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Food Chemistry 141 (2013) 3744–3751

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

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,**
Tocklai Experimental Station, Tea Research Association, Jorhat 785008, Assam, India
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-
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)

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

0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
L.P. Bhuyan et al. / Food Chemistry 141 (2013) 3744–3751 3745

People consider tea mostly as a pleasant drink only but the sci- nitrosamines (Chawla, Jo, Kang, Kim, & Byun, 2003). When food
entific community has recently discovered the therapeutic poten- containing nitrites is consumed, the formation of nitrosamine is
tial of this beverage. Theaflavins and thearubigins present in expected to take place in the stomach (Chawla et al., 2003). It
black tea are mainly responsible for the unique taste of tea and has been reported that phenolic compounds have higher nitrite
its health benefit. These compound possess a high antioxidant scavenging effects under low pH conditions (Kang, Park, & Lee,
capacity and biological activities like free radical scavenging activ- 1996). The free radicals are known to cause peroxidation of mem-
ity, superoxide scavenging activity, nitrite scavenging activity and brane lipids, leading to membrane damage. The antioxidants pres-
inhibition of lipid peroxidation (Dufresne & Farnworth, 2001; ent in tea scavenge the free radicals, thereby inhibiting lipid
Gardner et al., 2007; Higdon & Frei, 2003). In general, antioxidant peroxidation (Higdon & Frei, 2003; Mishra, Gautam, & Sharma,
activity may be closely related to preventive effects on various dis- 2006). Examination of the scavenging activity described here has
eases. Theaflavins, thearubigins as well as un-oxidised catechins been useful for providing basic information on the scavenging abil-
are the major polyphenol constituents of black tea mainly respon- ity of tea polyphenols and their structure–activity relationships
sible for antioxidant actions. Antioxidative properties of black tea (Nanjo et al., 1996). To enable more comprehensive comparison
are manifested by its ability to inhibit free radical generation, scav- between the investigated samples, the relative antioxidant activity
enge free radicals and chelate transition metal ions (Luczaj & index (RACI), based on calculation assigning equal weight to all
Skrzydlewska, 2005). Thus, they are capable of stabilizing or deac- antioxidant activity (AO) tests applied, has been introduced (Gorja-
tivating free radicals, which are generated by metabolic pathway novic et al., 2012).
within the body tissue. Studies have shown that the black tea quality is influenced by
DPPH assay is one of the widely used methods to test the anti- many factors such as season and altitude, genetic make-up of the
oxidative activity of the sample due to its stability, simplicity, and plant (Owuor & Obanda, 1995) and the region of production and
the short time required for analysis. Basically the antioxidant climate (Owuor, Obanda, Nyirenda, & Mandala, 2008). However,
capacity of the samples is measured through their ability to reduce there is not a great deal of information on the effects of these fac-
the DPPH by donating the hydrogen atom and can be determined tors on the total phenolic concentration in tea and subsequent
through the discoloration of the mixture using spectrophotometer. antioxidant activity in tea (Jayasekera, Molan, Garg, & Moughan,
However, even in the absence of active superoxide dismutase en- 2011). Therefore, we investigated the phenolic compounds and
zyme, tea extract can scavenge superoxide due to the presence of antioxidant activity of CTC black tea of six tea growing regions of
antioxidants (Marklund & Marklund, 1974). Nitrite as food preser- Assam and West Bengal, India. The relationship between the phe-
vative reacts with amines present in protein-rich foods in the nolic contents of tea infusions and antioxidant potential was also
acidic pH of the stomach, leading to formation of carcinogenic investigated. Another important objective of this research was to

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.
3746 L.P. Bhuyan et al. / Food Chemistry 141 (2013) 3744–3751

evaluate the impact of black tea phenolic compounds on antioxi- the variables. A Pearson-correlation analysis was carried out to
dant activity and tea taster’s quality. check the significances of the linear relations between obtained
2. Materials and methods The characterisation data were analysed through PCA using fac-
tor extraction with an eigenvalue > 1 and varimax rotation. All data
A total of 60 dryer mouth CTC (Crush Tear and Curl) black tea were log-transformed to achieve closeness to normal distribution
samples were collected from commercial gardens representing and all the variables were standardised by ‘z-score’ analysis to
six agro climatic regions of Assam (Upper Assam, South Bank, measure the similarity between the samples. Furthermore, we
North Bank and Barak valley) and West Bengal (Dooars and Terai), have done factorscore analysis to understand the source relevance
India, during 2011. of black tea samples collected from six different localities. The raw
Theaflavin (TF) and thearubigin (TR) were estimated by the data were used for graphical presentation (Box–Whisker diagram);
method of Ullah, Gogoi, and Baruah (1984). Total polyphenol con- whereas the transformed (log-transformed and standardised) ones
tent was determined using the method BS ISO 14502-1: 2005. were used for factor analysis (principal components analysis).
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 3. Results and discussion
the method described by Mishra et al. (2006).
The sensory tasting (organoleptic evaluation) was performed as Over the years several studies have established the relation-
described by Bhuyan et al. (2009). ships between biochemical components and quality of CTC black
Relative antioxidant activity index (RACI) was calculated from tea (Bhuyan et al., 2009; Senthil Kumar et al., 2011) The work on
the mean value of standard scores of different samples as proposed antioxidant activity of biochemical components of CTC black tea
by Gorjanovic et al. (2012). Standard scores were calculated by the is limited. Thus attempts have been made for determination of bio-
following equation chemical components, antioxidant activity and tasters’ evaluation
and their relationship in CTC black tea of six tea growing regions
xi  x
zi ¼ ð1Þ of Assam and West Bengal, India. As the samples were collected
from different regions, to understand the environmental impact,
where, zi = standard score of the sample i; xi = value of sample i; monthly average maximum and minimum temperatures and total
x ¼ mean; s = standard deviation. rainfall per month in 2011 along with a map of sample collection
Statistical analyses such as factor (principal component analy- areas are presented in Fig. 1. The average monthly maximum
sis) and least significant difference (ANOVA) were carried out using temperature of Barak Valley was higher than that of other regions.
SPSS statistical package. Analysis of variance and least significant Terai region also showed high temperature next to Barak Valley
difference (LSD) tests were applied to evaluate differences between during the harvesting period of tea, i.e. from March to September.

Fig. 2. Biochemical parameters of tea from different sampling sites (LSD = least significant difference at 5% level).
L.P. Bhuyan et al. / Food Chemistry 141 (2013) 3744–3751 3747

Although the rainfall of Terai and Dooars regions was higher, no respectively and comparable. The levels of Barak Valley, Terai
uniform trend for the regions was observed. and Dooars were 7.3–10.8 mg/g (mean 8.9 mg/g), 7.1–10.1 mg/g
Total polyphenol, theaflavin and thearubigin contents of CTC (mean 8.4 mg/g) and 7.6–10.2 mg/g (mean 8.7°mg/g) respectively.
black tea of Upper Assam, North Bank, South Bank, Barak Valley, Another quality parameter, thearubigins (TR) of Upper Assam,
Terai and Dooars are presented in Fig. 2. Significant variations of North Bank, South Bank and Dooars tea are comparable and their
the components amongst the regions were observed. The ranges levels were 118.3–172.5 mg/g (mean 146.4 mg/g), 102.9–
of total polyphenol content of Upper Assam, North Bank, South 177.9 mg/g (mean 151.4 mg/g), 92.4–196.9 mg/g (mean 151.7
Bank, Barak Valley, Terai and Dooars regions were 119.9– mg/g) and 112.0–173.7 mg/g (mean 144.1 mg/g) respectively. TR
178.8 mg/g (mean 145.0 mg/g), 116.1–159.5 mg/g (mean 134.8 of the Terai region tea contained the lowest (48.7–106.2 mg/g
mg/g), 105.3–191.2 mg/g (mean 143.4 mg/g), 100.2–148.1 mg/g and mean 67.0 mg/g) amongst the regions followed by Barak Val-
(mean 125.2 mg/g), 117.9–178.1 mg/g (mean 140.5 mg/g) and ley (60.3–127.8 mg/g and mean 99.2 mg/g). Total polyphenols
99.0–155.8 mg/g (mean 133.6 mg/g) respectively. The black tea and/or TF and TR are formed during fermentation (oxidation) stage
polyphenols are responsible for visual brightness and depth of col- of black tea processing and their precursors and the related en-
our and also perceived strength and mouth feel of the liquor (Bhu- zymes affecting their formation and degradation (Bhuyan et al.,
yan et al., 2009). Theaflavins (TF), the basic quality components of 2009). The variations in these chemicals of different regions might
CTC black tea of Brahmaputra Valley, i.e. Upper Assam, South Bank, be due to different raw materials, environment, soil status, manu-
and North Bank were higher than the regions of Barak Valley, Terai facturing processes, etc.
and Dooars, with levels of 9.7–13.7 mg/g (mean 11.4 mg/g), 8.9– Factor scores, calculated using factor loading matrix, represent
12.3 mg/g (mean 10.6) and 9.6–15.5 mg/g (mean 11.5 mg/g) relative intensities of each factor (Fig. 3). The tea samples from

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
3748 L.P. Bhuyan et al. / Food Chemistry 141 (2013) 3744–3751

Upper Assam, North Bank, and South Bank showed the highest Beek, 2004). This result is in agreement with Mello, Alves,
factor 1 and factor 2 scores, indicating the high content of theaf- Macedo, and Kubota (2004), who observed correlation between
lavins, thearubigins and their ratio. Tea samples from Barak Valley total polyphenol content and antioxidant activity which is very
and Terai region showed the lowest scores for factor 1 and factor good for black tea.
2, indicating lower theaflavin and thearubigin contents. Upper As- Four antioxidant activities namely free radical (DPPH) scaveng-
sam, North Bank and South bank are situated in geographically ing activity, superoxide scavenging activity, nitrite scavenging
and climatically similar regions. Their soil conditions are also activity and lipid peroxidation inhibition activity of CTC black tea
much similar (Mann & Gokhale, 1960). These are the responsible from Upper Assam, South Bank, North Bank, Barak valley, Terai
factors for tea quality. Although Terai region and Barak Valley and Dooars regions are presented in Fig. 4. Barak Valley and Terai
areas are far apart from each other, their climatic conditions are region tea had higher DPPH scavenging activity than tea from other
almost similar, which reflects that the tea samples from these regions, their levels being 85.38–89.03% (mean 86.73%) and
two regions are comparable with respect to biochemical parame- 84.50–88.03% (mean 86.37%) respectively. The mean value of
ters. Even though Dooars region is nearby the Terai region, they Dooars region tea was the lowest (77.57%) with a large range of
are climatically different from each other, which reflect in their minimum (69.57%) and maximum (87.91%) values. This may be
different tea qualities (Mcdowell et al., 1991; Owuor, Wachira, & due to high variation in total polyphenol contents of the samples
Ng’etich, 2010; Owuor et al., 2006). As observed in black tea, ex- The DPPH free radical scavenging activities of Upper Assam, North
tracts with higher antioxidant activity also had higher polyphenol Bank and South Bank were comparable, with the levels of
content, confirming that polyphenols are likely to contribute to 79.56–86.07% (mean 82.68%), 73.70–87.11% (mean 80.95%) and
the radical scavenging activity (Miliauskas, Venskutonis, & van 75.12–86.02% (mean 80.92%) respectively.

Fig. 4. Scavenging activities of tea from different sampling sites (LSD = least significant difference at 5% level).
L.P. Bhuyan et al. / Food Chemistry 141 (2013) 3744–3751 3749

Almost similar trend of superoxide scavenging activity with tea might be due to higher amount of theaflavin content than other
DPPH scavenging activity was observed amongst the regions. The regions. The inhibition ranges of lipid peroxidation of Upper As-
highest activity was observed in Barak Valley followed by Terai sam, North Bank, South Bank, Barak valley, Terai and Dooars re-
and other regions. The ranges of Upper Assam, North Bank, South gions tea were 85.68–98.01% (mean 90.78%), 70.57–89.50%
Bank, Barak Valley, Terai and Dooars regions were 58.43–73.15% (mean 84.67%), 70.21–93.33% (mean 86.20%), 72.33–86.41% (mean
(mean 66.19%), 58.77–73.49% (mean 66.84%), 67.36–89.97% (mean 81.13%), 72.06–87.96% (mean 78.96%) and 70.02–76.05% (mean
74.41%), 61.95–93.87% (mean 80.91%), 72.34–85.22% (mean 72.39%) respectively.
77.39%) and 63.72–74.12% (mean 68.45%) respectively. For the acceptance of tea and to understand the effect of bio-
Nitrite scavenging activity was found to be highest in South chemical components on quality, the role of tea taster is very
Bank followed by Dooars, Upper Assam, North Bank, Barak Valley important. Four liquor characteristics of tea, namely brightness,
and Terai, with levels of activity 90.34–99.52% (mean 98.06%), briskness, strength and quality of tasters’ assessment of black tea
96.16–98.18% (mean 97.44%), 93.42–98.90% (mean 96.55%), from Upper Assam, North Bank, South Bank, Barak Valley, Terai
87.29–96.01% (mean 92.18%), 88.83–98.88% (mean 92.16%) and and Dooars are presented in Fig. 5. South Bank showed the highest
80.39–89.18%, (mean 86.24%) respectively. The variations in the ni- of all the four Tea Tasters, characteristics followed by North Bank,
trite scavenging activities might be due to the variations in the lev- Upper Assam, Barak Valley, Dooars and Terai. Quality of tea charac-
els of theaflavin and thearubigin contents of black tea of different terised by Tea Tasters is the masking effect of biochemicals present
regions. in tea, out of which theaflavin and thearubigin are largely respon-
Lipid peroxidation inhibition of tea from Brahmaputra Valley sible for brightness, briskness and strength (Bhuyan et al., 2009).
i.e. Upper Assam, North Bank and South Bank was higher than The correlations between biochemical components, antioxidant
tea from other three regions i.e., Barak Valley, Terai and Dooars. activity and tasters’ quality are presented in Table 1. There is no
High activity of lipid peroxidation inhibition of Brahmaputra valley correlation between TP, TF or TR contents with DPPH activity. It

Fig. 5. Taster evaluation of processed tea from different sampling sites (LSD = least significant difference at 5% level).
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 have high antioxidant property. Determination of the theaflavin
DPPH scavenging activity of tea (Jayasekera et al., 2011). These re- profiles and elucidation of the role of the respective theaflavins
sults indicate that above polyphenols are not major contributors to on the antioxidant activity can be the future course of study.
the DPPH antioxidant capacity of fermented teas. Highly significant
positive correlations of theaflavins with two antioxidant activities Acknowledgements
like nitrite scavenging activity and lipid peroxidation inhibition
activity and tasters’ quality were observed. Significant positive cor- The authors are grateful to the Director, Tocklai Experimental
relations of thearubigins with nitrite scavenging activity and tast- Station, Tea Research Association for permission to publish the
ers’ quality were also observed. Significant positive correlation results and also wish to thank Dr. R.M. Bhagat, Deputy Director (re-
between theaflavins and tasters’ quality resembles the earlier find- search), Tocklai Experimental Station for providing meteorological
ings (Owuor et al., 2006; Senthil Kumar et al., 2011). On the basis of data of the regions. The authors are also grateful to Tea Board, India
results of all assays employed, the unit less relative antioxidant for financial assistance to carry out the work.
activity index (RACI) has been calculated by assigning equal weight
to each assay (Gorjanovic et al., 2012). Previously, RACI was effi-
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