CONTENTS

Page No.

1. Introduction 1

2. Aims and objectives 2

3. Theory 2

4. Reagents 4

5. Apparatus 4

6. Procedure 4

7. Results and Discussions 6

8. Conclusion 7

9. References 7

10. Tables (1-5) 8

11. Figures (1-5) 12

 FLOWERS, LEAVES AND FRUITS OF TEA PLANT

INTRODUCTION:
According to the ancient Chinese, Tea is a “gift from God”. Tea is an important beverage
and ranks second only to water in consumption. The fragrant brew is prepared from the
leaves of the plant Camellia Sinensis. Through cultivation tea has become an important
agricultural product, throughout the world particularly in regions close to the equator. Tea
is manufactured into a consumable product in tea factories. An outline of the tea
manufacturing processes generally adopted is shown in Fig.1.

The processing, chemistry and physiological functionality of tea beverages have intrigued
scholars and tea drinkers over the millennia. The leaves of Camellia Sinensis are similar
to most plants in general morphology and contain all the standard enzymes and structures
associated with plant cell growth and photosynthesis. Unique to tea plants are large
quantities of polyphenols, such as flavanoids, and methylxanthines that impart unique
flavour and functional properties of tea. The general composition of fresh tea leaves is
presented in Table 1.

The general characteristics of some of the components of the tea leaves are given below -
Polyphenols (Flavonoids): Green tea leaves contain many types of flavonoids, the most
important of which are the flavanols (catechins), the flavonols, and the flavanol
glycosides. Tea catechins are water – soluble, colorless substances, which impart the
bitter and astringent characteristic of green teas. Catechins are easily oxidized when
catalyzed by enzymes of the general class called oxidases and autoxidize in an alkaline
environment. The oxidation products of catechins are the red brown pigments found in
brewed and instant teas. The quality of tea infusion correlates with the flavanol content of
fresh tea leaves.
Other Phenolic compounds: There are several phenolic acids important to tea chemistry.
Gallic Acid and its quinic acid ester, theogallin have been identified in tea.
Caffeine and other Xanthines: Tea leaves contains 2.5 to 4.0% caffeine on a dry weight
basis and much smaller quantities of the related methylxanthine theobromine.
Lipids: These are water insoluble but soluble in organic solvents and include fats, oils,
triacylglycerols, fatty acids, glycolipids, phospolipids and steroids.
Theanine and other Amino Acids: Amino acids make up 4-8% of the soluble solids
found in brewed tea. There is an amino acid unique to tea, γ -N-ethylglutamine,
called theanine.
Minerals and Ash: Tea leaves are rich in potassium and contain significant quantities of
calcium, magnesium, aluminum and fluorides.
Volatiles or Aroma: The essential oil or aroma of tea provides much of the pleasing
flavour and scent of green and black tea beverages. Volatiles constitute less than 1 % of
the total mass of tea leaves and comprises of compounds like terpenes, terpene alcohols,
lactones, ketones, esters etc.
Enzymes: The enzymes most important to the chemistry and manufacturing of tea are
those responsible for the biosynthesis of tea flavonoids and those involved in the
conversion of fresh leaf into manufactured commercial teas. Alcohol dehydrogenase and
leucine α -ketoglutarate transaminase contribute to the development of aroma during
black tea manufacturing. Polyphenol oxidase and peroxidase are essential to the
formation of polyphenols unique to fermented teas.

Recently tea polyphenols (Fig. 2) have attracted considerable interest because of their
associated health properties. Green tea polyphenols (GTPs) are known for their
antioxidant properties. The polyphenols have the property of mopping up oxygen radicals
that can be generated in the body by partial reduction of molecular oxygen. Free radical
intermediates contribute to a wide range of diseases, including atherosclerosis,
emphysema, ulcerative colitis, diabetes, multiple sclerosis, rheumatoid arthritis,
Parkinson’s disease and cancer. Green tea shows higher antioxidative properties than
semi-fermented (Oolong tea) or fermented (Black) tea.

The study of extraction method and estimation of tea polyphenols from tea leaves,
therefore, have great importance in tea science.

AIMS and OBJECTIVES:

1. To extract and estimate polyphenol content in fresh tea leaves.

2. To investigate the effect of two different solvents (having different polarities),
viz., Methylene dichloride (higher dipole moment) and Chloroform (lower dipole
moment), used for Lipid and Caffeine removal, on polyphenol yield from tea
leaves.
3. To investigate the effect of multiple stage extraction on polyphenol yield from tea
leaves.

THEORY:

The polyphenols, as a whole, present in tea leaf or its unfermented product, green tea, are
called Green Tea Polyphenols (GTP). Structures of a few polyphenols are shown in Fig 2.

Based on the chemical properties of tea polyphenols, the common steps for extraction of
GTPs from tea products are – extraction by water in hot condition about 70-80 0C [Tepid
Water Extraction (TWE)], followed by removal of caffeine using suitable solvents like
Methylene chloride or Chloroform and extraction of GTPs by solvents like Ethyl acetate
etc. Finally the solvent in the final extract is removed and the product is dried.

The polyphenols are estimated by spectrophotometric method. The ability of an organic

compound to absorb ultraviolet radiation is dependent on its electronic structure. An
ultraviolet spectrum is a plot of the wavelength or frequency of absorption versus the
intensity of absorption (transmittance or absorbance). Fig. 3 shows the absorption
spectrum of a standard Gallic acid, a polyphenol.
The quantitative treatment of the absorption of radiant energy by matter depends on the
general principle known as Beer’s Law. Beer’s Law states that, “the rate of decrease in
intensity of radiation absorbed is proportional to the thickness of the medium and also to
the concentration of the solute”.

That is,

dI/dl = klc ---------- (1)

Where Io is the intensity at length = 0, and, I is the intensity at length = l and c is the
concentration of the solution, and k is the ‘molar absorption co-efficient’ and is
characteristic of the substance.

On integration of equation (1) we get -

I = Ioe-klc
ε
or I = Io 10- lc, where ε = 0.4343 k and is called ‘molar extinction co-efficient’

or log I = log Io – ε lc

or log Io – log I = ε lc

or log (Io/I) = ε lc , where log (Io/I) is called the ‘optical density’ or the ‘abrorbance
of the medium at the concentration ‘c’ and thickness of the medium ‘l’.

A plot of the ‘absorbance’ against concentration of a solution, taken in a cell of definite

thickness, results in a straight line. From this straight line concentration of an unknown
solution of the same substance can be very easily determined if the absorbance of the
solution could be measured.

Fig. 4 shows the layout of a double beam Ultra violet – Visible (UV-VIS)
spectrophotometer. The spectrophotometer is used to determine the total polyphenol
content in the leaf tea colorimetrically using Folin-Ciocalteu reagent. The reagent
comprises of phospho-tungstic acids used as oxidants, which on reduction by phenolic
hydroxyl groups yield a blue colour with a broad maximum absorption at λ = 725 nm
due to the formation of tungsten and molybdenum blues.

REAGENTS:

1. Acetonitrile
 2. Methylene dichloride

3. Chloroform

4. Ethyl Acetate

5. Folin-Ciocalteu reagent

6. Sodium Carbonate solution – saturated solution

7. Gallic Acid working standard solution, corresponding to 1000 mg/litre

APPARATUS:

1. Analytical Balance, capable of weighing to an accuracy of ± 0.001 g

2. Drying oven
3. Round bottom flask of 500ml capacity fitted with reflux condenser
4. Heating mantle for above
5. Separating funnel of 500ml capacity
6. Water bath, capable of being maintained at 70 ± 1 0C
7. Dispenser, for extracting solvent and set at 5 ml
8. Spectrophotometer, set at λ = 725 nm
9. Vortex Mixer, for efficient mixing during extraction
10. Graduated tubes, glass 10 ml capacity with 0.1 ml graduations
11. Automatic pipettes

PROCEDURE:

A. Extraction of Polyphenols from Tea Leaves:

Fresh tealeaves were collected and weighed. The leaves were deactivated (enzyme
deactivation) by hot and boiling water. The deactivated tea leaves were dried in an oven
at 110 0C till the moisture content of the dried leaves is less than 6 %. The dried leaves
were ground and extracted with water under reflux conditions in a round-bottomed flask.
The extract was filtered through glass wool in a funnel and collected in a beaker. The
collected extract were decaffeinated by liquid – liquid extraction with solvents like
chloroform, methylene dichloride etc. in a separating funnel. Caffeine was absorbed into
the organic layer and polyphenols remained in the aqueous layer. The polyphenols were
further extracted by liquid – liquid extraction using ethyl acetate. The ethyl acetate is
distilled off to get the golden yellow crystals and stored in refrigerator. The polyphenol
content of these crystals were estimated spectrophotometrically.

B. Estimation of Polyphenols:

Standard solution preparation:

0.1, 0.2, 0.3, 0.4, 0.5 ml of gallic acid standard solution, corresponding to 1000 mg/litre is
pippetted out in duplicate into graduated tubes. All are diluted to 0.5 ml with water. A
blank solution containing no gallic acid is also prepared.

Standard curve:

From each tube of standard solution 0.5 ml is pipetted out in duplicate into separate
disposable tubes and diluted to 5 ml with water. 0.2 ml of Folin-Ciocalteu reagent is
added to each tube, followed by 0.5 ml of sodium carbonate solution. The contents of all
the tubes are diluted to 10 ml by adding 4.3 ml water, stoppered, and mixed well. All the
solutions are allowed to stand at room temperature for 60 minutes, and, then the optical
densities (absorbance) are measured on the spectrophotometer set at λ = 725 nm with a
10 mm path length cell, with the blank solution as reference zero. The standard curve is
obtained by plotting the absorbance versus the concentrations (Fig. 5).

Preparation of Test Solution and estimation of polyphenols:

0.5 g of the yellow crystals is weighed out in duplicate into 50 ml volumetric flasks. 25
ml of warm water (about 60 0C) is added to dissolve the sample. The solution is allowed
to cool to room temperature. 5 ml of acetonitrile is added, diluted to 50 ml and mixed
well. 0.2 ml of sample extract is pippetted out into separate graduated tubes, diluted to 10
ml, and mixed well. 0.5 ml of this solution was then pipetted out and the colour
developed as per the procedure used for the standard solution. The absorbance at λ = 725
nm were measured and then the concentration of polyphenols in the prepared solution
were obtained from the standard curve. The total amount of polyphenol content in the
yellow crystalline solid is then calculated. From this the yield of polyphenol with respect
to the tea leaves were calculated.

RESULTS AND DISCUSSIONS:

Fig. 3. shows the absorption spectrum of standard Gallic acid solution in the visible
range. The spectrum shows a maximum absorption at λ = 725 nm. The absorbance
obtained at this wave length is further used to draw the standard curve and estimate the
polyphenol contents in the extract. The standard curve (calibration curve) drawn using a
standard Gallic acid solution is shown in Fig. 5. The curve is almost a straight line and is
used for determining the concentration of polyphenols from the extracts.

Fresh tea leaves were collected from two tea gardens (viz. garden 1 and garden 2). Table
3 shows that under identical conditions of extraction of polyphenols, the yield of
polyphenols varies depending on the source of the leaves. This variation is due to the
difference in agrochemical and environmental conditions like age of the tea plant, amount
of rainfall, nature and type of soil etc. However, the crude extracted material from garden
2 is purer in nature (Table 5).

Table 3 and Table 4 shows that when other conditions are maintained identical and only
the solvent in the 2nd step (used for removal of caffeine and lipids) is varied (exp. no. 1
and 3), the yield is also varied. When Chloroform is used in place of Dichloromethane
(Methylene dichloride), the yield increased from 12.18 to 20.56 %. This variation is due
to (a) the difference in polarity of the solvents, and (b) the difference in solublity of the
solvents in water. Dichloromethane, comparatively with higher polarity (dipole moment
µ = 1.54 Debye), dissolves and removes some of the polyphenols (which are also polar
compounds) along with the caffeine and lipids. This lowers the yield of polyphenols when
Dichloromethane is used in the 2nd step. The dipole moment of Chloroform is 1.02 Debye
and hence its polarity is less than Dichloromethane. Therefore, when Chloroform is used
in the 2nd step the amount of polyphenols dissolved and removed is much less, resulting in
increased yield of polyphenols. Again, the solubility of water in Dichloromethane is 1.40
g/Kg, while the solubility of water in Chloroform is 0.061 g/Kg. In step 1 of Table 3 and
Table 4 water dissolves the polyphenols, caffeines and lipids. In step 2, when water
solution comes in contact with the solvent for removal of caffeine and lipids, water
solution gets dissolved in higher amount when the solvent is Dichloromethane, then when
it is Chloroform. The water dissolving in the solvent carries along with it some amount of
dissolved polyphenols. These factors lead to increase in the yield of polyphenols when
Chloroform is used as solvent compared to Dichloromethane.

A study of Table 3, 4 and 5 shows that the polyphenol crystals prepared are not the pure
product but contain impurities. The impurities consist of minute amounts of amino acids,
organic acids, monosaccharides, polysaccharides, protein, lignin, lipids and mineral
matters etc.
Table 4 also show that when stages of extraction with water in the 1 st step, stages of
extraction with Choloroform in the 2nd step and volume of Ethyl acetate used in the 3rd
step is increased (exp. no. 4 & 5), the yield of polyphenols increased from 20.56 % to
26.80 %. The purity of the extracted crude product also increased from 95.63 % to 97.45
% due to increase in the stages of extraction (Table 5).

CONCLUSION:

• The yield of polyphenols from fresh tea leaves depends upon agrochemical and
environmental conditions.
• In the extraction of polyphenols, when solvents of low polarity and low solubility
in water are used for caffeine and lipids removal, the loss of polyphenols is less and yield
increases.
• The yield and purity of polyphenols increases whenthe no of stages of extraction
are increased.
• Green Tea polyphenols are receiving increasing importance worldwide, and
therefore more studies need to be done on their biochemistry.

REFERENCES:

1. Wang, H., Provan, G.J.,and Helliwell,K., “Tea flavonoids: their functions,

utilization and analysis”, Trends in Food Science and Technology”, 11(2000) 152-
160
2. Balentine, D., “Tea”, Encyclopaedia of Chemical Technology,4th Ed., 23, Ex Ed –
J Kroskwitz, M H Grant.
3. Ewing, G.W., “The Absorption of Radiation” Instrumental Methods of Chemical
Analysis, 2nd Ed, McGraw-Hill Book Co., London
Table 1. The general composition of fresh Tea Leaves

Components Quantity (wt. %)

Flavanols 25.00
Flavonols and Flavonol Glycosides 3.00
Polyphenolic acids and Depsides 5.00
Other Polyphenols 3.00
Caffeine 3.00
Theobromine 0.20
Amino Acids 4.00
Organic Acids 0.50
Monosaccharides 4.00
Polysaccharides 3.00
Cellulose 7.00
Protein 15.00
Lignin 6.00
Lipids 3.00
Chlorophyll and other Pigments 0.50
Ash 5.00
Volatiles 0.10
 Table 2. Calculation of total Polyphenol content from the absorbance value

Exp. Absorbance Concentration of Polyphenols content Amount of Total Polyphenol

n Polyphenols in the in the 50 ml of crude yellow content in the
o diluted solution (500 solution (i.e. 0.5 g of crystalline extracted material
times diluted) from the the material) material
standard curve extracted
(mg/litre) (g) (g) (g)
[a] [b] = [a X 0. 25] [c] [d] = [{b X c}/0.5]

1 0.2783 18.740 0.4685 1.430 1.340

2 0.2799 18.844 0.4711 1.162 1.095

3 0.2841 19.124 0.4781 1.075 1.028

4 0.2894 19.488 0.4872 1.375 1.340

 Table 3. Using Dichloromethane for Lipid and Caffeine removal

Exp. Fresh 1st Step 2nd Step 3rd Step Yield of Yield of
no. Leaves crude polyphenols wrt
Solid-liquid Lipid + Caffeine Liquid-liquid yellow fresh leaves
extraction from removal from the extraction from crystals
leaves filtrate of 1st step water layer of 2nd
step

g Solvent ml Solvent ml Solvent ml g g %

1. 11.00 Water 3× 15 Dichloro- 3× 15 Ethyl 6× 200 1.430 1.340 12.18

0 methane 0 Acetate
(From
garden 1)

2. 14.00 Water 3× 15 Dichloro- 3× 15 Ethyl 6× 200 1.162 1.095 7.82

0 methane 0 Acetate
(From
garden 2)

Table 4. Using Chloroform for Lipid and Caffeine removal

Exp. Fresh 1st Step 2nd Step 3rd Step Yield of Yield of
no. Leaves crude polyphenols
Solid-liquid Lipid + Caffeine Liquid-liquid yellow wrt fresh
extraction from removal from the extraction from crystals leaves
leaves filtrate of 1st step water layer of 2nd
step

g Solvent ml Solvent ml Solvent ml g g %

3. 5.00 Water 3× 15 Chloroform 3× 15 Ethyl 6× 200 1.075 1.028 20.56

0 0 Acetate
(From
garden 1)

4. 5.00 Water 4× 15 Chloroform 4× 15 Ethyl 6× 300 1.375 1.340 26.80

0 0 Acetate
(From
garden 1)
 Table 5. Purity of the extracted material

Exp. no Total amount of crude Total Polyphenol Purity of the

yellow crystalline content in the extracted
material extracted extracted material material
(g) (g) (%)
[c] [d] [d/c] X 100

1 1.430 1.340 93.71

2 1.162 1.095 94.23

3 1.075 1.028 95.63

4 1.375 1.340 97.45

 Fresh Leaves

Outdoor withering Indoor withering

Steaming, parching for 0.5 – 1hour without rolling
(deactivation of enzymes)

Indoor withering with Rolling

gentle hand rolling for
6-8 hours
Fermentation

Parching

Final Firing
(Inactivation of
endogenous
enzyme)
Rolling & Drying

Final Firing

Black Tea
(FULLY FERMENTED)

Sen-Cha Pouchong Tea

Chinese Green Tea Oolong Tea
(NON-FERMENTED) (SEMI FERMENTED)

Fig. 1. An outline of Tea manufacturing process

 OH
OH HO
HO
HO OH
HO OH
O
O OH
OH
OH H
OH H OH
OH
Epicatechin (EC) Epicatechin gallate (ECG)
2,3-Cis

OH OH
HO HO
OH OH
HO OH HO OH
O
OH O OH
OH H OH H
OH
Epigallocatechin (EGC) OH
Epigallocatechin Gallate (EGCG)

Flavanols

OH
HOOC OH
H OH
HO O
OH
OH
H OH
Gallic Acid
Catechin (2,3-trans)

Fig. 2. Molecular structures of some polyphenolic compounds

 0.9
Absorbance

0.7

0.5
425 525 625 725 825
Wave length (nm)

Fig. 3. UV Spectrum of standard Gallic acid solution (50 mg/litre)

 0.9

0.8

0.7

0.6
Absorbance

0.5

0.4

0.3

0.2

0.1
0 10 20 30 40 50 60
Concentration in mg/litre

Fig. 5. Standard curve for Gallic acid at Maximum Absorbance