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, -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 -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

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

or log Io – log I = lc or log (Io/I) = 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 = 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 0C

7. Dispenser, for extracting solvent and set at 5 ml

8. Spectrophotometer, set at = 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 = 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 = 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 = 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 = 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. no

Absorbance Concentration of Polyphenols in the diluted solution (500 times diluted) from the standard curve

(mg/litre)

[a]

Polyphenols content in the 50 ml of solution (i.e. 0.5 g of the material)

(g)

[b] = [a X 0. 25]

Amount of crude yellow crystalline material extracted

(g)

[c]

Total Polyphenol content in the

extracted material

(g)

[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. no.

Fresh Leaves

1st Step 2nd Step 3rd Step Yield of crude yellow crystals

Yield of polyphenols wrt fresh leavesSolid-liquid

extraction from leaves

Lipid + Caffeine removal from the filtrate of 1st step

Liquid-liquid extraction from water layer of 2nd

step

g Solvent ml Solvent ml Solvent ml g g %

1. 11.00

(From garden 1)

Water 3150

Dichloro- methane

3150

Ethyl Acetate

6 200 1.430 1.340 12.18

2. 14.00

(From garden 2)

Water 3150

Dichloro- methane

3150

Ethyl Acetate

6 200 1.162 1.095 7.82

Table 4. Using Chloroform for Lipid and Caffeine removal

Exp. no.

Fresh Leaves

1st Step 2nd Step 3rd Step Yield of crude yellow crystals

Yield of polyphenols wrt fresh leaves

Solid-liquid extraction from leaves

Lipid + Caffeine removal from the filtrate of 1st step

Liquid-liquid extraction from water layer of 2nd step

g Solvent ml Solvent ml Solvent ml g g %

3. 5.00

(From garden 1)

Water 3150

Chloroform 3150

Ethyl Acetate

6 200 1.075 1.028 20.56

4. 5.00

(From garden 1)

Water 4150

Chloroform 4150

Ethyl Acetate

6 300 1.375 1.340 26.80

Table 5. Purity of the extracted material

Exp. no Total amount of crude yellow crystalline material extracted

(g)

[c]

Total Polyphenol content in the

extracted material

(g)

[d]

Purity of the extracted material

(%)

[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 LeavesFresh Leaves

Steaming, parching (deactivation of enzymes)

Outdoor withering for 0.5 – 1hour

Indoor withering without rolling

Indoor withering with gentle hand rolling for

6-8 hours

Parching

Rolling

Fermentation

Final Firing(Inactivation of endogenous enzyme)

Rolling & Drying

Final Firing

Sen-ChaChinese Green Tea(NON-FERMENTED)

Pouchong TeaOolong Tea

(SEMI FERMENTED)

Black Tea(FULLY FERMENTED)

Fig. 1. An outline of Tea manufacturing process

Fig. 2. Molecular structures of some polyphenolic compounds

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

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