INTRODUCTION

Tea is the most commonly and widely used soft beverage in the household.
It acts as a stimulant for central nervous system and skeletal muscles. That
is why tea removes fatigue, tiredness and headache. It also increases the
capacity of thinking. It is also used for lowering body temperature. The
principal constituent of tea, which is responsible for all these properties, is
the alkaloid-caffeine. The amount of caffeine in tea leavers varies from
sample to sample.

Originally it was thought that caffeine is responsible for the taste and
flavour of tea. But pure caffeine has been found to be a tasteless while
substance. Therefore, the taste and flavour of tea is due to some other
substance present in it. There is a little doubt that the popularity of the
xanthenes beverages depends on their stimulant action, although most
people are unawareof any stimulation. The degree to which an individual is
stimulated by given amount of caffeine varies from individual to individual.

For example, some people boast their ability to drink several cups of coffee
in evening and yet sleep like a long, on the other hand there are people who
are so sensitive to caffeine that even a single cup of coffee will cause a
response boarding on the toxic. The xanthene beverages also create a
medical problem. They are dietary of a stimulant of the CNS. Often the
physicians face the question whether to deny caffeine-containing beverages
to patients or not.

In fact children are more susceptible than adults to excitation by xanthenes.

For this reason, tea and coffee should be excluded from their diet. Even
cocoa is of doubtful value. It has a high tannin content may be as high as 50
mg per cup. After all our main stress is on the presence of caffeine in
xanthene beverages and so in this project we will study and observe the
quantity of caffeine varying in different samples of tea leaves.
Theory

The most important methylated alkaloid that occurs naturally is caffeine. Its
molecular formula is CsH10N4O2. Its IUPAC name is 1, 3, 7-
trimethylxanthene and common name is 1-methylated thiobromine. Purely
it is white, crystalline solid in the form of needles. Its melting point is 1230c.
It is the main active principle component of tea leaves.

It is present in tea leaves up to 3% and can be extracted by first boiling the

tea leaves with water which dissolves many glycoside compounds in addition
to caffeine. The clear solution is then treated with lead acetate to precipitate
the glycoside compounds in the form of lead complex. The clear filtrate is
then extracted with extracts caffeine because it is more soluble in it then
water.
Uses of Caffeine :

l. In medicine, it is used to stimulate, central nervous system and to

increase flow of urine.

2. Because of its stimulating effects, caffeine has been used to relieve

fatigue. But it is dangerous and one may collapse if not consumes it under
certain limit.

3. Caffeine is also used in analgesic tablets, as it is believed to be a pain

reliever. It is also beneficial in migraines.

Effects of Caffeine

l. It is psycho - stimulant.
2. It improves physical and mental ability.

3. Its effect in learning is doubtful but intellectual performance may

improve where it has been used to reduce fatigue or boredom.

[Link] administered internally, it stimulates heart and nervous system and

also acts as diuretic. On the contrary their excessive use is harmful to
digestion and their long use leads to mental retardation.

Materials and methods

Sample preparation.

Five types of tea in loose leaf form: Pai Mu Tan-superior (white tea), Sencha
(green tea), Formosa Fine Oolong (oolong tea), Lingia (black tea), Yin Zhen
(yellow tea) and two types of loose leaf maté tea (maté tea and roasted maté
tea) were purchased on a local market. In order to simulate household
brewing conditions, teas were prepared using an aqueous extraction. Tea
samples (2.5 g) were poured with 200 ml of boiling water and stirred for 10
minutes. Extracts were filtered through a cotton wool, cooled at a room
temperature, diluted to 250 ml with distilled water, and used for
spectrophotometric analyses.

Caffeine isolation with chloroform.

The caffeine isolation procedure was performed according to a modified

method described by Rapić (1994). Briefly, 20 g of tea and 90 ml of distilled
water was refluxed for 30 min, and filtered under vacuum. The residue was
again refluxed and filtered. Obtained filtrates were combined, 12.5 ml of
Pb(CH3 COO)2 solution was added, boiled (5 min), and filtered through a
Büchner funnel with silica gel layer. The filtrate was extracted four times
with chloroform (40 ml). Combined chloroform phases were washed with
KOH solution and then with distilled water. Chloroform was removed from
extracts by rotary evaporator. After evaporation, extracted caffeine was
weighed and expressed in mg/l. Caffeine determination using the lead
acetate solution. This procedure is based on international standards with
some modifications (Yao et al. 1992, 1993). Tea extract was treated with HCl
solution (5 ml), Pb(CH3 COO)2 and H2 SO4 solution. Absorbance of
obtained extracts was measured at 274 nm. The content of caffeine (mg/l)
was calculated using a standard curve derived from caffeine (0–250 mg/l).
All measurements were performed in triplicate.

The micromethod for the determination of caffeine.

The teas were also analysed for their caffeine content according to the
method reported by Groisser (1978). Briefly, tea extracts (pH = 8–9) were
extracted with benzene and H2 SO4 . Absorbance of extracts was read at
273 nm against a blank (H2 SO4 ). Results, obtained from triplicate
analyses, were calculated using a standard curve and expressed as mg/l.

HPLC analysis of caffeine. Filtered tea extracts and caffeine solutions were
injected for HPLC analysis according to the method reported in our previous
study (Horžić et al. 2009). Equipment used consisted of a Varian Pro Star
Solvent Delivery System 230 and a Photodiode Array detector Varian Pro
Star 330 (Varian, Walnut Creek, USA) with a reversed-phase column
Pinnacle II C-18 (Restek, USA) (250 × 4.6 mm, 5 μm i.d.). Caffeine was
identified by comparing the retention times and spectral data with those of
authentic standards. All analyses were repeated three times. Antioxidant
capacity. Antioxidant capacity of tea extracts and caffeine solutions (100
mg/l) was determined using the ABTS radical scavenging assay according to
the method reported by Re et al. (1999), and ferric reducing/antioxidant
power (FRAP) assay, carried out according to the original method by Benzie
and Strain (1996).

HPLC analysis of caffeine. Filtered tea extractsand caffeine solutions were

injected for HPLC analysis according to the method reported in our previous
study (Horžić et al. 2009). Equipment used consisted of a Varian Pro Star
Solvent Delivery System 230 and a Photodiode Array detector Varian Pro
Star 330 (Varian, Walnut Creek, USA) with a reversed-phase column
Pinnacle II C-18 (Restek, USA) (250 × 4.6 mm, 5 μm i.d.). Caffeine was
identified by comparing the retention times and spectral data with those of
authentic standards. All analyses were repeated three times.
Antioxidant capacity.
Antioxidant capacity of tea extracts and caffeine solutions (100 mg/l) was
determined using the ABTS radical scavenging assay according to the
method reported by Re etal. (1999), and ferric reducing/antioxidant power
(FRAP) assay, carried out according to the original method by Benzie and
Strain (1996).
 Procedure

I. First of all, 50 grams of tea leaves were taken as sample and 150 ml
of water was added to it in a beaker.
II. Then the beaker was heated up to extreme boiling.
III. The solution was filtered and lead acetate was added to the filtrater,
leading to the formation of a curdy brown coloured precipitate.
IV. We kept on adding lead acetate till no more precipitate has been
formed.
V. Again solution was filtered.
VI. Now the filtrate so obtained was heated until it had become 50 ml
VII. Then the solution left was allowed to cool
VIII. After that, 20 ml. of chloroform was added to it.
IX. Soon after, two layers appeared in the separating funnel.
X. We separated the lower layer
XI. The solution then exposed to atmosphere in order to allow chloroform
to get evaporated.
XII. The residue left behind was caffeine.
XIII. Then we weighed it and recorded the observations.

Similar procedure was performed with different samples of

tealeaves and quantity of caffeine was observed in them

[Link] Label Tea (Brooke Bond) Weight of china dish 46.60gms

Weight of china dish with precipitate 47.20gms.

Amount of caffeine 0.60gms

[Link] Label Tea (Lipton) Weight of china dish 46.60gms

Weight of china dish with precipitate 47.15gms.

Amount of caffeine 0.55gms

1. Quantity of caffeine in Red label tea is 60mg. /sample of 50 gm. 2.
Quantity of caffeine in yellow label tea is 55mg./sample of 50 gm.
Quantity of caffeine in green label tea is 45mg./sample of 50 gm.