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~105~
Journal of Pharmacognosy and Phytochemistry 2014; 3(4): 105-116
E-ISSN: 2278-4136
P-ISSN: 2349-8196
JPP 2014; 3(4): 105-116
Received: 24-09-2014
Accepted: 09-10-2014
Karori S.M
Department of Biochemistry and
Molecular Biology, Egerton
University. P.O Box 536- 20115,
Egerton, Kenya.
Wachira F.N
Association for Strengthening
Agricultural Research in East and
Central Africa, P.O Box 765,
Entebbe, Uganda.
Ngure R.M
Department of Veterinary Clinical
Studies, Egerton University. P.O
Box 536-20115, Egerton, Kenya.
Mireji P.O
Department of Epidemiology of
Microbial Diseases, Yale School of
Public Health, 607 Laboratory of
Epidemiology and Public Health
,60 College St .New Haven, CT
06510, USA.
Correspondence:
Karori S.M
Department of Biochemistry and
Molecular Biology, Egerton
University. P.O Box 536- 20115,
Egerton, Kenya.
Polyphenolic composition and antioxidant activity of
Kenyan Tea cultivars
Karori S.M, Wachira F.N, Ngure R.M and Mireji P.O
Abstract
Polyphenolic fractions in tea are potent bioactive molecules. In this study, the polyphenolic composition
of 25 different types of Kenyan tea cultivars was determined using the HPLC and the Folins Ciocalteus
spectrophotometric methods. Total Polyphenols, Total Catechins, individual catechins and Antioxidant
Activity were significantly (P<0.05) different among tea varieties, with green tea had the highest levels
of Total Polyphenols ranging from (19.70-26.12%), TC (8.51%-17.60%), individual catechins, and AA
(86.65-94.50%). In vitro bioassay carried out using 2, 2’-diphenyl picrylhydrazyl radical showed
epigallocatechin gallate was the most potent catechin and the most potent in antioxidant activity
(r=0.968***). Epigallocatechin (r=0.659***, p<0.001), Epicatechigallate (r=0.454*, p<0.001) and
Epicatechin (EC) (r=0.780***, p<0.001) showed significant (p<0.05) antioxidant activity. Black tea
contained high levels of Theaflavins and Thearubigins (2.072% to 17.12%), respectively) which
accounted for its antioxidant activity (r=0.803*** and r=0.859***, respectively). Gallic acid also showed
significant (r=0.530*) contribution to the antioxidant activity in black tea. Data obtained from this study
reveals that different Kenyan tea cultivars have different polyphenolic composition which imparts on
their unique biochemical qualities. Green and white tea products are rich in catechins, black tea products
are rich in TFs and TRs while purple teas are rich in anthocyanins.
Keywords: Catechins, EGCG, Theaflavins, Thearubigens, Anthocyanins, Antioxidant Activity.
1. Introduction
Tea, from Camellia sinensis L.O Kuntze is one of the most widely consumed beverages in the
world and it was first introduced in Kenya in1904 by the British settlers. The crop has
expanded to cover an area of around 157,720 ha in the highlands East and West of the Great
Rift Valley in Kenya [1]. The tea plant is an evergreen bush that grows to 15 m high in the wild,
and 60–100 cm under cultivation. Tea in cultivation forms a table from which the young
leaves are harvested through and a cyclic pruning is carried out after every 3 to 4 years and
commercial harvesting is carried out either by hand or machine [2].
Young leaves of tea are processed into different types of products, the predominant ones being
black, green, white and oolong tea. Green tea is mainly consumed in China, Japan and the
Middle East, while black tea is mostly consumed in India, Sri-Lanka, European countries and
regions of Africa. Popularity of tea is due to its aroma, pleasant taste and medicinal benefit [3].
Tea from Kenya tea is better perceived since it is grown free of agrochemicals in an ideal
environment that naturally deters to pests and attack by plant diseases. This pleasant natural
condition guarantees the consumer the safest and most refreshing sought after health drink in
the world. Phytochemicals and functional components in tea are receiving a lot of attention
due to their potential benefits in health when consumed as part of a varied diet on a regular
basis and at effective levels. Many nutraceuticals, functional foods and naturally occurring
polyphenols have physiological and pharmacological activities including their well
characterized antioxidant properties [4, 5]. Since the scientific community and food industry
communities share a common goal of extending the quality of human life, through
development of viable options for the management of chronic diseases through the use of
nutraceuticals, functional foods have become a potential starting point. This is because
functional foods are fairly affordable, readily available and extremely active, have profound
effect on cell metabolism and often demonstrate few side effects [6]. It is evident that
nutraceuticals offer a selective advantage over synthetic drugs necessitating need to investigate
their usefulness to human health.
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Journal of Pharmacognosy and Phytochemistry
Despite the use of tea in food and drinks, it has increasingly
been put to many uses. For example, numerous
environmentally friendly industrial cleaning agents,
deodorizers and anti-microbial agents have been formulated
using tea [7, 8]. However, data to support the view that tea is
pharmacologically active has been generated particularly
using green tea, which is widely consumed in Asia [9, 10]. As a
result, green tea has been widely marketed as a health
product since the chemical composition is well characterized.
However, the much information on the chemical composition
of black aerated or fermented tea, the principle type of tea
product consumed in Kenya is based broadly on the
theaflavins and thearubigens but none of the theaflavins
fractions. In addition, information on the chemical
composition of purple tea, a novel product released recently
by the Tea Research Institute (TRI) is lacking. There is need
therefore to conduct systematic research on the
phytochemical composition of Kenyan tea cultivars
especially black tea and purple teas in order to generate
requisite data which can be used to understand the
pharmacological activity of tea.
2.1 Tea Products and Determination of Flavonoid Levels
2.1.1 Tea samples
The tea samples were sourced from Tea Research
Institute (TRI), Timbilil Estate, Kericho (latitude 0˚ 22’S,
longitude 35˚ 21’E, altitude 2180 m above mean sea level
and processed at the TRFK miniature factory as described by
Karori et al., [5].
2.1.2 Biochemical profiling of the tea extracts based on
Catechins
A modified method of Zuo et al., [11] which is based on high
performance liquid chromatography was used to assay for the
tea catechins of as described by Kerio et al., [12].
2.1.3 Determination of total polyphenols in the tea
extracts
The Folin-Ciocalteu phenol reagent method was used to
determine total polyphenols in the tea extracts according to
ISO (BS ISO 14502-1: 2005(E)).
2.1.4 Analysis of the content of total theaflavins and
individual theaflavin fractions content in the tea samples
Black, green, purple and white teas were also assayed for
total theaflavins (TF) using the flavognost method of Hilton
and Palmer Jones [13] whereas individual theaflavins fraction
ratio were determined as described by Kilel et al., [14].
2.1.5 Spectrophotometric determination of total
thearubigins in the tea samples
Total thearubigins (TRs) were determined in the tea samples
using the method of Roberts and Smith [15].
2.1.6 Determination of total monomeric anthocyanin
content
The total monomeric anthocyanin content in the processed
aerated, unaerated purple tea samples was determined in
duplicate using the pH differential method Kerio et al., [12].
2.1.7 Determination of antioxidant activity of tea
The stable DPPH (2,2-diphenyl-1-picrylhydrazyl) radical
was used for determination of free radical scavenging of tea
extracts using a modified method of Brand-Williams et al.,
[16]
2.1.8 Statistical analysis
All statistical analysis was carried out using MSTAT-C
statistical software. ANOVA was used to determine the
means, coefficient of variation and any differences between
the samples. Least Significance Difference (LSD) was used
separate means. The probability limit was set at p≤0.05
significant level. Results of the parameter determined were
expressed as a mean of the triplicate determination.
3. Results and Discussion
3.1 Chromatographic and Spectrophotometric Analysis
3.1.1 Tea polyphenols
This study compared the total polyphenols levels in tea
samples processed from Kenyan germplasm using the Folins-
Ciocalteus method. The green (unaerated), black (aerated),
white and purple teas samples analyzed differed significantly
in the levels of total polyphenols (p≤0.05). Kenyan green teas
were rich in total polyphenols with their levels ranging from
the highest amount of 26.1% for cultivar Ejulu-L to the
lowest of 19.7% for cultivar TRFK 430/12 as shown in Table
1. Black teas had a lower total polyphenol concentration
compared to green teas with cultivar Ejulu-L having 21.2%
and cultivar TRFK 301/4 the lowest value of 19.7%. It was
however evident, that some black teas from Kenya had a
higher polyphenolic concentration than green teas. Cultivar
TRFK 6/8, a high quality Kenyan genotype used in this study
as an internal standard for black tea quality, recorded a total
polyphenol content of 25.13% and 20.72% for unaerated and
aerated tea respectively, which was not significantly different
(p≤0.05) from tea cultivar Ejulu-L. Total polyphenol content
of aerated and anaerated teas processed from purple coloured
leaf cultivars was 20.03% and 21.90% respectively while
white teas processed from plucked shoots of cultivar AHP
S15/10 was 22.43% (Table 1).
Polyphenols which are constituents of secondary metabolism
in plants play a role in plant defense mechanism against
insects, birds and animals. This study revealed that these
chemicals are retained almost intact in unaerated processed
teas. Unaerated Green tea is made without enzymatic auto-
oxidation of polyphenols, since the enzyme polyphenol
oxidase is inactivated by heat during the early stages of
processing [17]. This process ensures that polyphenols present
in green tea are nearly the same as those found in fresh tea
leaves. In a broad sense, green tea polyphenols consist of
simple and complex compounds, the large majority of which
are the flavonoid monomers catechins, catechin gallates and
flavonols [18, 19].
Polyphenols occurring in black tea usually consist of residual
green tea polyphenols such as catechins flavonols and
oxidation products of green tea polyphenols such as
theaflavins and thearubigins [19]. Some catechins and catechin
gallates may be epimerized or degallated during the
processing of black tea. Most of the catechins and their
gallates undergo known enzymatic oxidation to form more
polymeric polyphenols that are characteristic of black tea,
namely theaflavins and thearubigins [19, 20]. Therefore, the
amount of polyphenols in green tea is higher than that of
black teas since the auto-oxidation process results in a
significant conversion of green tea polyphenols to highly
polymerized substances such as theaflavins which contribute
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Journal of Pharmacognosy and Phytochemistry
to the characteristic bright orange color of black tea and
thearubigens which are more chemically heterogeneous and
tend to be brownish-red [20, 21]. However, despite this
observation, the exact contribution of inter-flavonoid
linkages and the general structure of thearubigins to the
above quality parameters remains ambiguous and their
structures remain speculative. There is need therefore to
elucidate the thearubigins structure to help in optimizing the
tea processing parameters which might contribute to the
customer’s needs.
Table 1: Total polyphenols (TP) (%) and Total Catechins (TC) (%) levels of different tea products used in this study
Clone Green Tea (TP) Black Tea (TP) Green Tea (TC) Black Tea (TC)
TRFK 301/4 22.64de
f
14.96h 16.14cde
f
2.650kl
TRFK 301/5 22.07defg 15.91gh 16.42bcde 3.515hijkl
TRFK 303/216 21.42def 16.41fgh 15.78fgh 2.635l
TRFK 303/231 25.61ab 18.85cde 15.41ghi 3.680hijkl
TRFK 303/577 23.21def 23.21def 16.60bc 7.215ab
TRFK 303/745 20.85ghi 18.35de 11.26
m
4.305efgh
TRFK 337/138 20.85ghi 19.76abcd 17.60a 6.335bc
TRFK 371/3 24.75ab 20.38abc 14.93i 4.25efghi
TRFK 430/3 22.74de 18.22de
f
14.20j 3.820ghij
TRFK 430/4 19.98hi 17.56efg 9.52n 3.225ijkl
TRFK 430/12 19.70i 16.15gh 11.90l 3.900fghij
TRFK 430/63 21.26fgh 15.98gh 15.31hi 3.700ghijk
TRFK 430/90 23.25cd 19.65abcd 15.89efg 8.115a
TRFK 524/170 21.84defg 17.56efg 11.90l 3.82ghij
TRFK 524/48 22.70de 17.52efg 16.39bcde 3.665hijkl
BBK35 24.55
b
c 21.03a 16.04de
f
3.760ghij
EPKC12 22.03defg 20.38abc 12.14l 5.215de
EPK D 99/10 21.59efg 17.39efg 14.15j 3.150jkl
EJULU-L 26.12a 21.22a 16.53
b
c
d
5.850c
d
AHPS 15/10 22.43def 18.94bcde 15.99ef 5.000de
EPK TN 14/3 21.92defg 17.94efg 10.82
m
3.565hijkl
TRFK 6/8 25.13ab 20.72ab 16.85b 6.775bc
TRFK 31/8 21.94defg 20.73ab 14.31j 4.755efg
TRFK 31/11 22.85de 20.68abc 12.77
k
4.890de
f
TRFK 100/5 23.07d 19.09bcde 15.91efg 3.555hijkl
TRFK 306/1
21.90defg
LSD=1.436
CV=3.07%
Mean=22.7
20.03abcd
LSD=1.871
CV=4.87%
Mean=18.6
8.51o
LSD=1.436
CV=1.80%
Mean=14.3
5.235de
LSD=1.871
CV=11.46%
Mean=4.48
Means within a column followed by the same letter are not significantly different at p≤0.05 according to DMRT. Purple tea; TRFK 306/1;
White tea; AHPS 15/10
3.1.2 Total catechin content
Results of the total catechins levels in green, black and white
tea products processed from the 25 tea cultivars assayed in
this study are presented in Table 1. The tea cultivars
produced tea products that differed significantly (p≤0.05) in
total catechin content. Non-aerated (green) teas contained
significantly (p≤0.05) higher amounts of total catechins of
17.60% to 8.51% than aerated (black) teas which ranged
from 8.115% to 3.150%. These results demonstrated clearly
that the degree of auto-oxidation ‘’fermentation’’ during the
manufacturing process had an influence on the catechin
content of the final product. During this processing, the
polyphenol oxidase enzyme catalyzes the oxidation of
catechins into quinones by molecular oxygen [20, 22]. The
quinones generated from the oxidation of the B-ring in the
dihydroxylated catechins condense with the quinones from
the B-ring of the trihydroxy related catechins to form
theaflavins. Since there is a difference in the reduction
potential, quinones will also take part in the redox
equilibrium at fermentation and this explains the depletion of
catechins at different rates [19, 20]. White tea comes from the
same plant Camellia sinensis as the case of green and black
teas. It is processed from the young buds and/or young leaves
and the descriptive term “white” stems from the high
proportion of silvery buds harvested from the plants to
produce the tea. The buds to manufacture this type of tea are
picked then rapidly steamed and dried, without fermentation,
rolling or roasting. Minimal processing not only protects the
delicate, light and slightly sweet flavor of white tea, but also
enables the retention of high levels of phytochemicals [18, 19].
This explains why white tea had high levels of total catechins
despite being processed only the bud. The composition of tea
leaves varies significantly with shoot maturity and season.
Young leaves are composed of polyphenols in the following
order EGCG > EGC > ECG > EC. As for mature leaves, it is
EGC > EGCG > ECG > EC while old leaves composition
was EGC > EGCG > EC > ECG. It is therefore important to
use young leaves and obey plucking standards to achieve
optimum quality especially for black tea. However, since
mature and old tea leaves possess high amounts of EGC, it
would be advisable to research more on ways they can
utilized for further applications.
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Journal of Pharmacognosy and Phytochemistry
Purple leaf coloured cultivar TRFK 306/1 recorded a very
low concentration of total catechins of only 8.51% for
processed green tea. In order to establish why the purple teas
recorded low levels of catechins, an analysis of the total
monomeric anthocyanin content and a fractionation of
anthocyanin by HPLC were carried out. Results from this
experiment revealed that purple tea was instead rich in
anthocyanins which are important phytochemicals found in
the novel purple-pigmented cultivars and not catechins as
earlier thought. Anthocyanins are a sub-class of flavonoids
synthesized via the phenylpropanoid pathway and are wide
spread in the animal kingdom where they present diverse
biological and biochemical interests [23]. The anthocyanidins
fractions in the processed unaerated and aerated tea products
from the purple coloured tea cultivar 306/1 were identified
and quantified by HPLC using pure anthocyanidin
/anthocyanin standards. The order of elution of the
anthocyanidin/anthocyanin was cyanidin-3-O-galactoside <
cyanidin-3-O-glucoside < delphinidin < cyanidin <
pelargonidin < peonidin < malvidin with malvidin being the
most predominant anthocyanin as shown in a representative
HPLC chromatogram of aerated tea from a purple tea cultivar
(Figure 1). The results obtained from this study collaborated
with those of Kerio et al., [12].
Retention time, min
Fig 1: A representative HPLC chromatogram of processed black tea sample from purple colored cultivar TRFK 306/1
KEY: X-axis = Retention time (min) Y-axis = Peak area
However, it was noted that anthocyanidins levels were
significantly higher (p≤ 0.05) in unaerated than in aerated
tea. This can be attributed to the tea processing procedures
where conversion of fresh tea leaf to the aerated tea
decreases the total monomeric anthocyanins. Kerio et al., [12]
observed similar results while characterizing anthocyanins in
Kenyan teas and attributed this to anthocyanin degradation
during the manufacture of black teas. Although no
mechanism has been developed so far on the anthocyanin
degradation, studies in other plants such as strawberry have
been attempted [24]. In their work Liu et al., [24], found out
that anthocyanins were rapidly degraded by PPO in the
presence of other polyphenol compounds such as catechins.
For example, cyaniding-3-O-rutinoside is degraded by PPO
in the presence of (-)-epicatechin and this is responsible for
the formation of dehydroepicatechin A [12, 24]. The coupled
oxidation reactions can be used to explain the sudden
reduction of anthocaynins in the aerated teas. However, this
is not the case in the unaerated teas since PPO is deactivated
by steaming freshly plucked tea leaf and therefore the
formation of the reactive O-quinones is stopped and
subsequently, anthocyanins are not degraded. This is a
hypothesis which needs further studies to establish the exact
cause of degradation.
3.1.3 Catechin fractions
The catechins identified in tea cultivars were EC, EGC, ECG
and EGCG. The two main gallated catechins present were
EGCG and ECG while the others were non-gallated. Beside
the main peaks identified, several minor peaks were also
detected, which indicated that other unidentified catechin
compounds existed in the tea extracts (Figure 2). There was
however great similarity in the HPLC chromatographic
pattern which indicated the close similarity in catechin
profiles in the teas studied.
Catechin fractions assayed in this study were statistically
different (p<0.05) as shown in Table 2. The results obtained
revealed that black (aerated) teas had lower catechin levels
than the green and white (non-aerated) teas (Table 2 and 3).
Individual catechins varied significantly (p<0.05) among the
teas with EGCG, GC and EGC levels being the highest and
+C, ECG and EC being less abundant. These results are
similar to those of Karori et al., [5]. The reduction in the
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Journal of Pharmacognosy and Phytochemistry
catechin content in black teas compared to the green tea due
to the monomeric flavan-3-ols undergoing polyphenol
oxidase-dependent polymerization. This results in the
formation of theaflavins, thearubigins, bisflavanols and other
complex oligomers [25, 26, 27].
Retention time, min
Fig 2: A representative high performance liquid chromatogram of green tea cultivar
Table 2: Individual catechin (%) levels of different green tea products analyzed
Individual Catechins
Clone EGCG% EGC% EC% ECG% C%
TRFK 301/4 3.800
m
2.725j 2.745
b
4.585ab 2.690a
TRFK 301/5 3.105n 4.000fg 3.280a 4.180c 1.850
b
TRFK 303/216 4.870hi 5.770
b
c 1.960cde 2.035ijkl 1.075de
TRFK 303/231 5.555de
f
4.410e
f
1.485fghij 2.465e
f
0.5150hij
TRFK 303/577 5.185fgh 3.695gh 1.775cdefg 7.215ab 1.240c
d
TRFK 303/745 3.830l
m
3.370hi 1.355ghij
k
1.455n 1.250c
d
TRFK 337/138 5.725cde 4.040fg 1.440fghij
k
2.275fghi 1.280c
d
TRFK 371/3 5.525de
f
4.585e 1.770cdefg 1.990kl 0.9600e
f
TRFK 430/3 4.990ghi 5.130
d
1.725cdefg 3.820ghij 0.3200j
TRFK 430/4 3.000n 2.600j 1.275hijkl 1.665mn 1.295c
d
TRFK 430/12 3.920kl
m
3.225i 1.460fghij 2.650e 0.6400ghi
TRFK 430/63 5.040ghi 5.790
b
c 1.695defg 2.275ffghi 0.5100hij
TRFK 430/90 5.305fg 3.215i 1.870cde
f
2.185ghij
k
0.7250fgh
TRFK 524/170 3.660
m
3.470hi 1.600efghi 2.380fg 0.785fg
TRFK 524/48 6.030
b
c 5.715
b
c 1.935cde 2.295fgh 0.4200ij
BBK35 5.825c
d
6.075ab 1.835cde
f
3.400
d
0.7050fgh
EPKC12 4.235kl 3.250i 1.555efghij 2.385fg 0.7100fgh
EPK D 99/10 4.305j
k
5.530c
d
1.475fghij 1.992l 0.9150e
f
EJULU-L 6.625a 5.800
b
c 2.145c 4.775a 1.310c
d
AHPS 15/10 5.855c
d
5.540c
d
1.635efghi 2.380fg 0.5800ghij
EPK TN 14/3 3.755
m
3.560hi 1.225ijkl 1.915l 0.3650j
TRFK 6/8 6.420ab 6.255a 2.120c
d
4.415
b
c 1.1495c
TRFK 31/8 5.350efg 5.710
b
c 1.025kl 1.865l
m
0.3600j
TRFK 31/11 4.885hi 4.005fg 1.135jkl 2.350fg 0.4250ij
TRFK 100/5 4.695ij 5.405c
d
1.365ghij
k
2.025jkl 0.4900hij
TRFK 306/1
2.58o
LSD=0.4119
CV=4.18%
Mean=4.771
1.490
k
LSD=0.4119
CV=4.56%
Mean=4.398
0.8450l
LSD=0.432
CV=12.41%
Mean=1.682
0.975o
LSD=0.2437
CV=4.61%
Mean=2.601
0.4750hij
LSD=0.2605
CV=14.20%
Mean=0.899
Means within a column followed by the same letter are not significantly different at p≤0.05 according to DMRT.
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Journal of Pharmacognosy and Phytochemistry
White tea which is predominantly manufactured from the young
apical hairy bud only, showed high levels of EGCG and ECG that
are present in higher amounts in fresh young leaves. This result
corroborates the findings of Saijo et al., [28] who determined the
chemical constituents of young tea leaves and the change occurring
during leaf development. The decrease in the gallic acid esters of
catechin such as EGCG and ECG during leaf development means
that there is a slow biosynthesis of gallic acid moiety in each
catechin gallate compared with dry matter production. Since
catechin biosynthesis is slower than dry matter production from
young leaves to the less young leaves, it is apparent that there is no
weight increase in the less young and mature leaves and as a result
catechin moves to other young leaves or are metabolized to other
products. This accounts for the change in catechin levels in various
leaf developmental stages and hence the levels of residual catechins
in tea manufactured from different leaf ages as exemplified in the
differences in catechin levels between white tea and the other types
of teas in this study. Consequently, because of the different rates of
growth among different cultivars, clones will accumulate varying
amounts of catechins in their leaves [4]. Cultivar AHP S15/10, from
which white tea is processed, is a fast growing clone compared to
cultivar TRFK 6/8 from which green and black teas were produced
[29]. The lower total polyphenol content in the white tea than in the
green teas in our assay can be ascribed to the fast growth of cultivar
AHP S15/10 compared to other cultivar such as TRFK 6/8.
Table 3: Individual catechin (%) levels of different black tea products analyzed
Individual Catechins
Clone EGCG% EGC% EC% ECG% C%
TRFK 301/4 0.2950j 0.5050j 0.4250
b
cdefg 0.8900defgh 0.0650
d
TRFK 301/5 0.6050gh ij 0.9450hij 0.6050
b
0.9700defg 0.3900c
d
TRFK 303/216 0.8600efgh 0.6700ij 0.2600fg 0.6850fghij 0.1650c
d
TRFK 303/231 0.8100efgh 1.590efghi 0.1750g 0.9650defg 0.2400c
d
TRFK 303/577 1.330ab 4.065a 0.5050
b
cde
f
1.095cde 0.4150c
d
TRFK 303/745 0.9000defgh 2.530cde 0.2efgh 0.4950ij 0.1150
d
TRFK 337/138 1.010
b
cde
f
2.965
b
c
d
0.5550
b
cde 1.020de
f
0.3950c
d
TRFK 371/3 1.010
b
cde
f
1.260ghij 0.3400cdefg 1.100cde 4.25efghi
TRFK 430/3 0.9150cdefgh 1.515fghi 0.4200
b
cdefg 0.8350efghi 0.1350
d
TRFK 430/4 0.5550hij 1.655efgh 0.3200defg 0.4250j 0.2500c
d
TRFK 430/12 0.8450efgh 1.695efgh 0.5800
b
c
d
0.6900fghij 0.2200c
d
TRFK 430/63 0.7050fghi 1.955efg 0.4350
b
cdefg 0.4350j 0.3250c
d
TRFK 430/90 1.295abc
d
3.805ab 1.300a 1.210
b
c
d
2.235a
TRFK 524/170 0.3450ij 1.150ghi j 0.5200
b
cde
f
0.9850defg 0.7300
b
c
d
TRFK 524/48 0.7250fghi 1.450fghij 0.5100
b
cde
f
0.6900fghij 0.8900abc
d
BBK35 0.5700hij 1.975efg 0.2950efg 0.8750defg 1.045abc
d
EPKC12 1.010
b
cde
f
2.055defg 0.4850
b
cde
f
1.105cde 5.215de
EPK D 99/10 0.7950efgh 1.130ghij 0.4850
b
cde
f
0.6900fghij 0.7450
b
c
d
EJULU-L 1.315ab 3.190abc 0.5900
b
c 1.810a 1.545abc
AHPS 15/10 1.000
b
cdefg 2.065defg 0.2750fg 0.5350hij 1.415abc
d
EPK TN 14/3 0.7900efgh 1.550fghi 0.3200defg 0.6300ghij 0.9000abc
d
TRFK 6/8 1.315ab 3.830ab 0.6050
b
1.405
b
c 1.900ab
TRFK 31/8 1.485a 4.755efg 0.3400cdefg 0.8250efghi 1.450abc
d
TRFK 31/11 1.170abcde 2.375cde
f
0.4700
b
cde
f
0.8750defgh 1.255abc
d
TRFK 100/5 0.8150efgh 1.730efgh 0.4700
b
cde
f
0.6900fghij 0.8900abc
d
TRFK 306/1
1.300abc
LSD=0.3962
CV=21.15%
Mean=0.914
5.235de
LSD=0.9682
CV=24.02%
Mean=1.956
0.4300
b
cdefg
LSD=0.2605
CV=27.29%
Mean=0.461
1.505ab
LSD=0.3684
CV=19.71%
Mean=0.901
1.185abc
d
LSD=1.401
CV=15.59%
Mean=0.795
Means within a column followed by the same letter are not significantly different at p≤0.05 according to DMRT.
3.1.4 Gallic acid
The results on gallic acid (3, 4, 5-trihydroxybenzoic acid) are
presented in Table 4. The results show significant (p≤0.05)
differences in gallic acid content between the black (aerated),
green (non-aerated) and white (non-aerated) teas. This can be
attributed to the fermentation reaction where a considerable
quantity of EGC, EC, EGCG and ECG are oxidized to form
theaflavins and their gallates and gallic acid is an important
molecule in this reaction. Despite the formation of free gallic
acid during fermentation through the process of degallation
of EGCG, the enhanced utilization of gallic acid in the
formation of TFs and TRs contributes to the decline of gallic
acid though Muthumani and Kumar [30] in their study argued
that the decline is not significant. The formation pathways of
gallic acid have been shown to include the hydration of
epigallocatechin gallate and degradation from the dimer of
epigallocatechin gallate [31]. However, since these catechins
are vital in black tea formation, the hydration that leads to the
formation of gallic acid is therefore suppressed at the
expense of theaflavins and thearubigin formation. The levels
of gallic acid and individual catechins in the black tea have
been shown to decrease with an increase in fermentation
temperature and time for different clones [19, 20, 27].
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Journal of Pharmacognosy and Phytochemistry
Table 4: Gallic acid (GA) (%) levels of different tea products
analyzed
Clone Green Tea Black Tea
TRFK 301/4 1.075a 0.1350n
TRFK 301/5 0.4500jkl 0.1550mn
TRFK 303/216 0.5300ghi 0.2350jlk
TRFK 303/231 0.5250ghi 0.2900hij
TRFK 303/577 0.5900efg 0.3450fgh
TRFK 303/745 0.4650ijkl 0.3050ghi
TRFK 337/138 0.6250def 0.4250de
TRFK 371/3 0.4850hijk 0.2750ijk
TRFK 430/3 0.4900hij 0.2750ijk
TRFK 430/4 0.4100l 0.1700lmn
TRFK 430/12 0.4500jkl 0.1850lmn
TRFK 430/63 0.8650b 0.5350bc
TRFK 430/90 0.4800hijk 0.4600d
TRFK 524/170 0.7600c 0.4800cd
TRFK 524/48 0.7750c 0.3600efg
BBK35 0.8000bc 0.6050a
EPKC12 0.6650d 0.6250a
EPK D 99/10 0.4250jkl 0.1650mn
EJULU-L 0.8200bc 0.4750cd
AHPS 15/10 0.6750d 0.4600d
EPK TN 14/3 0.5650fg 0.210klm
TRFK 6/8 0.7800c 0.5950ab
TRFK 31/8 0.5450gh 0.3800ef
TRFK 31/11 0.5700fg 0.2800hijk
TRFK 100/5 0.6400de 0.3150fghi
TRFK 306/1
0.4200kl
LSD=0.05613
CV=4.20%
Mean=0.353
0.4200de
LSD=0.05613
CV=7.18%
Mean=0.611
Means within a column followed by the same letter are not
significantly different at p≤0.05 according to DMRT.
3.1.5 Total theaflavins and total thearubigens levels of
black, green and white tea products
There was a significant difference in the total TFs and TRs
levels for Kenyan teas. Black tea had the highest levels of
total TFs and total TRs which ranged from 2.072% to
17.12%, respectively (Table 5). However, results from the
present study clearly showed that TRs were present in green
tea products and black and white (unaerated) tea products
from the purple coloured leaf tea clones. Further observations
revealed that in green and white teas, TRs were formed in the
presence of low levels of TFs unlike in black tea where the
TFs levels were slightly higher. Black tea therefore has high
levels of TFs and TRs that are the main fermentation
products as evident in table 5 below.
The variation in the polyphenolic composition of the
different tea products resulted from the leaf maceration
during manufacturing. The rolling and cutting of the tea
shoots in non-orthodox manufacture causes a release of
polyphenol oxidase which interacts with phenolic
compounds, one simple catechin and one gallocatechin, to
produce, theaflavins and thearubigins that possess a
benzotropolone skeleton [32, 33]. Owuor and Obanda [34]
investigated the use of green tea flavan-3-ols in predicting
black tea quality potential and revealed that a correct balance
of the trihydroxylated flavan-3-ols and dihydroxylated
flavan-3-ols was necessary to ensure maximum formation of
the theaflavins. The trihydroxylflavan-3-ols are oxidized
faster during the fermentation phase of black tea processing
explaining the high levels of EGCG and EGC in green tea
and the subsequent reduction in black tea. Theaflavins are
further oxidized to form thearubigins that are heterogeneous
in nature and contribute significantly towards taste, color and
body of tea [13, 19 & 35].
Results from the present study however clearly showed that
TRs were present in green tea. Further observation revealed
that in green tea, TRs were formed in the presence of low
levels of TFs unlike in black tea where the levels were almost
similar. This may suggest that theaflavins are not the only
source of thearubigins. Wilson and Clifford [36] explained the
factors affecting the formation and degradation of theaflavins
and thearubigins in black tea and observed that maximum
synthesis of theaflavins occurs when oxygen is in excess to
support benzotropolone ring formation. However, under
limiting oxygen concentration, polyphenol oxidase, which
has a high affinity for the substrate, has a preferential
demand for oxygen and theaflavins formation is suppressed
at the expense of catechin quinone formation. This
competition for oxygen is particularly noticeable during the
early stages of fermentation when the concentration of the
catechins is at its highest and enzyme turnover is unimpeded
by substrate availability. This occurs during green tea
manufacture since the enzyme is active before deactivation
through steaming. For this reason, high enzyme activity in an
already low oxygen concentration creates almost total
anaerobiosis, which suppresses benzotropolone ring
formation. Consequently as a result of this, thearubigens are
formed, mainly from gallocatechins since the simple
catechins are unable to react in benzotropolone ring
formation. Moreover, it might be possible to minimize
thearubigins formation by deactivating the enzyme
immediately after plucking through a steaming procedure
although this is hardly achievable during commercial tea
processing. Further research is desirable to explain in details
the existence of these thearubigins in green tea and the
importance of steaming during tea processing [14, 19, 26, 37, 39, 40].
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Journal of Pharmacognosy and Phytochemistry
Table 5: Total theaflavins and total thearubigins (%) levels of black, green, purple and white tea products processed from different cultivars
Clone Green Tea Black Tea
TF% TR% TF% TR%
TRFK 301/4 0.8500cde 9.705de
f
0.96k
r
13.11hij
TRFK 301/5 0.6750defghij 10.02cde 1.555jkl
m
15.55
b
c
d
TRFK 303/216 0.5950efghij 9.775cde
f
1.275q
r
14.59cde
f
TRFK 303/231 0.6500efghij 8.700hi 1.665defg 13.680hijkl
TRFK 303/577 1.025
b
c 11.48
b
1.525klmn 15.16
b
cde
TRFK 303/745 0.8000cdefgh 9.835cde
f
1.655efgh 13.41fghij
TRFK 337/138 0.6800defghij 8.880hi 1.800
b
16.26ab
TRFK 371/3 0.5750fghij 12.15a 1.685cde
f
15.54
b
c
d
TRFK 430/3 0.6550defghij 6.935
k
1.280
q
14.12efgh
TRFK 430/4 1.685a 9.74de
f
1.750
b
c 16.02ab
TRFK 430/12 0.8450cde 8.865ij 1.395
p
12.35j
TRFK 430/63 0.5500ghij 8.68hi 1.475no 12.74ij
TRFK 430/90 0.6850defghij 9.280fgh 1.605ghij 13.22ghij
TRFK 524/170 0.4800ij 9.800cde
f
1.21q
r
14.07efghi
TRFK 524/48 0.5350hij 10.14cde 1.415op 14.00efghi
BBK35 0.6200efghij 8.980ghi 1.590hij
k
13.30fghij
EPKC12 0.8100cdefg 10.18c
d
1.725c
d
14.56cdefg
EPK D 99/10 0.9200
b
c
d
12.00ab 1.695cde 17.12a
EJULU-L 1.135
b
10.37c 1.735
b
c 16.03ab
AHPS 15/10 0.7200defghi 10.17c
d
1.505mn 13.40fghij
EPK TN 14/3 0.8250cde
f
10.15c
d
1.515lmn 12.72ij
TRFK 6/8 1.150
b
12.28a 2.072a 16.13ab
TRFK 31/8 0.6750defghij 9.600de
f
1.470no 14.28defgh
TRFK 31/11 0.7350defghi 9.535efg 1.625fghi 13.90efghi
TRFK 100/5 0.4450j 8.05j 1.580ijkl 13.86efghi
TRFK 306/1
0.5350hij
LSD=0.2685
CV=3.43%
Mean=0.764
8.865hi
LSD=0.6144
CV=3.06%
Mean=9.767
1.240qr
LSD=0.0651
CV=2.25%
Mean=1.538
15.68bc
LSD=1.372
CV=4.61%
Mean=14.48
Means within a column followed by the same letter are not significantly different at p≤0.05 according to DMRT.
3.1.6 Theaflavin fractions
Theaflavins present in the assayed samples were fractionated
and found to contain the following fractions; theaflavin-3-
monogallate, theaflavin-3’-monogallate b, theaflavin-3, 3’-
digallate and theaflavin-3, 3- digallate. These fractions were
significantly different (p< 0.0001) in all tea cultivars. The
fractions differ in structure and previous studies have
elucidated them as shown in Figure 3.
Fig 3: Molecular structure of theaflavins. theaflavin (TF): R’= R’’=
OH, theaflavin-3-gallate (TF-3-G): R’=H, R’’= galloyl; theaflavin-
3’-gallate (TF-3’-G): R’ galloyl, R’’ H; theaflavin-3, 3’-digallate
(TF-dG): R’=R’’=galloyl.
Green tea catechins are oxidized and dimerized during the
manufacture of black tea to form orange red pigments
namely theaflavins (TF), which is a mixture of theaflavins
(TF1), theaflavin-3-gallate (TF2A), theaflavin-3’-gallate
(TF2B) and theaflavin-3,3’-digallate (TF3). These molecules
have recently aroused a lot of interest since they are thought
to have stronger biological properties than free theaflavins
due to the presence of gallic acid residues. In this study, the
theaflavin fractions correlated significantly well with each
other (Table 6) and this might explain why the activity of
black teas observed could not be entirely attributed to the
presence of theaflavins alone. The strong correlation of the
digillate fractions is a clear indication that they synergize
with other polyphenols to enhance the bioactivity. Obanda et
al., [20] observed that theaflavin gallate is the most astringent
and has been estimated to be 6.4 times more astringent than
simple theaflavin, and 2.88 times more astringent than either
theaflavin-3-monogallate or theaflavin-3’-monogallate. The
formation of a single theaflavin molecule requires a
dihydroxy and a trihydroxy flavan-3-ol, as follows:-
Epicatechin (EC) + Epigallocatechin (EGC) = simple
theaflavin (TF); EC + Epigallocatechin gallate (EGCg) =
Theaflavin-3-gallate (TF-3-g); Epicatechin gallate (ECG) +
EGC = Theaflavin-3’-gallate (TF-3’- g);ECG + EGCg =
Theaflavin-3, 3’-digallate (TF dg) [14]. Since theaflavin
fractions are proving to be essential biologically active
molecules, it is important to understand how the correct ratio
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Journal of Pharmacognosy and Phytochemistry
of dihydroxyl flavan-3-ols to trihydroxyl flavan-3-ols can be
utilized to produce value added teas with enhanced quality
and biological use.
Table 6: Correlation coefficient matrix analyses between various
theaflavin fractions
TF3MG TF3’MG TF33DG TF33’DG
1.000 0.962*** 0.873*** 0.961*** TF3MG
1.000 0.823** 0.908*** TF3’MG
1.000 0.937*** TF33DG
1.000 TF33’DG
** - Correlation significant at the p< 0.01 level
*** - Correlation significant at the p< 0.001 level
3.1.7 Antioxidant activity of green, black, purple and
white tea products
The polyphenolic composition of tea and especially its
catechins has aroused interest in their potential as radical
scavenging compounds. Data on antioxidant capacity is
presented in Table 7. Overall, green and white teas’ had
significantly (p< 0.05) higher antioxidant activity compared
to black tea. However, some black teas from cultivars such as
Ejulu-L, TRFK 6/8 and TRFK 306/1 had a higher antioxidant
activity compared to some unaerated green teas.
Table 7: Percent antioxidant capacity (AA) of green, black and
white tea products analyzed
Clone Green Tea Black Tea
AA AA
TRFK 301/4 89.80defgh 87.55de
TRFK 301/5 88.30h 84.10
f
TRFK 303/216 90.55cdefgh 88.90c
d
TRFK 303/231 90.75cdefgh 89.15
b
c
TRFK 303/577 90.85cdefg 89.65abc
TRFK 303/745 90.55cdefgh 88.75c
d
TRFK 337/138 91.25cde
f
90.00abc
TRFK 371/3 91.35cde
f
88.85c
d
TRFK 430/3 91.70
b
cde 88.45c
d
TRFK 430/4 89.10fgh 88.70c
d
TRFK 430/12 92.05abcde 88.80c
d
TRFK 430/63 88.65gh 86.00e
TRFK 430/90 89.60efgh 89.10
b
c
d
TRFK 524/170 91.90abcde 89.55abc
TRFK 524/48 91.15cdefg 89.60abc
BBK35 91.70
b
cde 89.15
b
c
EPKC12 91.90abcde 88.90c
d
EPK D 99/10 91.90abcde 88.55c
d
EJULU-L 94.05ab 91.10a
AHPS 15/10 92.50abc 89.80abc
EPK TN 14/3 89.00fgh 89.60abc
TRFK 6/8 94.30a 91.05a
TRFK 31/8 92.20abc
d
88.90c
d
TRFK 31/11 92.45abc 89.30
b
c
TRFK 100/5 91.40cde
f
89.70abc
TRFK 306/1
92.35abc
LSD=2.505
CV=1.33%
Mean=91.21
90.65ab
LSD=1.871
CV=11.46%
Mean=88.94
+Data has been arcsine transformed.
Means within a column followed by the same letter are not
significantly different at p≤0.05 according to DMRT.
Table 8 presents data on the correlation between tea
polyphenols contents and the antioxidant activity of different
types of tea products. Total catechins significantly (p<0.001)
correlated with antioxidant activity (r=0.909***). EGCG was
identified as the most potent antioxidant (r=0.968***,
p<0.001). EGC (r=0.659***), EC (r=0.780***), ECG
(r=0.454*) and GA (r=0.530*) contents also showed
significant influence on the antioxidant activity. Therefore,
the antioxidant activity was higher in tea extracts containing
high levels of EGCG, EGC, and ECG. These results are
similar to those of Gramza et al., [41] and Karori et al., [5].
This antioxidative effect of polyphenols has been attributed
to the phenolic hydroxyl groups in their structures that make
them potent free radical scavengers [42]. The antioxidative
properties of catechins are marked particularly by their
ability to inhibit free radical generation, scavenging free
radicals and chelate transition metal ions mainly, iron and
copper [43]. Nakagawa and Yokozawa [44] showed in their
study that green tea extracts significantly impaired nitrogen
oxide production in a concentration dependent manner and
showed a direct scavenging activity against super oxide
anion. On the basis of these results, it appears that the most
effective radical scavengers are catechins with a 3’, 4’ and
5’-trihydroxylated substitution pattern on the B ring and/or
hydroxyl group at C-3 position of the catechin structure
(Figure 3). This hydroxylation confers a higher degree of
stability on the catechin phenoxyl radical by participating in
electron delocalisation that is an important feature of the
antiradical potential. A study using electron spin resonance
showed that the presence of 3’, 4’, and 5’-trihydroxyl groups
attached to the B-ring of the flavan skeleton enhanced the
radical scavenging efficiency displayed by catechins,
compared to those with 3’, 4’-dihydroxyl groups. At the
same time the insertion of a galloyl moiety into three
positions of the C-ring exerted a synergistic impact on
superoxide anion scavenging activity [43, 45]. This explains
why radical scavenging is high in the gallocatechins namely
EGCG and EGC that are potent antioxidants [42, 46, 47].
To support this observation that EGCG and ECG were potent
antioxidants, the findings correlated well with a study
conducted in the Republic of Korea that showed that EGC
has the highest specific total oxy-radical scavenging capacity
against peroxyl radicals, hydroxyl radicals and peroxynitrite,
while ECG was the least effective among other catechins [48].
Raza and John [49] reported that tea catechins prevent
molecular degradation in oxidative stress conditions by
directly altering the subcellular ROS production, glutathione
metabolism and cytochrome P450 2E1 activity. These results
are promising for the chemotherapeutic use of tea catechins
in oxidative stress-related diseases.
Black teas analyzed in this study exhibited some antioxidant
activity with a high DPPH radical scavenging activity though
less than that of green, white and purple teas. During black
tea manufacture, the gallocatechins are first to be oxidized
and dimerised to TFs and TRs because of their high
oxidation potential and high concentration in the leaves.
These major phenolic compounds in black tea also
contributed significantly to the radical scavenging activity
namely TFs (r=0.803***, p<0.001), TRs (0.859***,
p<0.001) and GA (r=0.530*, p<0.05). Interestingly, TFs,
which are the major phenolic products in black tea, had a
higher radical scavenging activity compared to some of its
precursors ECG, EGC and EC (Table 8). This confirms that
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Journal of Pharmacognosy and Phytochemistry
conversion of catechins to TFs during black tea process did
not affect the radical scavenging potency. These observations
are consistent with those of Leung et al. [21]; Karori et al., [5]
and Wachira et al., [12] who showed that black tea posses
more or less the same antioxidant potency as catechins
present in green tea. EGCG and EGC contribute significantly
to the formation of TFs. These are B ring trihydroxylated
catechins, which are oxidized at a much faster rate than the B
ring dihydroxylated catechins including EC, ECG and +C
due to their lower oxidation potential [24]. TFs formed from
this reaction have hydroxyl groups (OH) considered
necessary for free radical scavenging activity. These
additional groups increase the total number of phenyl
hydroxyl groups and make the gallate containing catechins
and TFs more able to donate protons due to resonance
delocalization thereby expressing the observed antioxidant
activity of black tea. Similarly, gallic acid contributed
significantly to the radical scavenging activity in black tea
because it is a potent hydrogen donator to DPPH.
Table 8: Correlation coefficient matrix analyses between various tea chemical parameters
TP TFs TRs EGC EGCG ECG +C GA AA TC EC
1.00 0.818*** 0.663*** 0.718*** 0.803*** 0.715** 0.520** 0.626** 0.895*** 0.830*** 0.681*** TP
1.00 0.791*** 0.732*** 0.852*** 0.632* 0.452* 0.584* 0.803*** 0.808*** 0.689*** TFs
1.00 0.686** 0.843*** 0.619** 0.393* 0.378* 0.859*** 0.826*** 0.733*** TRs
1.00 0.847*** 0.505** 0.291* 0.552** 0.659*** 0.856*** 0.688*** EGC
1.00 0.719** 0.444*** 0.656** 0.968*** 0.956*** 0.777*** EGCG
1.00 0.744* 0.637*** 0.454* 0.8300*** 0.814*** ECG
1.00 0.472 0.232* 0.608*** 0.690*** +C
1.00 0.530* 0.685** 0.628** GA
1.00 0.909*** 0.780*** AA
1.00 0.895*** TC
1.00 EC
Additionally, the present study provided evidence of the
contribution of TRs towards the antioxidant activity of black
tea (r=0.803***, p<0.001). The antioxidant activity of TRs
can be explained by the presence of 3-OH groups, which are
more or less esterified by gallic acid in the TRs structure.
However, this is a highly speculative hypothesis since to date
there is no definite data on TRs structures [21, 50].
Thearubigins are assumed to be formed by the tea plant as a
defense mechanism [35]. Plants are thought to utilize the
strategy of plant browning as a defense tool. Therefore, the
action of a polyphenol oxidase enzyme on phenolic
secondary metabolites to produce a brownish coloration is
aimed at deterring pest organisms. The tea plant uses a
similar process to oxidize flavan-3-ols to thearubigins using
tea polyphenol oxidase to gain an evolutionary advantage by
deterring pest organisms [51]. Thearubigins account for
around 60% to70% of the dry weight of a typical black tea
infusion, and any attempt to understand the numerous
beneficial health effects of this beverage must take this class
of compounds into consideration. However, only a few
studies on the biological effects of thearubigins are available.
The reason for this lack of knowledge is obvious because
TRs have been mysterious for decades, with no clear
structural picture and only vague and sometimes
contradictory knowledge available. Therefore, there are no
defined compounds from the TR fractions that can be used
for biological testing, hence no standardized method for
obtaining extracts for biological testing. Despite these
limitations, TRs are useful molecules that need a detailed
study to establish their role in disease prevention. An attempt
so far made in this field has established that TRs extracts
lowered the expression of superoxide dismutase, a free
radical scavenger, in contrast to theaflavins, TR extracts were
able to inhibit DNA synthesis in U-937 leukemia cell lines,
giving a rationale for the anti-cancer activity of TRs [52]. Lin
et al., [3] showed that TR extracts were able to block nitric
oxide synthase in macrophages and therefore suppress the
anti-inflammatory response and multiple stages of
carcinogenesis. Data obtained from this study reveals that
different tea cultivars have different polyphenolic
composition which imparts on their unique biochemical
qualities.
4. Acknowledgement
We acknowledge the Tea Research Institute (TRI) and the
Division Research and Extension-Egerton University.
5. References
1. Wachira FN. Topical issue. TRFK Quarterly Bulletin
2002; 9:2-8.
2. Vo TD. An assessment on genetic diversity in Vietnam
tea using morphology, ISSR and microsatellites.
Dissertation 2006.
3. Lin YS, Tsai YJ, Tsay JS, Lin JK. Factors affecting the
levels of tea polyphenols and caffeine in tea leave.
Journal of Agricultural Food Chemistry 2003; 51:1864-
1873.
4. Wachira FN, Kamunya S. Kenyan teas are rich in
antioxidants. Tea 2005; 26:81-89.
5. Karori SM, Wachira FN, Wanyoko JK, Ngure RM.
Antioxidant capacity of different types of tea products.
African Journal of Biotechnology 2007; 6:2287-2296.
6. Mandel S, Packer L, Moussa YB, Weireb O. Proceedings
from the third international conference on mechanism
and action of nutraceuticals. Journal of Nutritional
Biochemistry 2005; 16:513-520.
7. Magoma GN, Wachira FN, Obanda M. The
pharmacological potential and catechins diversity
inherent in Kenyan tea. Tea 2001; 22:83-93.
8. Wachira FN, Ronno W. Tea research along value chains;
Constraints and potential areas of inter-institutional
collaboration. Tea 2005; 26:19-31.
~115~
Journal of Pharmacognosy and Phytochemistry
9. Picard D. The biochemistry of green tea polyphenols and
their potential application in human skin cancer.
Alternative Medical Review 1996; 1:31-42.
10. Lekh R, Okubo JT, Unlocking the secrets of green tea
and its health benefits in humans. Proceedings of
International Conference of Tea Culture and Science,
Shizuoka Japan 2004; 367-370.
11. Zuo Y, Chen H, Deng Y. Simultaneous determination of
catechins, caffeine and gallic acids in green, oolong,
black and Pu-err teas using HPLC with a photodiode
array detector. Talanta 2002; 57:307-316.
12. Hilton PJ, Palmer-Jones R. Relationship between the
flavanol composition of fresh tea shoots and theaflavin
content of manufactured tea. Journal of Science of Food
and Agriculture 1973; 24:813-818.
13. Kerio LC, Wachira FN, Wanyoko JK, Rotich MK.
Characterization of anthocyanins in Kenyan teas:
Extraction and identification. Food Chemistry 2012;
131:31-38.
14. Kilel EC, Wanyoko JK, Faraj AK, Wachira FN.
Variation in theaflavins and catechin contents in
processed leaf from the purple tea varieties in Kenya.
Tea 2012; 33:97-106.
15. Brand-Williams W, Cuvellier M, Berset C. Use of free
radical method to evaluate antioxidant activity.
Lebensmitted Wissen schafund Technologie 1995;
28:25-30.
16. Roberts EA, Smith RF. Spectrophotometric
determination of theaflavins and thearubigins in black
tea liquors in assessments of quality in tea. The Analyst,
1961; 86:94-98.
17. Ingrid P. Tea Flavonols; An overview. Victor Preedy
(EDS). Tea in health and disease prevention. Elsevier,
London, 2012, 73-79.
18. Jenny T. Mao MD. White tea: The plants, Processing,
Manufacturing and potential health benefits. In: Victor
Preedy (EDS), Tea in health and disease prevention.
Elsevier, London, 2012, 3-16.
19. Shitandi A, Ngure FM, Mahungu SM. Tea Processing
and its Impact on Catechins, Theaflavin and Thearubigin
Formation. Victor Preedy (Eds). Tea in Health and
Disease Prevention. Elsevier, London 2012; 193-206.
20. Obanda M, Owuor P, Mang’oka R. Changes in the
chemical and sensory quality parameters of black tea due
to variations of fermentation time and temperature. Food
Chemistry 2001; 75:395-404.
21. Leung LK, Yakun S, Chen R, Zhang Z, Hang YU, Chen
ZY et al. Theaflavins in black tea and catechins in green
tea are equally effective in antioxidant activity. Journal
of Nutrition 2001; 131:2248-2251.
22. Selena A, Steep JR. Green tea; the processing,
manufacturing and production. Victor Preedy (EDS).
Tea in health and disease prevention. Elsevier, London.
2012; 19-32.
23. He F, Pan QH, Ying S, Duan CQ. Biosynthesis and
Genetic Regulation of Proanthocyanidins in Plants.
Molecules 2008; 13:2674-2703.
24. Liu L, Cao S, Xie B, Sun Z, Wu J. Degradation of
cyanidin 3-rutinoside in the presence of (_)-epicatechin
and litchi pericarp polyphenol oxidase. Journal of
Agricultural and Food Chemistry 2007; 55:9074-9078.
25. Robertson A. The chemistry and biochemistry of black
tea production, the non-volatiles. In: Wilson, K.,
Clifford, M.N. (Eds.), Tea. Cultivation to consumption.
Chapman and Hall, London, UK, 1992, 55-601.
26. Mario V, Theo P, Mulder J, Molhuizen H. Theaflavins
from black tea, especially theaflavin-3-gallate, reduce
the incorporation of cholesterol into mixed micelles.
Journal of Agriculture and Food Chemistry 2008;
56:12031-12036.
27. Ngure M, Wanyoko K, Mahungu M, Shitandi A.
Catechins depletion patterns in relation to theaflavin and
thearubigins formation in tea. Journal of Food Chemistry
2009; 115:8-14.
28. Saijo R, Kato M, Takeda Y. Changes in the contents of
catechins and caffeine during leaf development.
Proceedings of International Conference of Tea Culture
and Science, Shizuoka Japan, 2004; 251-254.
29. Wachira FN, Kamunya S, Karori S, Chalo R, Maritim.
Tea Plants: botanical Aspects. Victor Preedy (EDS). Tea
in health and disease prevention. Elsevier, London,
2012, 3-16.
30. Muthumani T, Senthil KR. Influence of fermentation
time on the development of compounds responsible for
quality in black tea. Food Chemistry 2006; 89:98-102.
31. Chen CY, Ren JL, Viola SY, Lee JD, Preedy VR, Jason
TC et al. Gallic Acid in Old Oolong Tea. Victor Preedy
(EDS), Tea in health and disease prevention, Elsevier,
London, 2013, 447-550.
32. Reeves SG, Owuor PO, Othieno CO. Biochemistry of
black tea manufacture. Tropical Science 1987; 27:121-
133.
33. Mahanta PK, Hemanta BK. Theaflavins pigment
formation and polyphenol oxidase activity as a criterion
of fermentation in orthodox and CTC Teas. Journal of
Agriculture and Food Chemistry 1992; 40:860-863.
34. Owuor PO, Obanda M. The use of green tea (Camellia
sinensis) leaf flavan-3-ol composition in predicting plain
black tea quality potential. Food Chemistry 2006;
100:873-884.
35. Harbowy ME, Balentine DA. Tea chemistry. Critical
Review on Plant Science, Vol 16, Taylor and Francis,
1997, 415-480.
36. Wilson KC, Clifford MN. Tea cultivation to
consumption. Chapman and Hall, London, 1992, 553-
593.
37. Bradfield AE. Some recent developments in the
chemistry of tea. Chemical Indica 1946; 65:242-246.
38. Bokuchava MA, Skobeleva NI. The chemistry and
biochemistry of tea and tea manufacture. Advanced
Food Research 1969; 17:215-292.
39. Owuor PO, Horita H, Tsushida T, Murai T. Comparison
of the chemical composition of black teas from main
black tea producing parts of the world. Tea 1986a; 7:71-
78.
40. Owuor PO, Mutea MS, Obanda AM, Reeves SG. Effects
of withering on some quality parameters of black tea.
Preliminary results. Tea 1986b; 7:13-17.
41. Gramza A, Khokhar S, Yoko S, Swiglo GA, Hes M,
Korczak J et al. Antioxidant activity of tea extracts in
lipids and their correlation with polyphenolic content.
European Journal of Science and Technology 2006;
108:351-362.
42. Amie D, Amie DD, Beslo D, Trinajstie D. Structure-
radical scavenging activity relationships of flavonoids.
Croatica Chemica Acta 2003; 76:55-61.
~116~
Journal of Pharmacognosy and Phytochemistry
43. Bahorun T, Vidushi NB, Naushad AT, Jhoti S, Amitabye
LR, Okezie IA et al. Bioactive Phytophenolics and
Antioxidant Functions of Aqueous and Organic Tea
Extracts. Victor Preedy (Eds). Tea in health and disease
prevention. Elsevier, London, 2013, 233-246.
44. Nakagawa T, Yokozawa T. Direct scavenging of nitric
oxide and superoxide by green tea. Food and Chemical
Toxicology 2002; 40:1745-1750.
45. Unno T, Yayabe F, Hayakawa T, Tsuge H. Electron spin
resonance spectroscopic evaluation of scavenging
activity of tea catechins on superoxide radicals generated
by a phenazine methosulfate and NADH system. Food
Chemistry 2002; 76:259-265.
46. Rao TP, Lekh RJ, Takado Y. Green tea catechins against
oxidative stress of renal diseases: Protective Effects of
Tea on Human Health. Edited by Navendev, K. Jain,
Maqsood Siddiqi and John Weis Burger. London: CAB.
2006; 2:109-119.
47. Kang R, Livesey KM, Zeh HJ, Loze MT, Tang D.
HMGB1: A novel Beclin 1-binding protein active in
autophagy. Autophagy 2010; 6:1209-1211.
48. Raza H, John A. In vitro protection of reactive oxygen
species-induced degradation of lipids, proteins and 2-
deoxyribose by tea catechins. Food and Chemical
Toxicology 2007; 45:1814-1820.
49. Li D, Wang N. Wan X. Thearubigins: The complicated
polyphenols in black tea. International Symposium on
Innovation in Tea Science and Sustainable Development
in Tea Industry, 2005, 647-658.
50. Martinez MV, Whitaker JR. The biochemistry and
control of enzymatic browning. Trends in Food Science
and Technology 1995; 6:195-200.
51. Das M, Chaudhuri T, Goswami SK. Studies with black
tea and its constituents on leukemic cells and cell lines.
Journal of Experimental and Clinical Cancer Research
2002; 21:563-568.
