stable 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) was used
(21.7), GC (6.1) and GA (4.3) (Figure 2). Previously,
for the determination of free radical scavenging of the tea extracts
results for green tea (Ferruzzi and Green, 2005) and for
using a modified method of Brand-Wil iams et al. (1995). 5 g of tea
clonal fresh leaves (Ender et al., 2004) were comparable
was infused in 100 ml of boiling double-distil ed water fol owed by
stirring with a magnetic stirrer and additional steeping for 30 min at
to the observed results.
room temperature. The extracts were strained through a nylon
The total catechin content was significantly higher
mesh (120 µm) fol owed by a filter paper (Whatman No. 54). Aliq-
(P<0.05) in green tea than oolong and black tea as
uots of the extracts were kept frozen (-1oC) until further use. The
indicated in Table 1. This demonstrates clearly that the
soluble solid extract was standardized to give stock solutions of 50
degree of fermentation during the manufacturing process
mg soluble solids per 100 ml of 50% methanol. A methanolic
had an influence on the Catechin content of the final
solution (50 µl) of the antioxidant was placed in a cuvette and 6.0 x
10 - 5 M methanolic solution of DPPH (2 ml) was added (DPPH
product. Black tea is obtained by a post-harvest fermen-
solution was made using 80 % methanol). The decrease in absor-
tation which is an auto-oxidation reaction catalyzed by
bance at 517 nm was determined using a CE 393 digital grating
the enzyme polyphenol oxidase, whereas green teas are
spectrophotometer until the absorbance stabilized. Reading was
steamed to inactivate oxidation. Oolong tea is obtained
done at 15 or 30 min interval. The DPPH solution was prepared
by a partial oxidation of the leaf, an intermediate process
afresh and kept in the dark to minimize the loss of free radical stock
solution. Al determinations were performed in duplicate. The (%) in-
between green and black tea manufacture (Peterson et
hibitation of DPPH radical was calculated from the absorbance data
al., 2005). White tea is a product of partial steaming and
according to (Yen and Duh, 1994).
air-drying of the hairy tips. This unique processing pre-

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Karori et al. 2291





Figure 2. A representative high performance liquid chromatogram of green BP CTC tea.



serves most of the catechins in white tea (Table 1).
during manufacturing. The rol ing and cutting of the tea
Individual catechins varied significantly (P<0.05) among
shoots in non-orthodox manufacture causes a release of
the teas with EGCG, GC and EGC levels being the
polyphenol oxidase which interacts with phenolic com-
highest and +C, ECG and EC being less abundant.
pounds, one simple catechin and one gal ocatechin, to
These results are similar to those of (Ender et al., 2004).
produce theaflavins and thearubigins that posses a
White tea which is predominantly manufactured from the
benzotropolone skeleton (Reeves et al., 1987; Mahanta
young apical hairy bud only showed high levels of EGCG
and Hemanta, 1992). Owuor and Obanda (2006) investi-
and ECG that are present in higher amounts in fresh
gated the use of green tea flavan-3-ols in predicting black
young leaves. This latter result corroborates the result by
tea quality potential and revealed that a correct balance
Saijo et al. (2004) who determined the chemical consti-
of the trihydroxylated flavan-3-ols and dihydroxylated
tuents of young tea leaves and the change occurring
flavan-3-ols was necessary to ensure maximum forma-
during leaf development. The decrease in the gal ic acid
tion of the theaflavins. The trihydroxylflavan-3-ols are
esters of catechin such as EGCG and ECG during leaf
oxidized faster during the fermentation phase of black tea
development means that there is a slow biosynthesis of
processing explaining the high levels of EGCG and EGC
gal ic acid moiety in each catechin gal ate compared with
in green tea and the subsequent reduction in black tea.
dry matter production. Since catechin biosynthesis is
Theaflavins are further oxidized to form thearubigins that
slower than dry matter production from young leaves to
are heterogeneous in nature and contribute significantly
the less young leaves, it is apparent that there is no
towards taste, color and body of tea (Obanda et al., 2004;
weight increase in the less young and mature leaves and
Li et al., 2005). Black tea therefore has high levels TFs
as a result catechin moves to other young leaves or are
and TRs that are the main fermentation products as
metabolized to other products. This accounts for the
evident in Table 1.
change in catechin levels in various leaf developmental
Results from the present study however clearly showed
stages and hence the levels of residual catechins in tea
that TRs were present in green tea (Table 1). Further
manufactured from different ages as exemplified in the
observation revealed that in green tea, TRs were formed
differences in catechin levels between white tea and
in the presence of low levels of TFs unlike in black tea
other types of teas in our study (Table 1).
where the levels were almost similar (Table 1). This may
The variation in the polyphenolic composition of the
suggest that theaflavins are not the only source of thearu-
different tea products resulted from the leaf maceration
bigins. Wilson and Clifford (1992) explained the factors

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2292 Afr. J. Biotechnol.



Table 1. Phenolic composition and antioxidant activity (%) of various teas.


Total
Total
Individual catechins



Sample
polyphenols
catechins
EGC
EGCG
GC
ECG
+C
EC
TFs
TRs
AA+
Kenyan teas
Black PD (CTC)
20.65cd, 7
5.91e,9
1.12 de, 9
2.82de, 9
4.88a, 1
0.99b, 3
0.37b, 6
0.62g, 9
11.61c, 3
10.30d, 4
73.31d, 6
Black BP (CTC)
17.45f, 11
3.07f,14
0.63e, 11
1.43ef, 11
3.60cd, 4
0.57cde, 9
0.20 de, 11
0.25h, 11
18.75a, 1
15.65a, 1
71.19fg, 10
Green PF (CTC)
26.85a, 2
14.93a, 1
5.47a, 1
6.78a, 1
3.26cd, 5
0.75bc, 4
0.43 b, 2
1.56ab, 2
0.46fg, 8
5.59h, 11
77.22a, 1
Green BP (CTC)
25.70a, 4
10.04d, 7
5.17ab, 2
3.11cd, 8
2.45ef, 9
0.28 e, 12
0.30 b, 4
1.21cde, 5
1.63e, 6
8.38f, 8
73.31d, 5
Black orthodox
22.25b,5
4.62ef, 10
1.20de, 10
1.87def, 10
3.00de, 7
0.62cd, 8
0.33 bc, 8
0.79fg, 9
13.80b, 2
11.51c, 3
71.52ef, 9
Green orthodox
27.10 a, 1
11.06cd, 4
4.91ab, 4
4.72b, 5
3.84bc, 3
0.35de, 11
0.38b, 5
0.98ef, 7
0.41fg, 10
5.77h, 10
75.64b, 3
Oolong orthodox
26.15a, 3
9.49d, 8
2.31c, 7
4.37bc, 7
3.01de, 6
1.03b, 2
0.57a, 1
1.30 bcd, 4
6.81d, 5
8.75ef, 7
74.78c, 4
White tea/ Silvery tip
21.30c, 6
10.20cd, 6
1.73cd, 8
5.53ab, 2
4.46ab, 2
1.505a, 1
0.41b, 3
1.00def, 6
0.85f, 7
1.75i, 12
76.01b, 2
Other ungraded teas










Japanese teas
Green CTC cultivar
19.36de,9
12.69ab, 2
4.75b, 5
5.05b, 3
1.83fg, 10
0.70bc, 7
0.35bc, 7
1.85a, 1
0.25g, 12
9.06 e, 6
71.76ef, 8
Yabukita
Green CTC cultivar
19.78de,8
12.24bc, 3
4.97ab, 3
4.18b, 4
1.79fg, 11
0.74bc, 5
0.23cde, 10
1.50bc, 3
0.45fg, 9
7.69g, 9
70.54g, 11
Yutakamidori
Chinese teas
Green CTC cultivar
18.82ef,10
11.19cd, 5
4.65b, 6
4.64b, 6
2.48ef, 8
0.71bc, 6
0.31bcd, 9
0.90fg, 8
0.31fg, 11
9.30e, 5
69.21 h, 13
Hanlu
Green CTC cultivar
11.42g,12
3.31f, 12
0.60e, 12
0.78f, 12
1.20g, 12
0.36de, 10
0.14e, 12
0.22h, 12
11.57c, 4
13.55 b, 2
67.62 i, 14
Yinghong
Fresh unprocessed vegetables
Spinach
-
-
-
-
-
-
-
-
-
-
69.34h, 12
Onion
-
-
-
-
-
-
-
-
-
-
72.35e, 7
Grand mean
21.48
9.95
3.12
3.82
2.98
0.72
0.34
1.01
5.62
8.88
72.41
LSD (P<0.05)
1.423
1.856
0.6995
1.423
0.7431
0.3265
0.1206
0.2953
0.5697
0.5947
0.9021
C.V (%)
3.01
8.30
10.20
16.92
11.34
20.58
4.59
13.26
16.93
3.04
0.55

DMRT ranking - Means within a column fol owed by the same letter are not significantly different at P=0.05 according to Duncan’s Multiple Range Test –DMRT. Numerical ranking is from the highest
value of the parameter to the lowest.
+Data has been arcsine transformed.
Tea grades: PD, pekoe dust; BP, broken pekoe; PF, pekoe fanning’s; CTC, curl tear crush.
EGC, Epigal ocatechin; EGCG, Epigal ocatechingal ate; GC, Gal ocatechin; ECG, Epicatechin gal ate; +C, Catechin; EC, Epicatechin; TFs, Theaflavins; TRs, Thearubigins; AA Antioxidant Activity.

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Karori et al. 2293






affecting the formation and degradation of theaflavins and
that are potent antioxidants (Zhu et al., 2001; Amie, et al.,
thearubigins in black tea and observed that maximum
2003, and Rao, et al., 2006).
synthesis of theaflavins occurs when oxygen is in excess
Black teas analyzed in this study exhibited some antio-
to support benzotropolone ring formation. However,
xidant activity with a high DPPH radical scavenging acti-
under a limiting oxygen concentration, polyphenol oxid-
vity though less than that of green, white and oolong tea.
ase, which has a high affinity for the substrate, has a
During black tea manufacture, the gal ocatechins are first
preferential demand for oxygen and theaflavins formation
to be oxidized and dimerised to TFs and TRs because of
is suppressed at the expense of catechin quinone forma-
their high oxidation potential and high concentration in
tion. This competition for oxygen is particularly noticeable
the leaves. These major phenolic compounds in black tea
during the early stages of fermentation when the concen-
also contributed significantly to the radical scavenging
tration of the catechins is at its highest and enzyme
activity that is, TFs (r = 0.920, P<0.001) and TRs (0.807,
turnover is unimpeded by substrate availability. This
P<0.001) and GA (r = 0.530, P<0.05). Interestingly, TFs,
occurs during green tea manufacture since the enzyme is
which are major phenolic product in black tea, had a
active before deactivation through steaming. For this
higher radical scavenging activity compared to some of
reason, high enzyme activity in an already low oxygen
its precursors ECG, EGC and EC (Table 2). This con-
concentration creates almost total anaerobiosis, which
firms that conversion of catechins to TFs during black tea
suppresses benzotropolone ring formation. Consequently
processing did not affect the radical scavenging potency.
as a result of this, thearubigins are formed, mainly from
These observations are consistent with those of Leung et
gal ocatechins since the simple catechins are unable to
al. (2001) who showed that black tea posses more or less
react in benzotropolone ring formation. Moreover, it might
the same antioxidant potency as catechins present in
be possible to minimize thearubigins formation by deac-
green tea. EGCG and EGC contribute significantly to the
tivating the enzyme immediately after plucking through a
formation of TFs. These are B ring trihydroxylated cate-
steaming procedure although this is hardly achievable
chins, which are oxidized at a much faster rate than the B
during commercial tea processing. Further research is
ring dihydroxylated catechins (EC, ECG and +C) due to
desirable to explain in details the existence of this thearu-
their lower oxidation potential (Owuor and Obanda,
bigins in green tea and the importance of steaming during
2006). TFs formed from this reaction have hydroxyl
tea processing.
groups (OH) considered necessary for free radical
The polyphenolic composition of tea and especial y its
scavenging activity. These additional groups increase the
catechins has aroused interest in their potential as radical
total number of phenyl hydroxyl groups and make the
scavenging compounds. Data on antioxidant capacity is
gal ate containing catechins and TFs more able to donate
presented in Table 1. Overal , green and white teas’ had
protons due to resonance delocalization thereby expres-
significantly (p< 0.05) higher antioxidant activity com-
sing the observed antioxidant activity of black tea.
pared to black tea. There was no significant difference in
Similarly, gal ic acid contributed significantly to the radical
the antioxidant capacity of black tea manufactured using
scavenging activity in black tea because it is a potent
orthodox and non-orthodox methods. Table 2 presents
hydrogen donator to DPPH. Additional y, our study pro-
data on the correlation between tea polyphenols contents
vided evidence of the contribution of TRs towards the
and the antioxidant activity of different types of tea
antioxidant activity of black tea. A significant correlation
products. Total catechins significantly (p<0.001) corre-
(r=0.807, P<0.001) was observed (Table 2). The antio-
lated with antioxidant activity (r = 0.959). EGCG was
xidant activity of TRs can be explained by the presence
identified as the most potent antioxidant (r = 0.989,
of 3-OH groups, which are more or less esterified by
P<0.001). EC, EGC, +C and ECG contents also showed
gal ic acid in the TRs structure. However, this is a highly
significant influence on the antioxidant activity. Therefore,
speculative hypothesis since to date there is no definite
the antioxidant activity was higher in tea extracts contain-
data on TRs structures (Li et al., 2005). Oolong (semi-
ning high levels of EGCG, EC, EGC, +C and ECG. These
fermented) tea which was intermediate between green
results are similar to those of Gramza et al. (2006). This
and black tea did not contain high levels of the major anti-
antioxidative effect of polyphenols has been attributed to
oxidative gal ocatechins and also did not yet contain a
the phenolic hydroxyl groups in their structures that make
great amount of theaflavins and thearubigins which are
them potent free radical scavengers (Amie et al., 2003).
found in ful y fermented black tea. Consequently, this
On the basis of these results, it appears that the most
type of tea had an antioxidant activity that was inter-
effective radical scavengers are catechins with a 3’, 4’
mediate of that of green and black tea (Table 1).
and 5’-trihydroxylated substitution pattern on the B ring
A comparison of the antioxidant activity of the Kenyan
and/or hydroxyl group at C-3 position of the catechin
commercial teas with those of Japan and China was
structure. This hydroxylation confers a higher degree of
carried out to determine the effect of the variety from
stability on the catechin phenoxyl radical by participating
which the tea products were processed on antioxidant
in electron delocalisation that is an important feature of
activity. This study revealed that Kenyan tea products
the antiradical potential. This explains why radical sca-
both green and black were rich in total polyphenols as
venging is high in the gal ocatechins i.e. EGCG and EGC
shown in Table 1. The high polyphenol content in Kenyan

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2294 Afr. J. Biotechnol.



Table 2. Correlation coefficient matrix analyses between various tea chemical parameters.

TP
TFs
TRs
EGC
EGCG
ECG
+C
EC
GC
GA
AA
TC

1.00
0.881***
0.734***
0.919***
0.791***
0.530*
0.832***
0.920***
0.675**
0.637**
0.807***
0.899***
TP

1.00
0.952***
0.894***
0.908***
0.262
0.766***
0.885***
0.308
0.584*
0.920***
0.955***
TFs


1.00
0.706**
0.922***
0.658**
0.738***
0.803***
0.450
0.378
0.930***
0.892***
TRs



1.00
0.782***
0.674**
0.624**
0.860***
0.644**
0.815**
0.787***
0.919***
EGC




1.00
0.597*
0.769***
0.873***
0.333
0.505*
0.989***
0.965***
EGCG





1.00
0.578*
0.261
0.757***
0.771***
0.579*
0.255
ECG






1.00
0.887***
0.401
0.442
0.766***
0.794***
+C







1.00
0.627**
0.449
0.860***
0.937***
EC








1.00
0.184
0.400
0.369
GC









1.00
0.530*
0.645**
GA










1.00
0.959***
AA











1.00
TC

*Correlation significant at the P< 0.05 level.
**Correlation significant at the P< 0.01 level.
***Correlation significant at the P< 0.001 level.

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Karori et al. 2295






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