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					                                                                               Sujitha et al.




                                                                      ISSN 0976-2272
J. B io s c i. Re s ., 2011. Vol. 2(4):176-182

      Extraction of polyphenols from green tea and their external addition during
                               fermentation on black tea quality
                   SUGUMAR DAISYLIN ANBU SUJITHA, aRAMASAMY
    SHANMUGASUNDARAM SENTHIL KUMAR, bGOVINDASAMY KOTTURc*
                          AND SUBRAMANIAN MURUGESANd
  a
    Dept. of Chemistry, Sri Sai Ram Institute of Technology, West Tambaram, Chennai – 44
      b
        Process Chemistry, Harrisons Malayalam Limited, Coimbatore, Tamil Nadu, India
         c*
            Tea Technology Division,UPASI Tea Research Foundation, Nirar Dam BPO,
                              Valparai – 642 127, Tamilnadu, India
             d
               T.Stanes &Co. Ltd, Phyto Pharma Testing Lab, Coimbatore, Tamilnadu
Abstract
The experiment on polyphenol addition on black tea quality had been carried out using
crop shoots of UPASI-9. Polyphenol was extracted from enzyme-inactivated dried green
leaf and the extracted polyphenol was added to the cut dhool during fermentation at varying
proportions (2, 4 and 6% on made tea basis) and processed into black tea and the teas were
analysed for their quality, antioxidant activities (reducing power and DPPH (2, 2 - diphenyl
-1- picryl hydrazyl radical) radical scavenging activity) and total polyphenols. The
polyphenol enriched black teas had higher theaflavin (TF) and total liquor colour (TLC)
over the untreated control. Total polyphenols and radical scavenging activity were higher
when the crude polyphenol was added at 4% as compared to 2% and 6% addition. The
present study revealed that the quality constituents of black teas such as theaflavins, water
extract (WE) and the antioxidant activities were enhanced by the addition of polyphenols to
cut dhool during fermentation and the optimum concentration of polyphenols was found to
be 4%.
 Key words: polyphenols, theaflavins, reducing power, water extract
For correspondence: kotturg@gmail.com

Introduction                                     Depending on the degree of fermentation,
        Tea is defined as the hot aqueous        teas are categorized into three major types;
infusion of the dried leaves of the plant        green, oolong and black tea. The
Camellia sinensis. It is one of the most         unfermented green tea and semi fermented
popular beverages worldwide, and its             oolong tea are popular mainly in China and
consumption is second, next to water             Japan. However, in the rest of the world the
(Graham, 1992) due to its health–enhancing       fully fermented black tea is the regular
properties. Many of the studies suggest that     choice. India is proud to be the second
tea is protective against certain cancers,       largest producer and has captured premier
cardiovascular          diseases        and      position in black tea production and
neurodegenerative diseases (Mendel and           domestic consumption and third place in
Youdim, 2004). Therefore, tea extracts have      export market where India caters 27% of
gained popularity as ingredients in dietary      the global demand (Kidwai, 1999). Black tea
supplements and functional foods.                manufacture involves four major steps, viz.,
                                                 withering, leaf distortion or cutting,
JOURNAL OF BIOSCIENCES RESEARCH 2(4):176-182                                             176
                                                                                     Sujitha et al.

oxidation of polyphenols (fermentation) and      Materials and methods
firing. Polyphenols are the major flavanoids     Extrac tio n o f p o ly p h e n o ls fro m e n zy m e
present in green leaf, accounting for up to      in ac tiv ate d d rie d le av e s an d th e ir
30% on the dry weight basis. Catechins are
                                                 ad d itio n to b lac k te a
the integral part of polyphenols and best
known for their potent antioxidant activities,   Tea shoots of UPASI–9 comprising of
being more effective radical scavengers than     apical bud and two leaves were harvested at
vitamins C and E. Catechins prevent skin         UPASI TRI experimental Farm, Valparai
from harmful UV-rays and protect against         located in the Anamallais of Western Ghats.
hyper pigmentation and hence can be used         Fresh leaf samples were steamed for 7 min
in cosmetics. It includes (-)-epicatechin        and then placed in a fluid bed drier at 800 C
(EC), (-)-epigallocatechin (EGC), (-)-
                                                 for 30 min. About 100 g of dried green tea
epicatechin gallate (ECG), (+) - Catechins
(+C) and (–) - epigallocatechin gallate          leaves was extracted with 70 % ethanol for
(EGCG). EGCG is the most abundant                one hour and the extract was filtered.
catechins and accounts for 65% of the total      Extraction was carried out for three times
catechin content in green tea (Zaveri, 2006).    and concentrated using a rotary vacuum
On the other hand, black tea has                 evaporator. The extract was partitioned with
comparatively low catechin content but with      an equal volume of chloroform to eliminate
significant amount of theaflavins and
                                                 caffeine (Kim et al, 2002). The aqueous layer
thearubigins (conversion products from
catechins during processing) which are           was again extracted with equal amount of
absent in green tea (Graham, 1992). To           ethyl acetate for three times. The ethyl
meet the taste preferences of consumers and      acetate layer was dried and the crude
increase tea consumption, certain studies on     polyphenols were collected and used for the
external addition of both enzymes and            study. The complete extraction procedure
substrates are needed. Available literature
                                                 for polyphenol is given in Fig. 1. During
revealed that addition of enzymes during
fermentation resulted in an improvement in       black tea manufacturing process crude
the overall quality of black tea. Mariya John    polyphenol was added at various
et al, (2006) studied the effect of external     concentrations (2, 4 and 6% on made tea
addition of different substrates on total        basis) to cut dhool during fermentation and
catechin content and they found that             processed in to black tea.
shikimic acid was the substrate that             Exp e rim e n tal CTC b lac k te a
accounted for the maximum enhancement
                                                 m an u fac tu re
in synthesis of total catechins. However,
published information on the influence of        About 2 kg of green leaves of UPASI-9
external      added      substrates    (crude    were spread on the withering troughs.
polyphenol) on quality and antioxidant           Ambient air was passed with a velocity of 45
activities in tea is limited. Generally green    cfm (cubic feet per meter) through the
teas have more antioxidant property and          leaves for a period of 16 hours to reduce the
hence to increase the medicinal value of         moisture content and to bring about
black tea, polyphenol enrichment has been
attempted on normal black tea. This shall        adequate physical and chemical wither.
ensure a product which has the normal            Withered leaves were passed through
characters of black tea together with the        miniature CTC (cut, tear and curl) machine
health benefits of green leaf polyphenols. In    five times to get adequate maceration. The
this context, exogenous addition of              cut dhool (polyphenol addition stage) was
substrates (crude polyphenols) at different      allowed to ferment in a chamber where
concentration with particular reference to       temperature was maintained at 25°C and 95
quality parameters and antioxidant activities
of black teas were studied.                      % relative humidity. Fermentation period

JOURNAL OF BIOSCIENCES RESEARCH 2(4):176-182                                                     177
                                                                                                      Sujitha et al.
was specific to each cultivar because of                     Es tim atio n o f re d u c in g p o w e r
variations in their oxidation potential.                      Exactly 1 mL of the extract was mixed with
Optimum fermentation time was assessed                       2.5 mL of phosphate buffer (0.2 M, pH 6.6)
by adopting method as reported by Ullah                      and 2.5 mL of potassium ferricyanide (1%).
(1977). Fermented dhool was dried in a                       The mixture was incubated at 50o C for 30
miniature fluid bed drier with a blast of                    minutes.          Afterwards         2.5     mL      of
120°C hot air for 15 minutes which reduces                   trichloroacetic acid (10%) was added to the
the moisture content to less than 3 %. Tea                   mixture, which was then centrifuged at 3000
samples thus manufactured were sorted                        rpm for 10 minutes. Finally, 2.5 mL of the
using Endecott sieve shaker and the pekoe                    upper layer was pipetted out and mixed with
fanning’s (PF) grade was selected for the                    2.5 mL of distilled water and 0.5 mL of
analysis of quality parameters and                           ferric chloride (0.1%) was added. The
antioxidant activities. (Senthil Kumar, 2004).               absorbance was measured at 700 nm using a
An aly s is         of     q u ality      p aram e te rs ,   Perkin Elmer Lambda 35 UV-Visible
p o ly p h e n o ls an d c ate c h in frac tio n s           Spectrophotometer. The intensity of
 Quality parameters namely theaflavins,                      reducing power is directly proportional to
thearubigins and total liquor colour were                    the absorbance of the reaction mixture
estimated by the method of Thanaraj and                      (Yildirim et.al, 2001).
Seshadri (1990). Total polyphenols were                      Statis tic al an aly s is
estimated by using the procedure of ISO                      The experiment was repeated thrice and the
14502-1 (2005) and the catechin fractions                    results were statistically analysed (Gomez
were analysed by using the method of ISO                     and Gomez, 1984).
14502-2 (2005). Water extract was estimated                  Results and discussion
as per Indian Standard IS 13862:1999.                        Extrac tio n o f p o ly p h e n o ls fro m g re e n
De te rm in atio n o f an tio xid an t ac tiv itie s         te a, p h y s ic al an d c h e m ic al
Es tim atio n o f DPPH rad ic al s c av e n g in g           c h arac te rizatio n
ac tiv ity                                                   Crude polyphenols were extracted from
 Free radical scavenging effect was estimated                enzyme-inactivated dried green leaf and the
according to the method of Blois (1958) as                   compounds present in crude polyphenols
modified by Zhu et.al, (2002). Briefly, a                    and their physical characterization were
1mM solution of DPPH radical solution in                     given in Table 1. The observation revealed
ethanol was prepared, and then 1mL of this                   that the polyphenol extracted from dried
solution was mixed with 3 mL of extract                      green tea leaves showed fine crystals like
solution in ethanol containing 50-500g of                   texture. The recovery percentage obtained
dried extract; the mixture was then vortexed                 was 54.40 and this varies with the method
vigorously and left for 30 min at room                       of extraction and purification. High
temperature in the dark and the absorbance                   recovery percentage indicates effectiveness
was measured at 517 nm with a                                of the method of extraction. The colour of
spectrophotometer and is calculated as                       the crude polyphenols were reddish brown
DPPH Scavenging % = [(Control                                and soluble in water, acetone and ethyl
Absorbance – Extract Absorbance)/Control                     acetate. Moisture of the crude polyphenol
Absorbance] x 100. For control 3 mL of                       obtained varied between 2 to 3 percent.
distilled water was added to 1mL of 1mM                      Yield is the next important parameter and
solution of DPPH radical solution and the                    for the commercial products it varies from
rest of the procedures remain the same.                      22 to 25% dry weight basis. At the
                                                             laboratory level we have achieved a yield of

JOURNAL OF BIOSCIENCES RESEARCH 2(4):176-182                                                                   178
                                                                                           Sujitha et al.
16% polyphenols with a purity of 85%.                      Addition of crude polyphenols during
Among the catechin fractions, EGCG                         fermentation increased the content of
accounted high contribution over the rest of               theaflavins, total liquor colour and water
the catechin fractions whereas the total                   extract over the untreated control (Table 2).
catechin content was found to be 65.77%.                   Similar results were observed in the case of
                                                           polyphenols, DPPH radical scavenging
          Dried green tea leaves                           activity and reducing power (Table 3).
                   (100 g)                                 Identical results were noticed when the
                                                           antioxidant activities were estimated in their
                                                           brew also (Table 4). Among the three
                                                           treatments studied, 4% polyphenol addition
         Extraction for 1hr with
              ethanol (70%)                                recorded higher amount of quality
                                                           parameters and antioxidant activities,
                                                           whereas addition of polyphenols at 6%
                                                           decreased the contents of TF, TLC and WE.
            Separation by filter                           For quality assessment of tea, quantification
                                                           of catechin content are reported to be the
                                                           most reliable biochemical methods that
                                                           manifested on the black tea pigments,
               Clarification by                            theaflavins (TF) and thearubigins (TR).
                centrifugation
                                                           Attractive colour of tea infusion is due to
                                                           theaflavins and it emerged as a quality
                                                           measure of tea. Increase in quality
               Separation with                             parameters viz., TF and TR could be due to
                  chloroform                               primary     reaction     occurring     during
                                                           fermentation is the oxidation of tea
                                                           flavanols (catechins) by the action of
            Separation with ethyl
                                                           polyphenol oxidase which transform the
                    acetate                                polyphenols and catechins in to black tea
                                                           pigments namely theaflavins, thearubigins
                                                           (Ravichandran and Parthiban, 1998). These
                                                           two pigments are positively correlated to the
            Evaporation of ethyl                           black tea quality and correlated well with the
                 acetate layer
                                                           price realization (Hilton and Ellis, 1972).
                                                           Total liquor colour is a measure of strength
                                                           of tea liquor which showed same trend as
             Crude polyphenols                             that of TF. Water extract refers to the total
                                                           water soluble solids present in black tea
                                                           water extract which should be above 32%
Figure 1. Schematic diagram of and is directly related to the Cuppage which
polyphenol extraction from dried green is a measure number of cups per kilogram
tea leaves                                                 of tea (Ramaswamy et al, 1993). Higher
In flu e n c e o f ad d itio n o f p o ly p h e n o ls o n values of water extract are associated with
liq u o r p aram e te rs an d an tio xid an t the higher/better quality of south Indian
ac tiv itie s o f b lac k te a an d its b re w             plain black teas (Senthil Kumar, 2004). The
                                                           increase in water extract in polyphenols

JOURNAL OF BIOSCIENCES RESEARCH 2(4):176-182                                                        179
                                                                                 Sujitha et al.
enriched tea is because of the water soluble    Research John Wiley and Sons, New
character of polyphenols.                       York, 20–30.
The high antioxidant activities (DPPH and     Graham,        H.N.,     1992.      Green      tea
reducing power) of polyphenols enriched         composition,          consumption,          and
teas might be due to the di and mono            polyphenol chemistry. Prev. Med. 21: 334-
hydroxyl substitutions in the aromatic ring     350.
which possess potent hydrogen donating           Hilton, P.J., and Ellis, R., 1972.
abilities as described by Shimada et al,        Estimation of the market value of Central
(1992). Antioxidant are believed to intercept   African tea by theaflavin analysis. J. Sci.
the free radical chain of oxidation and to      Food Agric. 23: 227-232.
donate hydrogen from the phenolic             IS 13862:1999. Determination of water
hydroxyl radical groups, thereby forming a      extract (first revision), in Indian Standards
stable end product, which does not initiate     Specification for Tea, Indian Standard
or propagate further oxidation lipids. The      Institution (BIS), New Delhi.
study carried out by Liang et al, (1999) in    ISO 14502–1: 2005. Determination of
pineapple juice also showed similar             substances characteristics of green and
observation. According to them the addition     black tea–Part–1: Content of total
of polyphenols improved the thermal             polyphenols in tea – Colorimetric method
stability of tea polyphenols complexed          using Folin-Ciocalteu’s reagent.
bromelain and increased the antioxidant       ISO 14502–2: 2005. Tea–Method for
activity after forming a complex with tea       determination            of         substances
polyphenols.                                    characteristics of green and black tea –
Conclusion                                      Part – 2: Content of catechins in green tea
The present study has revealed a simple and     – Method using high-performance
efficient procedure for the extraction of       chromatography.
polyphenols from the green tea. The             Kidwai, S. M., 1999. Tea industry. The
optimum concentration of polyphenols was        telegraph daily. Calcutta: Anand.
found to be four percent on made tea basis.   Kim J.I., Hong, S.B., and Row, K.H, 2002.
The extracted crude polyphenols can be          Effect of particle size in preparative
used in the production of polyphenol            reversed-phase high-performance liquid
enriched black teas which enhances the          chromatography on the isolation of
quality and antioxidant activities of teas and  epigallocatechin gallate from Korean
also their price level in commercial market.    green tea. J Chromatogr, 949: 275-280.
Acknowledgement                               Liang, H.H., Huang, H.H., and Kwok, K.C.,
         The authors are grateful to            1999. Properties of tea-polyphenol
Dr.N.Muraleedharan,           Adviser,     and  complexed bromelain. Food Res Int, 32:
Dr.P.Mohan Kumar, Director of UPASI             545-551.
Tea      Research      Institute   for        Mariya John, K.M., Sasikumar, R., Deepu
                                          their
encouragements.                                 Vijayan, Saravanan, M., Rahul, P.R., and
                                                Raj Kumar, R., 2006. Influence of
References                                      externally added substrates on total
Blois,       M.S.,      1958.     Antioxidant   catechin content in tea leaves (Camellia
  determinations by the use of a stable free    sp.). Asian J Plant Sci, 5 (1): 116-119.
  radical. Nature, 26: 1199-1200.             Mendel, S., and Youdim, M.B., 2004.
Gomez, K.A., and Gomez, A.A., 1984.             Catechin polyphenols: Neurodegeneration
  Statistical Procedures for Agricultural

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  disease. Free Radic. Biol. Med, 37: 304-317.
Ramaswamy, V., Rajendiran, A.R., and Raju,        Thanaraj, S.N.S., and Seshadri, R., 1990.
  K., 1993. To add or not to add. UPASI           Influence of polyphenol oxidase activity and
  Tea Scientific Department Bulletin, 46: 132-    polyphenol content of tea shoot on quality
  143.                                            of black tea. J. Sci. Food Agric. 51: 57-69.
Ravichandran, R., and Parthiban, R., 1998.        Ullah, M.R., 1977. Alternative test for
  Changes in enzyme activities (polyphenol          assessment of fermentation. In: Proceedings
  oxidase and phenylalanine ammonia lyase)          of Tocklai Conference, 29: 75-77.
  with type of tea leaf and during black tea      Yildirim, A., Mavi, A., and Kara, A.A., 2001.
  manufacture and effect of enzyme                  Determination of antioxidant and
  supplementation of dhool on black tea             antimicrobial activities of Rumex crispus
  quality. Food Chem, 62: 277–281.                  L.extracts. J. Agric. Food Chem. 49: 4083-
Senthil Kumar, R.S., 2004. Studies on               4089.
  improving quality of south Indian black         Zaveri, N.T., 2006. Green tea and its
  teas. Final Technical Report submitted to Tea     polyphenolic catechins: Medicinal uses in
  Board, Kolkata, India, 1-71.                      cancer and noncancer applications. Life
  Shimada, K., Fujikawa, K., Yahara, K.,            Science, 78: 2073-2080.
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  Table 1. Product details of crude polyphenols extracted from dried green leaves
       Texture                                    Fine crystals
       Solubility                                 Water, Acetone and Ethyl acetate
       Colour                                     Reddish brown
       Moisture (%)                               2.80
       Yield (%)                                  16.00
       Recovery (%)                               54.40
       Polyphenols (%)                            85.00
       Gallic acid (%)                            0.82
        (-)- Epigallocatechin (%)                 10.23
       (+) – Catechin (%)                         1.48
       (-)-Epicatechin (%)                        2.80
       (–) - Epigallocatechin gallate (%)         41.82
       (-) – Epicatechin gallate (%)              9.44
       Total Catechins (%)                        65.77




JOURNAL OF BIOSCIENCES RESEARCH 2(4):176-182                                              181
                                                                          Sujitha et al.
Table 2 . Effect of external addition of polyphenols on black tea quality parameters
                             *
     Addition of              TF               TR                 HPS            TLC               WE
    Polyphenols (%)         (%)               (%)                (%)                                (%)
    Control                 1.40              11.24              10.03           3.47             38.40
    2.0                     1.51              13.44              10.18           4.12             39.40
    4.0                     1.60              13.75              10.53           4.63             40.94
    6.0                     1.55              14.31              10.55           4.56             40.57
     SEm±                    0.01              0.05               0.022           0.02             0.05
     CD @ P=0.05             0.03              0.13               0.04            0.05             0.12
     CD @ P= 0.01            0.05              0.20                 0.05           0.08             0.18
*
TF = theaflavins; TR = thearubigins; HPS = highly polymerised substances;
TLC = total liquor colour; SEm = mean standard error; CD – critical difference

    Table 3 Effect of polyphenol addition on polyphenol content and antioxidant
                               activities of black tea
Addition of           Polyphenols            DPPH radical            Reducing power
Polyphenols (%)         (%)                  scavenging activity (%) (in OD units)
Control               16.73                    53.82                    0.420
2.0                   19.08                   57.44                     0.461
4.0                   19.80                   59.30                     0.568
6.0                   18.32                   57.10                     0.548

SEm±                       0.11                         0.29                              0.01
CD @ P=0.05                0.27                          0.71                             0.01
CD @ P= 0.01               0.41                          1.07                             0.02
DPPH = 2, 2 - diphenyl -1- picryl hydrazyl radical; OD= optical density;
SEm = mean standard error; CD – critical difference
    Table 4 Effect of external addition of polyphenols on polyphenol content and
                          antioxidant activities of tea brew
Addition of           Polyphenols           DPPH radical              Reducing power
Polyphenols (%)        (%)                  scavenging activity (%) (in OD units)
Control               15.90                   48.71                     0.402
2.0                   16.66                  55.46                      0.415
4.0                   17.16                  56.55                      0.430
6.0                   16.62                  55.45                      0.428

SEm±                       0.08                         0.53                               0.00
CD @ P= 0.05                0.20                        1.30                              0.01
CD @ P= 0.01                0.31                        1.97                               0.01
DPPH = 2, 2 - diphenyl -1- picryl hydrazyl radical; OD= optical density;
SEm = mean standard error; CD – critical difference.




JOURNAL OF BIOSCIENCES RESEARCH 2(4):176-182                                                        182

				
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