Food Chemistry 141 (2013) 3744–3751

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Food Chemistry
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Polyphenolic compounds and antioxidant activity of CTC black tea

of North-East India
Lakshi Prasad Bhuyan a,⇑, Santanu Sabhapondit a, Binoti Devi Baruah a, Cinmoy Bordoloi a,
Ramen Gogoi a, Pradip Bhattacharyya b,**
a
Tocklai Experimental Station, Tea Research Association, Jorhat 785008, Assam, India
b
Indian Statistical Institute, North-East Center, Tezpur 784028, Assam, India

a r t i c l e i n f o a b s t r a c t

Article history: Sixty black tea samples from different agro climatic zones of northeast India were assessed for biochem-
Received 16 April 2013 ical constituents that determine quality and also influence organoleptic perception. The antioxidant
Received in revised form 14 June 2013 activities such as a,a-diphenyl-b-picrylhydrazyl (DPPH) free radical scavenging, lipid peroxidation inhi-
Accepted 18 June 2013
bition, nitrite scavenging and super oxide scavenging, of the collected samples were analysed. Out of the
Available online 27 June 2013
four antioxidant activities, the super oxide scavenging activity was lowest and nitrite scavenging activity
was highest. Theaflavin was significantly and positively correlated with nitrite scavenging and lipid per-
Keywords:
oxidation inhibition activities. Thearubigins showed a significant positive correlation only with nitrite
CTC black tea
Chemical composition
scavenging activity. Correlations between relative antioxidant capacity index (RACI) and TF, TR and tast-
Antioxidant activity ers’ quality were positive and significant. Tea tasters’ parameters were significantly and positively corre-
Northeast India lated with each other. Principal component analysis showed that Upper Assam, North Bank and South
Bank produced better quality tea than other regions with respect to TF, TR, RACI and tasters’ quality.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction theaflavins (golden yellow) and thearubigins (orange brown)

Tea (Camellia sinensis) is a perennial crop and the second most
popular drink after water, consumed by over two-third of the
world’s population (Bhuyan et al., 2009; Gupta, Saha, & Giri,
2002). It is grown in over thirty countries, exclusively in the sub-
tropical and tropical zones. India is one of the main tea growers,
exporting more than 12% of the world’s tea and with over
521,000 ha under cultivation (Gohain et al., 2012).
Black tea quality depends mainly on the components of the tea
infusions and tea prices vary mostly depending on the quality
which has traditionally been assessed by a tea taster who has
developed a language of his own to describe various quality attri-
butes of a tea infusion. In assessment of tea quality or price, profes-
sional tea tasters mainly consider the tea liquor characteristics
(Bhuyan et al., 2009). Quality of black tea is a complex phenome-
non and depends on inherent chemical composition of green leaf
and chemical components produced during processing. The quality
of CTC (crush, tear and curl) black tea mainly depends on flavanol
composition present in the cell vacuole of green leaf, oxidised dur-
ing processing leading to the formation of black tea pigments,
⇑ Corresponding author.
E-mail addresses: bhuyanlp@yahoo.com (L.P. Bhuyan), b_pradip@rediffmail.com
(P. Bhattacharyya).
⁄⁄
Co-corresponding author.
which are largely responsible for brightness, briskness, colour
and strength (Bhuyan et al., 2009; Robertson, 1992; Senthil Kumar
et al., 2011). The typical composition of black tea beverage contains
8% catechins, 10% flavonol glycosides, 12% theaflavins, and 70%
thearubigins (Wiseman, Waterhouse, & Korver, 2001). Liang, Lu,
Zhang, Wu, and Wu (2003) showed that theaflavin makes a greater
contribution to the brightness of black tea infusion.
Tea has long been reported as a rich source of antioxidants. The
antioxidant activity of black tea, attributed to its polyphenols, may
account for prevention of chronic diseases (Gardner, Ruxton, &
Leeds, 2007). Theaflavins content in black teas possess antioxida-
tive properties. Theaflavins have more hydroxyl groups, which
are considered to be necessary for exerting scavenging activity
(antioxidative properties) (Luczaj & Skrzydlewska, 2005). The for-
mation of theaflavins depends upon geographical area of produc-
tion and genetic variations of cultivar. Theaflavins formation is
also dependent upon the environmental condition (McDowell, Fea-
kes, & Gay, 1991). The relationship of theaflavins with tea tasters’
quality is also varied. Roberts (1962) reported a very good correla-
tion of theaflavin with high cash value of CTC black tea. Tea com-
position and also the taste and aroma vary with climate, soil,
cultural practices, cultivar (variety), season etc. (Sarmah & Rao,
2009). There is a significant relationship between theaflavins and
tasters’ quality (Owuor et al., 2006).

0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.06.086
 L.P. Bhuyan et al. / Food Chemistry 141 (2013) 3744–3751
People consider tea mostly as a pleasant drink only but the sci-
entific community has recently discovered the therapeutic poten-
tial of this beverage. Theaflavins and thearubigins present in
black tea are mainly responsible for the unique taste of tea and
its health benefit. These compound possess a high antioxidant
capacity and biological activities like free radical scavenging activ-
ity, superoxide scavenging activity, nitrite scavenging activity and
inhibition of lipid peroxidation (Dufresne & Farnworth, 2001;
Gardner et al., 2007; Higdon & Frei, 2003). In general, antioxidant
activity may be closely related to preventive effects on various dis-
eases. Theaflavins, thearubigins as well as un-oxidised catechins
are the major polyphenol constituents of black tea mainly respon-
sible for antioxidant actions. Antioxidative properties of black tea
are manifested by its ability to inhibit free radical generation, scav-
enge free radicals and chelate transition metal ions (Luczaj &
Skrzydlewska, 2005). Thus, they are capable of stabilizing or deac-
tivating free radicals, which are generated by metabolic pathway
within the body tissue.
DPPH assay is one of the widely used methods to test the anti-
oxidative activity of the sample due to its stability, simplicity, and
the short time required for analysis. Basically the antioxidant
capacity of the samples is measured through their ability to reduce
the DPPH by donating the hydrogen atom and can be determined
through the discoloration of the mixture using spectrophotometer.
However, even in the absence of active superoxide dismutase en-
zyme, tea extract can scavenge superoxide due to the presence of
antioxidants (Marklund & Marklund, 1974). Nitrite as food preser-
vative reacts with amines present in protein-rich foods in the
acidic pH of the stomach, leading to formation of carcinogenic
Fig. 1. Locations of study areas: (1) Upper Assam, (2) North Bank, (3) South Bank and (4) Barak valley from Assam and (5) Terai and (6) Dooars from West Bengal, India.
3745
nitrosamines (Chawla, Jo, Kang, Kim, & Byun, 2003). When food
containing nitrites is consumed, the formation of nitrosamine is
expected to take place in the stomach (Chawla et al., 2003). It
has been reported that phenolic compounds have higher nitrite
scavenging effects under low pH conditions (Kang, Park, & Lee,
1996). The free radicals are known to cause peroxidation of mem-
brane lipids, leading to membrane damage. The antioxidants pres-
ent in tea scavenge the free radicals, thereby inhibiting lipid
peroxidation (Higdon & Frei, 2003; Mishra, Gautam, & Sharma,
2006). Examination of the scavenging activity described here has
been useful for providing basic information on the scavenging abil-
ity of tea polyphenols and their structure–activity relationships
(Nanjo et al., 1996). To enable more comprehensive comparison
between the investigated samples, the relative antioxidant activity
index (RACI), based on calculation assigning equal weight to all
antioxidant activity (AO) tests applied, has been introduced (Gorja-
novic et al., 2012).
Studies have shown that the black tea quality is influenced by
many factors such as season and altitude, genetic make-up of the
plant (Owuor & Obanda, 1995) and the region of production and
climate (Owuor, Obanda, Nyirenda, & Mandala, 2008). However,
there is not a great deal of information on the effects of these fac-
tors on the total phenolic concentration in tea and subsequent
antioxidant activity in tea (Jayasekera, Molan, Garg, & Moughan,
2011). Therefore, we investigated the phenolic compounds and
antioxidant activity of CTC black tea of six tea growing regions of
Assam and West Bengal, India. The relationship between the phe-
nolic contents of tea infusions and antioxidant potential was also
investigated. Another important objective of this research was to
3746 L.P. Bhuyan et al. / Food Chemistry 141 (2013) 3744–3751
evaluate the impact of black tea phenolic compounds on antioxi-
dant activity and tea taster’s quality.
2. Materials and methods
A total of 60 dryer mouth CTC (Crush Tear and Curl) black tea
samples were collected from commercial gardens representing
six agro climatic regions of Assam (Upper Assam, South Bank,
North Bank and Barak valley) and West Bengal (Dooars and Terai),
India, during 2011.
Theaflavin (TF) and thearubigin (TR) were estimated by the
method of Ullah, Gogoi, and Baruah (1984). Total polyphenol con-
tent was determined using the method BS ISO 14502-1: 2005.
Four antioxidant activities, viz. free radical (DPPH) scavenging
activity, super oxide scavenging activity, lipid peroxidation inhibi-
tion activity and nitrite scavenging activity were determined by
the method described by Mishra et al. (2006).
The sensory tasting (organoleptic evaluation) was performed as
described by Bhuyan et al. (2009).
Relative antioxidant activity index (RACI) was calculated from
the mean value of standard scores of different samples as proposed
by Gorjanovic et al. (2012). Standard scores were calculated by the
following equation
xi  x
zi ¼
s
where, zi = standard score of the sample i; xi = value of sample i;
x ¼ mean; s = standard deviation.
Statistical analyses such as factor (principal component analy-
sis) and least significant difference (ANOVA) were carried out using
SPSS statistical package. Analysis of variance and least significant
difference (LSD) tests were applied to evaluate differences between
Fig. 2. Biochemical parameters of tea from different sampling sites (LSD = least significant difference at 5% level).
the variables. A Pearson-correlation analysis was carried out to
check the significances of the linear relations between obtained
variables.
The characterisation data were analysed through PCA using fac-
tor extraction with an eigenvalue > 1 and varimax rotation. All data
were log-transformed to achieve closeness to normal distribution
and all the variables were standardised by ‘z-score’ analysis to
measure the similarity between the samples. Furthermore, we
have done factorscore analysis to understand the source relevance
of black tea samples collected from six different localities. The raw
data were used for graphical presentation (Box–Whisker diagram);
whereas the transformed (log-transformed and standardised) ones
were used for factor analysis (principal components analysis).
3. Results and discussion
Over the years several studies have established the relation-
ships between biochemical components and quality of CTC black
tea (Bhuyan et al., 2009; Senthil Kumar et al., 2011) The work on
antioxidant activity of biochemical components of CTC black tea
is limited. Thus attempts have been made for determination of bio-
chemical components, antioxidant activity and tasters’ evaluation
and their relationship in CTC black tea of six tea growing regions
ð1Þ of Assam and West Bengal, India. As the samples were collected
from different regions, to understand the environmental impact,
monthly average maximum and minimum temperatures and total
rainfall per month in 2011 along with a map of sample collection
areas are presented in Fig. 1. The average monthly maximum
temperature of Barak Valley was higher than that of other regions.
Terai region also showed high temperature next to Barak Valley
during the harvesting period of tea, i.e. from March to September.
 L.P. Bhuyan et al. / Food Chemistry 141 (2013) 3744–3751
Although the rainfall of Terai and Dooars regions was higher, no
uniform trend for the regions was observed.
Total polyphenol, theaflavin and thearubigin contents of CTC
black tea of Upper Assam, North Bank, South Bank, Barak Valley,
Terai and Dooars are presented in Fig. 2. Significant variations of
the components amongst the regions were observed. The ranges
of total polyphenol content of Upper Assam, North Bank, South
Bank, Barak Valley, Terai and Dooars regions were 119.9–
178.8 mg/g (mean 145.0 mg/g), 116.1–159.5 mg/g (mean 134.8
mg/g), 105.3–191.2 mg/g (mean 143.4 mg/g), 100.2–148.1 mg/g
(mean 125.2 mg/g), 117.9–178.1 mg/g (mean 140.5 mg/g) and
99.0–155.8 mg/g (mean 133.6 mg/g) respectively. The black tea
polyphenols are responsible for visual brightness and depth of col-
our and also perceived strength and mouth feel of the liquor (Bhu-
yan et al., 2009). Theaflavins (TF), the basic quality components of
CTC black tea of Brahmaputra Valley, i.e. Upper Assam, South Bank,
and North Bank were higher than the regions of Barak Valley, Terai
and Dooars, with levels of 9.7–13.7 mg/g (mean 11.4 mg/g), 8.9–
12.3 mg/g (mean 10.6) and 9.6–15.5 mg/g (mean 11.5 mg/g)
Fig. 3. (A) Factor loading pattern of studied parameters after varimax rotation; (B) a bi-plot factor score for each site. Values within bracket indicate eigenvector of individual
variables.
3747
respectively and comparable. The levels of Barak Valley, Terai
and Dooars were 7.3–10.8 mg/g (mean 8.9 mg/g), 7.1–10.1 mg/g
(mean 8.4 mg/g) and 7.6–10.2 mg/g (mean 8.7°mg/g) respectively.
Another quality parameter, thearubigins (TR) of Upper Assam,
North Bank, South Bank and Dooars tea are comparable and their
levels were 118.3–172.5 mg/g (mean 146.4 mg/g), 102.9–
177.9 mg/g (mean 151.4 mg/g), 92.4–196.9 mg/g (mean 151.7
mg/g) and 112.0–173.7 mg/g (mean 144.1 mg/g) respectively. TR
of the Terai region tea contained the lowest (48.7–106.2 mg/g
and mean 67.0 mg/g) amongst the regions followed by Barak Val-
ley (60.3–127.8 mg/g and mean 99.2 mg/g). Total polyphenols
and/or TF and TR are formed during fermentation (oxidation) stage
of black tea processing and their precursors and the related en-
zymes affecting their formation and degradation (Bhuyan et al.,
2009). The variations in these chemicals of different regions might
be due to different raw materials, environment, soil status, manu-
facturing processes, etc.
Factor scores, calculated using factor loading matrix, represent
relative intensities of each factor (Fig. 3). The tea samples from
3748 L.P. Bhuyan et al. / Food Chemistry 141 (2013) 3744–3751
Upper Assam, North Bank, and South Bank showed the highest
factor 1 and factor 2 scores, indicating the high content of theaf-
lavins, thearubigins and their ratio. Tea samples from Barak Valley
and Terai region showed the lowest scores for factor 1 and factor
2, indicating lower theaflavin and thearubigin contents. Upper As-
sam, North Bank and South bank are situated in geographically
and climatically similar regions. Their soil conditions are also
much similar (Mann & Gokhale, 1960). These are the responsible
factors for tea quality. Although Terai region and Barak Valley
areas are far apart from each other, their climatic conditions are
almost similar, which reflects that the tea samples from these
two regions are comparable with respect to biochemical parame-
ters. Even though Dooars region is nearby the Terai region, they
are climatically different from each other, which reflect in their
different tea qualities (Mcdowell et al., 1991; Owuor, Wachira, &
Ng’etich, 2010; Owuor et al., 2006). As observed in black tea, ex-
tracts with higher antioxidant activity also had higher polyphenol
content, confirming that polyphenols are likely to contribute to
the radical scavenging activity (Miliauskas, Venskutonis, & van
Fig. 4. Scavenging activities of tea from different sampling sites (LSD = least significant difference at 5% level).
Beek, 2004). This result is in agreement with Mello, Alves,
Macedo, and Kubota (2004), who observed correlation between
total polyphenol content and antioxidant activity which is very
good for black tea.
Four antioxidant activities namely free radical (DPPH) scaveng-
ing activity, superoxide scavenging activity, nitrite scavenging
activity and lipid peroxidation inhibition activity of CTC black tea
from Upper Assam, South Bank, North Bank, Barak valley, Terai
and Dooars regions are presented in Fig. 4. Barak Valley and Terai
region tea had higher DPPH scavenging activity than tea from other
regions, their levels being 85.38–89.03% (mean 86.73%) and
84.50–88.03% (mean 86.37%) respectively. The mean value of
Dooars region tea was the lowest (77.57%) with a large range of
minimum (69.57%) and maximum (87.91%) values. This may be
due to high variation in total polyphenol contents of the samples
The DPPH free radical scavenging activities of Upper Assam, North
Bank and South Bank were comparable, with the levels of
79.56–86.07% (mean 82.68%), 73.70–87.11% (mean 80.95%) and
75.12–86.02% (mean 80.92%) respectively.
 L.P. Bhuyan et al. / Food Chemistry 141 (2013) 3744–3751
Almost similar trend of superoxide scavenging activity with
DPPH scavenging activity was observed amongst the regions. The
highest activity was observed in Barak Valley followed by Terai
and other regions. The ranges of Upper Assam, North Bank, South
Bank, Barak Valley, Terai and Dooars regions were 58.43–73.15%
(mean 66.19%), 58.77–73.49% (mean 66.84%), 67.36–89.97% (mean
74.41%), 61.95–93.87% (mean 80.91%), 72.34–85.22% (mean
77.39%) and 63.72–74.12% (mean 68.45%) respectively.
Nitrite scavenging activity was found to be highest in South
Bank followed by Dooars, Upper Assam, North Bank, Barak Valley
and Terai, with levels of activity 90.34–99.52% (mean 98.06%),
96.16–98.18% (mean 97.44%), 93.42–98.90% (mean 96.55%),
87.29–96.01% (mean 92.18%), 88.83–98.88% (mean 92.16%) and
80.39–89.18%, (mean 86.24%) respectively. The variations in the ni-
trite scavenging activities might be due to the variations in the lev-
els of theaflavin and thearubigin contents of black tea of different
regions.
Lipid peroxidation inhibition of tea from Brahmaputra Valley
i.e. Upper Assam, North Bank and South Bank was higher than
tea from other three regions i.e., Barak Valley, Terai and Dooars.
High activity of lipid peroxidation inhibition of Brahmaputra valley
Fig. 5. Taster evaluation of processed tea from different sampling sites (LSD = least significant difference at 5% level).
3749
tea might be due to higher amount of theaflavin content than other
regions. The inhibition ranges of lipid peroxidation of Upper As-
sam, North Bank, South Bank, Barak valley, Terai and Dooars re-
gions tea were 85.68–98.01% (mean 90.78%), 70.57–89.50%
(mean 84.67%), 70.21–93.33% (mean 86.20%), 72.33–86.41% (mean
81.13%), 72.06–87.96% (mean 78.96%) and 70.02–76.05% (mean
72.39%) respectively.
For the acceptance of tea and to understand the effect of bio-
chemical components on quality, the role of tea taster is very
important. Four liquor characteristics of tea, namely brightness,
briskness, strength and quality of tasters’ assessment of black tea
from Upper Assam, North Bank, South Bank, Barak Valley, Terai
and Dooars are presented in Fig. 5. South Bank showed the highest
of all the four Tea Tasters, characteristics followed by North Bank,
Upper Assam, Barak Valley, Dooars and Terai. Quality of tea charac-
terised by Tea Tasters is the masking effect of biochemicals present
in tea, out of which theaflavin and thearubigin are largely respon-
sible for brightness, briskness and strength (Bhuyan et al., 2009).
The correlations between biochemical components, antioxidant
activity and tasters’ quality are presented in Table 1. There is no
correlation between TP, TF or TR contents with DPPH activity. It
3750 L.P. Bhuyan et al. / Food Chemistry 141 (2013) 3744–3751

Table 1
Correlation coefficient amongst biochemical parameters, scavenging activities and tasters’ quality (n = 60).

Parameters DPPH Super oxide Nitrite Lipid peroxidation RACI TP TF TR Brightness Briskness Strength
scavenging scavenging scavenging inhibition activity
activity activity activity
TP 0.12 0.2 0.14 0.09 0.04
TF 0.14 0.21 0.39** 0.46** 0.26* 0.04
TR 0.38** 0.34** 0.70** 0.13 0.05 0.12 0.51**
Brightness 0.11 0.14 0.57** 0.44** 0.39** 0.04 0.54** 0.54**
Briskness 0.23 0.11 0.48** 0.41** 0.29* 0.20 0.43** 0.43** 0.83**
Strength 0.1 0.13 0.44** 0.48** 0.35** 0.07 0.44** 0.43** 0.83** 0.87**
Quality 0.17 0.11 0.46** 0.45** 0.32* 0.11 0.45** 0.43** 0.85** 0.96** 0.96**
*
Significant at 5% level.
**
Significant at 1% level.

may be that the above parameters did not have the effect of the
DPPH scavenging activity of tea (Jayasekera et al., 2011). These re-
sults indicate that above polyphenols are not major contributors to
the DPPH antioxidant capacity of fermented teas. Highly significant
positive correlations of theaflavins with two antioxidant activities
like nitrite scavenging activity and lipid peroxidation inhibition
activity and tasters’ quality were observed. Significant positive cor-
relations of thearubigins with nitrite scavenging activity and tast-
ers’ quality were also observed. Significant positive correlation
between theaflavins and tasters’ quality resembles the earlier find-
ings (Owuor et al., 2006; Senthil Kumar et al., 2011). On the basis of
results of all assays employed, the unit less relative antioxidant
activity index (RACI) has been calculated by assigning equal weight
to each assay (Gorjanovic et al., 2012). Previously, RACI was effi-
ciently used to compare the antioxidant activities of numerous
samples obtained from multiple assays (Gorjanovic et al., 2012).
Here, it was introduced to enable more comprehensive comparison
between investigated samples as well as comparison between
applied assays. Like nitrite and lipid scavenging activities, RACI
was also higher in upper Assam, South Bank and Barak Valley tea
extracts compared to other regions. This high content of RACI
may be due to the corresponding theaflavin contents. This is also
supported by the significant positive correlation of RACI with
theaflavins (0.26⁄) (Table 1). RACI also had positive significant cor-
relation with Tasters’ quality (0.32⁄).
Linear correlation analysis showed that there were significant
linear relationships between the individual tea quality attributes,
and the total quality score was significantly correlated to the indi-
vidual tea quality attributes (Table 1). This suggests that a good tea
should have good individual quality attributes.
4. Conclusion
The study demonstrates considerable variations in polyphenol
contents and antioxidant properties of CTC black tea samples of
the regions. These variations may be due to differences in cultivar
variety, climate, geographical location, soil condition, processing
conditions etc. The variations in TF and TR contents are responsible
for quality differences of black tea from the regions. These two
components of the three regions of Brahmaputra Valley (Upper As-
sam, North Bank and South Bank) are comparable and higher than
the other regions. The tea tasters’ sensory assessment is in confor-
mity with these biochemical findings. TF content is significantly
correlated with nitrite scavenging activity and lipid peroxidation
inhibition activity of black tea, whereas TR content is significantly
correlated with nitrite scavenging activity only. RACI is also signif-
icantly and positively correlated with TF, TR and tasters quality
parameters. Hence, TF and TR are the key components of CTC black
tea which not only influence the tasters sensory evaluation but also
have high antioxidant property. Determination of the theaflavin
profiles and elucidation of the role of the respective theaflavins
on the antioxidant activity can be the future course of study.
Acknowledgements
The authors are grateful to the Director, Tocklai Experimental
Station, Tea Research Association for permission to publish the
results and also wish to thank Dr. R.M. Bhagat, Deputy Director (re-
search), Tocklai Experimental Station for providing meteorological
data of the regions. The authors are also grateful to Tea Board, India
for financial assistance to carry out the work.
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