1

EXTRACTIVE SPECTROPHOTOMETRIC DETERMINATION CAFFEINE (1, 3,

7 – TRIMETHYL, 1H – PURINE – 2, 6 [3H, 7H] – DIONE) IN SELECTED

CONSUMER PRODUCTS AND ITS DEPOSITION IN THE HEART, KIDNEY

AND LIVER

BY

AKAJAGBOR JAMES UWUBETINE PG/M.Sc/07/08/43707

FEBRUARY 2010

2

EXTRACTIVE SPECTROPHOTOMETRIC DETERMINATION CAFFEINE (1, 3,

7 – TRIMETHYL, 1H – PURINE – 2, 6 [3H, 7H] – DIONE) IN SELECTED

CONSUMER PRODUCTS AND ITS DEPOSITION IN THE HEART, KIDNEY

AND LIVER

BY

AKAJAGBOR JAMES UWUBETINE PG/M.Sc/07/08/43707

DR. P.O. UKOHA RESEARCH SUPERVISOR

A THESIS SUBMITTED TO THE DEPARTMENT OF PURE & INDUSTRIAL CHEMISTRY, FACULTY OF PHYSICAL SCIENCES,

UNIVERSITY OF NIGERIA,

NSUKKA

FEBRUARY 2010

3

CERTIFICATION

AKAJAGBOR JAMES UWUBETINE, a postgraduate student in the Department of Pure

and Industrial Chemistry with the registration number PG/M.Sc/07/08/43707 has

satisfactorily completed the requirement for the course and research work for the degree of

MSc in Analytical Chemistry. The originality of this project is not in doubt, as no part or

whole of it has been submitted for any Diploma or Degree of this University or any other

institution.

Supervisor ----------------------------------- Dr. P.O. Ukoha Signature/Date Head of Department ---------------------------------- Dr. P. O. Ukoha Signature/Date External Supervisor ---------------------------------- Signature/Date

4

DEDICATION

This thesis is dedicated to God Almighty

5

ACKNOWLEDGEMENTS

The God Almighty is highly acknowledged for the provision of unusual strength and good

health that sustained me throughout the programme

The unflinching support of my supervisor, Dr. P.O. Ukoha and his untiring guidance and

advice is highly appreciated.

Finally, I wish to thank my friends especially Mr. T.O. Oni, Mr. A. Dauda and staff of

Lighthouse Petroleum Laboratory who offered innumerable assistance when the analysis

of the work were being carried out.

AKAJAGBOR JAMES UWUBETINE

NSUKKA

2010

6

ABSTRACT

The max of caffeine at 1ppm was determined to be 276nm and all subsequent

measurements of absorbances were based on this max value. The standard calibration

curve for pure caffeine at concentrations ranging from 10-3 – 10-6 M was prepared based

on the max of 276nm. The Molar Absorptivity derived from the calibration curve was

found to be constant at 1922.3 which confirm that the curve is in obedience to Beer-

Lambert’s Law. The extraction of caffeine from the various food products was carried out

using Association of Official Analytical Chemists (AOAC) procedure (962.13), and the

caffeine content of these products were determined spectrophotometrically. Analysis

results of the food products show that Cola Acuminata (native Kola nut) contains 5.93mg

of caffeine per g, while Cola Nitida (gworo) contains 5.7mg of caffeine per g. Other

results recorded are as follow: Nescafe (1.89mg/g), Bitter kola (0.77mg/g), Bournvita

(0.02mg/g), Milo (0.02mg/g), Coca-cola (0.1mg/cm3), Fanta (0.03mg/cm3), Power Horse

(0.26mg/cm3), Red Bull (0.35mg/cm3. While Guinness small stout, Gulder, Chelsea

London Dry Gin and Bacchus Wine registered trace values for caffeine. Probable

deposition of caffeine on body organs resulting from the consumption of caffeinated

products was investigated. Thus pure caffeine or it’s equivalent amount in energy drink

(Power Horse) at doses of either 1mg per kg and 2mg per kg body weight were selectively

administered to male albino rats through their normal animal feed in groups A, B, C, D,

and E. Group D is the control group and the rats therein were placed on normal diet of

animal feed only. Treatment was for 14 days, after which the rats were euthanized. The

caffeine treatment significantly left some deposits of caffeine at 1.096mg, 1.62mg,

1.52mg, 0.003mg and 2.827mg in the respective livers of A, B, C, D & E. while caffeine

7

deposits in the kidney of groups A, B, C, D and E respectively indicated 0.290mg,

0.514mg, 0.417mg, 0.003mg and 0.591mg. Lastly the caffeine deposits in hearts of groups

A, B, C, D & E respectively showed 0.066mg, 0.092mg, 0.029mg, 0.002mg and 0.066mg.

Finally apparent change in weight was observed in the various groups. Group D (control

group) gained 11.07% weight, and those in A & E gained 3.89% and 6.27% respectively,

while group B & C recorded a weight loss of 1.48% and 1.98% respectively. Therefore

treating rats at low concentration (1mg/kg and 2mg/kg of body weight could leave

reasonable deposits of caffeine in the organs but with significant dehydration in some of

the rats. The health implications of the results are highlighted in the discussion.

8

TABLE OF CONTENTS

Title page I

Certification III

Dedication IV

Acknowledgements V

Abstract VI

Table of Contents VIII

List of Tables XIII

List of Figures XIV

CHAPTER ONE

1.0 Introduction 1

1.1 History 2

1.2 Purpose of Study 4

CHAPTER TWO

2.1 Chemistry of Caffeine 5

2.2 Determination of Structure 6

2.2.1 Position of the Methyl Group 7

2.2.2 Position of Oxygen Atom 8

2.3.0 Properties of Caffeine 10

2.3.1 Solubilities of Caffeine 10

2.3.2 Solubilities of Caffeine in Water from 0oC to 100oC 11

2.3.3 Chemical Test 11

9

2.3.4 Murexide Test 12

2.3.5 Tannic Acid Test 12

2.4 Pharmacological Actions of Xanthines 12

2.4.1 Metabolism 12

2.4.2 Effect of Caffeine on Central Nervous System (CNS) 15

2.4.3 Effect on the Heart 17

2.4.4 Effect of Caffeine on Children 18

2.4.5 Caffeine Intake During Pregnancy 19

2.4.6 Diuresis and Caffeine 19

2.4.7 The Mutagenic Activity of Caffeine in Man 20

2.4.8 The Effect of Caffeine on Skeletal Muscle 21

2.4.9 Caffeine’s Effect on Those with Hepatitis/Liver Disease 21

2.4.10 Parkinsons Disease 22

2.4.11 Effects of Caffeine on Memory and Learning 22

2.4.12 World Consumption of Caffeine 23

2.4.13 Therapeutic Effect of Caffeine 24

2.4.14 Adverse Effect of Caffeine 25

2.4.15 Production of Caffeine 26

2.4.15.1 Decaffeination 26

2.4.15.2 Supercritical Carbon Dioxide Extraction 26

2.4.15.3 Extraction by Nonharzardous Organic Solvents 27

2.4.16 Stereochemistry 27

2.4.17 Religious View on Caffeine 28

10

2.4.18 Spectrophotometric Determination of Caffeine 28

CHAPTER THREE

3.0 Experimental 30

3.1.0 Materials 30

3.1.1 Instruments/Apparatus 33

3.2.0 Methods 33

3.2.1 UV Spectra of Caffeine 33

3.2.2 Preparation of Calibration Curve from the Pure Caffeine 33

3.2.3 Caffeine Determination in Alcoholic/Non Alcoholic Beverages 34

3.2.4 Extraction of Caffeine from Kola nuts 34

3.2.5 Coffee (Product of Nestle (Nig.) Plc.) 35

3.2.6 Determination of Caffeine from Soft Drinks 35

3.2.7 Determination of Caffeine in Alcoholic Beverages 36

3.2.8 Determination of Caffeine Content in living tissues 36

3.2.9 Melting Point Determination 39

3.2.10 Murexide Confirmatory Test for Caffeine 39

3.2.11 Tannic Acid Confirmatory Test for Caffeine 39

CHAPTER FOUR

4.0 Results and Discussions 40

4.1 Physical Properties 40

4.1.1 Melting Point of Pure/Extracted Caffeine 40

4.1.2 Confirmatory Test for Caffeine 41

11

4.1.2.1 Murexide Test 41

4.1.2.2 Tannic Acid Test for Caffeine 42

4.2 Maximum Wavelength for 1ppm Caffeine Solution 43

4.3 Standard Calibration Curve for Caffeine 45

4.3.1 Estimation of Molar Absorptivity (∈) 46

4.3.2 Determination of Actual Concentration of Caffeine 46

4.3.3 Determination of Molar Absorptivity 47

4.4 Determination of Caffeine Content in Some Food Product 48

4.4.1 Caffeine Content in Red Bull 48

4.4.2 Caffeine Content in Power Horse 48

4.4.3 Caffeine Content in Coca-Cola 49

4.4.4 Caffeine Content in Fanta 50

4.4.5 Caffeine Content in Guinness Stout 50

4.4.6 Caffeine Content in Bacchus Tonic Wine 51

4.4.7 Caffeine Content in Gulder Beer 51

4.4.8 Caffeine Content in Chelsea London Dry Gin 52

4.4.9 Caffeine Content in Kola Nut (Cola Nitida) 52

4.4.10 Caffeine Content in Kola Nut (Cola Acuminata) 52

4.4.11 Caffeine Content in Kola Nut (Bitter Kola) 53

4.4.12 Caffeine Content in Nescafe Instant Coffee 53

4.4.13 Caffeine Content in Bournvita 53

4.4.14 Caffeine Content in Milo 54

4.4.15 Summary of Caffeine Content of Analyzed Food Products 55

12

4.5.0 Determination of Caffeine Content in the Livers, Kidneys and Hearts of

Experimental Rats 56

4.5.1.A Caffeine Content in the Heart of Group A Rats 56

4.5.1.B Caffeine Content in the Kidney of Group A Rats 56

4.5.1.C Caffeine Content in the Liver of Group A Rats 57

4.6. Discussion 61

4.7. Recommendation 63

4.8. Conclusion 64

References 66

Appendices 73

13

LIST OF TABLES

2.1 Determination of Caffeine Factor 31

3.1 Summary of Reagent used for the Extraction and Spectrophotometric Analysis 32

3.2 Summary of Analyzed Materials 33

3.3 Experimental Rats 34

3.4 Feeding Pattern and Weight of Experimental Rats 39

4.1 Summary of Melting and Boiling point of Caffeine 42

4.2 Summary of Murexide Confirmatory Test 43

4.3 Summary of Tannic Acid Confirmatory Test 43

4.4 Summary of Estimate of Molar Absorptivity (∈) Values 47

4.5 Concentration of Caffeine in some Food Products 56

4.6 Initial and Final Weights of Experimental Rats 79

4.7 Distribution of Caffeine in the Liver, Kidney and Heart of Experimental Rats 80

4.8 Anova Two Factors Statistical Analysis 81

14

LIST OF FIGURES

4.1 Maximum Wavelength for 1ppm Caffeine Solution 45

4.2 Standard Calibration Curve of Caffeine 46

15

CHAPTER ONE

1.0. Introduction

Caffeine is a white crystalline xanthine alkaloid1, found in many plant species such

as coffee, tea and to a lesser extent cocoa. Other less commonly used sources of

caffeine include the yerba mate2 and guarana plants, which are sometimes used in

the preparation of teas and energy drinks. Many natural sources of caffeine also

contain widely varying mixtures of other xanthine alkaloids, including the cardiac

stimulants theophylline and theobromine and other substances such as polyphenols

that can form insoluble complexes with caffeine3. The world’s primary source of

caffeine is the coffee bean (the seed of the plant from which coffee is brewed).

Caffeine content in coffee varies widely depending on the type of coffee bean and

method of preparation used4.

The structure of caffeine is as shown below

N

N

CH3

O

N

N

O

CH3

CH3

The IUPAC Name; 1,3,7-trimethyl-1H-Purine-2, 6 (3H, 7H) – dione. Other names

are; 1,3,7 – trimethyl xanthine, Theine, Methyl Theobromine.

16

Apart from coffee, tea is another common source of caffeine. Certain types of tea,

such as black and oolong, contain more caffeine than most other teas.

Caffeine is a common ingredient of soft drinks such as cola, originally prepared

from kola nuts. Soft drinks typically contain about 10 to 50 milligrams of caffeine

per serving. By contrast, energy drinks such as Red Bull contains as much as 80

milligrams of caffeine per serving5 the caffeine in these drinks either originate from

the ingredients used or is an additive derived from the product of decaffeination or

from chemical synthesis. Gurana, a prime ingredient of energy drinks, contains large

amount of caffeine with small amount of theobromine and theophylline in a natural

slow release excepient6.

1.1 History

Humans have consumed caffeine since Stone Age 7.

Early people found that chewing the seed, bark or leaves of certain plants had the

effect of easing fatigue, stimulating awareness, and elevating mood. Only much later

was it found that steeping such plants in hot water increased the effect of caffeine.

The history of coffee has been recorded as far back as the ninth century. During that

time, coffee beans were available only in their native habitat, Ethiopia. A popular

legend traces its discovery to goatherder named Kaldi, who apparently observed

goats that became elated and sleepless at night after browsing on coffee shrubs and,

upon trying the berries that the goats had been eating, experienced the same vitality.

In 1587, Malaye Jaziri recorded that one Sheikh, Jamal-ai-Din al –Dhabhani, Mufti

17

of Aden, was the first to adopt the use of coffee in 1454, and that in the 15th century

the Sufis of Yemen routinely used coffee to stay awake during prayers8.

Towards the close of the 16th century the use of coffee was recorded by a European

resident in Egypt and about this time it came into general use in the Near East. The

appreciation of coffee as a beverage in Europe, where it was first known as “Arabian

wine” dates from the 17th century. During this time, “coffee houses” were

established, the first being opened in Constantinople and Venice. In Britain, the first

coffee houses were opened in London 1652, at St. Michel’s Alley, Cornhill. They

soon became popular throughout Western Europe, and played a significant role in

social relations in the 17th and 18th centuries9.

Kola nut, like coffee berry and tealeaf, appears to have ancient origins. It is chewed

in many West African cultures, individual or in a social setting, to restore vitality

and ease hunger pangs.

In1819, the German chemist Friedrich Ferdinand Runge isolated relatively pure

caffeine for the first time. According to Runge he did this at the behest of Johann

Wolf gang Von. Goethe10. In 1927, Oudry isolated “Theine” from tea, but it was

later proved by Mulder and Jobao that theine was the same as caffeine11. Herman

Emil Fischer was the first to achieve its total synthesis12. This was part of the work

for which Fischer was awarded Nobel Prize in 1902.

Today, global consumption of caffeine has been estimated at 120,000 tones per

annum13 making it the world’s most popular psychoactive, substance.

18

1.2. Purpose of Study

Caffeine is a central nervous system and metabolic stimulant14 and is used

recreationally and medically to reduce physical fatigue and restore mental alertness

when unusual weakness or drowsiness occurs. However, some people do not take

coffee or kola nuts because of associated feelings of restlessness or sleeplessness

that do accompany consumption. Ironically these people consume other types of

beverages like Lipton, Bournvita, Ovaltine, Milo, soft drinks and more recently

energy drinks (Red Bull etc), not minding the caffeine contents of these products.

The purpose of this study is to carry out spectrophotometric analysis of various

consumer products with the view to determine the amount of caffeine content in

them and to further determine the concentration of caffeine deposit on vital human

organs such as heart, kidney and liver, as a result of consuming these caffeine

bearing products. Finally the physiological consequences of caffeine intake in the

body will be highlighted.

19

CHAPTER TWO

2.1. Chemistry of Caffeine

Caffeine belongs to family of purine, with specific relationship with theobromine

and theophylline. The structural relationship of caffeine to other members of purine

group, indicate a similar skeletal structure. This relationship is as shown below:

N

N

CH3

O

N

N

O

CH3

CH3

N

N

O

N

NH

N

N

HO

N

N

O

CH3

CH3

N

N

CH3

O

N

N

O

CH3

H

(1) Caffeine(2) Purine

(3) Theobromine(4) Theophylline

(3,7 –Dimethylxanthine) (1,3, dimethylxanthine)

From the structure of theobromine and theophylline, it is apparent that caffeine can

be prepared through methylation with one of several methylating agents. Example of

methylation process is the synthesis of caffeine from theophylline.

20

N

N

CH3

O

N

N

O

CH3

N

N

CH3

O

N

N

O

CH3

CH3

Caffeine

CH3I in Et OH

1 equivalentMeONa

Theophylline

2.2. Determination of Structure

Caffeine with the molecular formular C8H10N4O2 and molar mass of 194.19gmol-1 is

related to uric acid in the sense that on oxidation with potassium chlorate in

hydrochloric acid, gives dimethylalloxan and methyl urea in equimolar proportions.

The structure of dimethylalloxan is shown by its conversion to sym-dimethyurea and

mesoxalic acid on hydrolysis. The results of these reactions show that both caffeine

and uric acid have the same skeletal structure positions of two and also establishes

the methyl groups and one oxygen atom in caffeine.

N

NCH3

CH3

O

O

OO

CH3 NHCONH CH3 + HOOC.CO.COOHH2O

The next step is to establish the positions of the remaining methyl group and oxygen

atom.

21

ME

NO

N

NME

12 3 4

56 7 89

The above skeleton for caffeine summarizes the information, so far obtained. The

third methyl group can be at either position of or 9 while the remaining oxygen atom

may be at position 6 or 8.

2.2.1. Position of the Methyl Group

Caffeine ][O Dimethylalloxan + methyl urea + third product. The third product

on hydrolysis gives N – methylglycine, carbon dioxide and ammonia. Thus this

production must be N – methyl hydantoin.

NCH2

COC=O

Me

COOHCH2 NHCH3

NH3 + CO2+

H2O

NH

From the foregoing, caffeine contains two rings, one of dimethylalloxan, and the

other of methylhydanton thus two structures are possible for caffeine.

N

N N

NMe

Me

Me

O

N

N N

NMe

Me MeO

I II

22

Fisher still isolated a forth oxidation product, namely sqm-dimethyloxomaide

.33 NHCOCONHCHCH Examination of I and II above shows that only I can form

this oxamide and so I is caffeine skeleton.

2.2.2. Position of Oxygen Atom

From the determination of the position of the third methyl group, two possible

structure for caffeine fit the facts very well.

N

N N

NMe

Me

Me

O

O

N

N N

NMe

Me MeO

O

Me

III IV

By comparison with uric acid structure, (III) appears more likely. However, that III

is the structure of caffeine was shown by Fischer as follows:

CaffeineC8H10O2N4

Cl2 chloro caffeineC8H9O2N4Cl

MeOHNaOH

Methoxy caffeineC8H9O2N4OCH3

Dil.HClboil

Oxycaaffeine + CH3ClC8H10O3Na

Fischer further showed that oxycaffeine was identical to trimethyluric acid because

on methylation with CH3I in aqeous NaOH, oxycaffeine was converted to

tetramethyluric acid. Therefore, methoxycaffeine is either V or VI and oxycaffeine

either VII or VIII

23

N

N N

NMe

Me

Me

O

O

OMeN

N N

NMe

MeO

O

OMe

N

N N

NMe

Me

Me

O

O

OH N

N N

NMe

Me

Me

O

OH

O

V VI

VII VIII

or

or

The structure for caffeine has also been confirmed by various syntheses e.g.

N

N N

NH

H

H

O

O

O N

N N

NCH3

CH3

CH3

O

O

O

H

CH3I

NaOHPOCl3

Uric acidtrimethyl uric acid

VIII

1, 3, 7

N

N N

NCH 3

C H 3

C H 3

O

O

Cl N

N N

N

C H 3

O

O

C hloro c affe ine

C affe ine

HI

24

2.3. Properties of Caffeine

Molecular Formular C8H10N4O2

Molar Mass 194.19

Appearance Odourless, white needles or powder, with

bitter taste.

Density 1.29gcm-3

Melting Point 237oC

Boiling Point 178oC (sublimes)

Effects on litmus paper A saturated aqueous solution is neutral to

litmus.

It crystallizes from water as monohydrate and also from dry solvents as the

anhydrous material. The hydrate gradually looses its water on exposure and

completely at 100oC. The solubilities of caffeine vary with different organic solvents

and this is given below:

2.3.1. Solubilities of Caffeine in g/100cm3.

Solvent Temperature (25oC)

Acetone 2.0

Benzene 1.0

Chloroform 18.2

Ethanol 1.5

Water 2.2

Ethyl Ether 0.2

25

2.3.2. Solubilities of Caffeine in Water from 0oC to 100oC

Temperature 0oC Solubility of Caffeine in g/100cm3 Water

0 0.60

15 1.0

20 1.46

25 2.13

30 2.80

40 4.64

50 6.75

60 10.35

70 13.50

80 19.75

100 50.00

2.3.3. Chemical Tests

Different xanthines can be distinguished through tests that have been proposed.

Caffeine can be distinguished from theobromine through Nessler’s reagent or with

potassium dichromate / sulphuric acid reagent. Also theophylline responds to copper

sulphate / sodium hydroxide solution differently from caffeine and theobromine.

26

2.3.4. Murexide Test for Caffeine

In porcelain dish, if 1mL of hydrochloric acid is added to 5g of sample (caffeine),

followed by addition of 50mg of potassium dichlorate and then evaporate the content

to dryness. There after if the dish is inverted over a vessel containing a few drops of

10% by weight ammonium hydroxide solution. The presence of caffeine in the

residue will cause it to acquire a purple colour that disappears on treatment with a

solution of fixed alkali.

2.3.5. Tannic Acid Test

If a 10% ethanolic solution of tannic acid is added drop wise to a saturated solution

of caffeine, a precipitate forms which is soluble in excess reagent.

2.4. Pharmacological Actions of Xanthines

Caffeine is a central nervous system and metabolic stimulant. It stimulates the

central nervous system first at the higher levels, resulting in increased alertness and

wakefulness, faster and clearer flow of thought, increased focus, and better general

body co-ordination.

2.4.1. Metabolism

Caffeine is completely absorbed by the stomach and small intestine within forty-five

minutes of ingestion. The hydrophilic properties of caffeine allow its passage

through all biological membranes. There is no blood-brain barrier to caffeine in the

adult or the fetal animal. There is no placenta barrier to caffeine and unusually high

caffeine has been reported in premature infants born to women who are heavy

caffeine consumers. After ingestion, it is distributed throughout the tissues of the

27

body and is eliminated by first – order kinetic15. The half-life of caffeine i.e. the time

required for the body to eliminate one half of the total amount of caffeine consumed

at a given time, varies widely among individuals according to such factors as age,

liver function, pregnancy, some concurrent medications, and the level of enzymes in

the liver needed for caffeine metabolism. In healthy adults, caffeine’s half-life is

approximately 3 –4 hours. In women taking oral contraceptives, this is increased to 5

– 10 hours15 and in pregnant women the half-life is roughly 9-11 hours16. Caffeine’s

half-life can increase to 96 hours especially to individuals who have severe liver

disease17. It has been observed that infants and young children usually exhibit half –

life longer than adults, while half-life in a new baby may be as long as 30 hours.

Generally caffeine is metabolized in the liver by the cytochrome p450 oxidase

enzyme system (specifically the IA2 isozyme) into three metabolic

dimethylxanthines18 with each having its effect on the body.

28

N

NN

NO

CH3

OCH3

CH3

N

N NH

O

CH3O

NCH3

N

NN

NO

OCH3

CH3N

NN

NO

CH3

OCH3

Caffeine

Parnxanthine (84%)

Theobromine (12%)

Theophylline (4%)

1. Paraxanthine [84%]: Has the effect of increasing hypolysis, thereby leading to

elevated glycerol and free fatty acid levels in the blood plasma.

2. Theobromine [12%]: Dilates blood vessels and increases urine volume.

3. Theophylline [4%]: Relaxes smooth muscles of the bronchi, and is used to treat

asthma. The therapeutic dose of theophylline, however is many times greater than

the levels attained from caffeine metabolism.

29

2.4.2. Effect of Caffeine on Central Nervous System (CNS)

The caffeine’s effect on the central nervous system is derived substantially from its

purine structure that has close resemblance to the structure of adenosine, which has

been known to have overwhelming influence on the nervous system.

N

N NCH3

O

CH3O

NCH3

N

N

NNH2

N

Adenosine

O

OHOH

OH

Caffeine

Adenosine induces signal that one neuron can use to tell another to stop releasing

neurotransmitter because it can’t handle the stimulation. Adenosine is the final

breakdown product of adenosine triphosphate [ATP], which is the cellular currency

of energy. It so happens that when cells have used the energy of adenosine

triphosphate, it breaks into adenosine diphosphate which is then used for energy,

which will consequently lead to it’s further breakdown into adenosine

monophosphate. In the cell’s last attempt to squeeze molecular power from

adenosine monophosphate, its last phosphate bond is broken for the necessary

energy, which finally results in the adenosine monophosphate being broken down

into simple adenosine. At this point, the neuron has very little energy left for the

successful firing of an action potential. Adenosine from this process is then released

30

from the postsynaptic cell and binds to receptors of presynaptic cell. If the amount of

adenosine released is large enough this will translate into inhibitory effect on the

release of neurotransmitter from presynaptic neuron’s axon terminal. This no doubt

triggers a mechanism that inhibits further secretion of excitatory neurotransmitters

into the synapse especially acting on moderating effect of postsynaptic from the

presynaptic neuron in restriction of further stimulation of transmitters from the

presynaptic neuron. Arising from various chemical cascade as indicated above, and

given the depletion of energy that accompanies the formation of adenosine, it is

obvious that adenosine work generally to inhibit the activity of the central nervous

system.

Caffeine being a competitive inhibitor of adenosine, it binds to the adenosine

receptor, but does not trigger the chemical cascade that inhibits neurotransmitters

release and blocks the site, so it prevents the adenosine from binding with

consequent effect of preventing message to be related or passed through the synapse.

This action of caffeine no doubt allows continuous excitation of the central nervous

system and will result in continued stimulation of neurons that will hitherto not fire

or release neurotransmitter into the synapse19. Chronic caffeine use also alters the

release of other neurotransmitter in the brain such as dopamine and catecholamine.

The antagonistic effects of caffeine at the presynaptic adenosine receptor could

allow for increased activity of these neurotransmitters. An example of this is the

increased cholinergic activity in the hippocampus following caffeine ingestion20.

The psychostimulant effects of caffeine such as decreased fatigue and increase

wakefulness can be traced to this cholinergic pathway. However, these physiological

31

effects of caffeine, generally depends on amount ingested. A small amount, probably

less than 500mg may enhance psychomotor performance, improving the work

output. If the amount is more than 500mg, it may cause anxiety, restlessness,

insomnia, and elevated body temperature. In large amounts, and especially over

extended period of time, caffeine can lead to a condition known as caffeinism21.

Caffeinism usually combines caffeine dependency with a wide range of unpleasant

physical and mental conditions including nervousness, irritability, anxiety,

tremulousness, muscle twitching (hyper reflexia) insomnia, headache, respiratory

alkalosis and heart palpitations. Furthermore, because caffeine increases the

production of stomach acid, high usage over time can lead to peptic ulcers, erosive

esophagitis and gastroesophagent disease22.

2.4.3. Effect on the Heart

The important measures of cardiovascular function are the pressure of the blood as it

flows through the arteries (blood pressure) and the heart beat rate. Blood pressure is

of special concern because high blood pressure is an indicator of strain on the heart

and blood vessels and of possible obstruction somewhere in the circulatory system.

Anything that causes or adds to high blood pressure could be dangerous. A person’s

blood pressure at any given time depends on two things: The output of blood from

the heart and the resistance of the circulatory system to the flow of blood. The output

from the heart is determined in part by the rate at which the heart beats. When the

resistances to blood flow and the volume of the blood pumped through the system at

each heartbeat remain constant, blood pressure and heartbeat rise and fall together.

32

Increased heartbeat rate usually accompanies the use of caffeine, although the

change is generally small and not statistically significant. In some studies, reduced

heartbeat rate was found after caffeine administration. Other researchers have

reported that caffeine causes an initial decrease and then an increase in heartbeat

rate.

Generally it has been established from recent research that drinking caffeinated

beverages causes significant increase in blood pressure. High blood pressure is a

silent disease that can create a devastating complication, including hardening of the

arteries, kidney problems, poor eyesight and heart attacks.

Another study has reported that intake of caffeine bearing products elevate LDL

cholesterol level significantly within three months of consumption. A new study has

suggested that caffeine gives a troubling boost to ones biological indicator of poor

heart health, which has inadvertently increase blood concentrations of the amino

acid (homocysteine) which has been associated with elevated risk of heart attacks.

2.4.4. Effect of Caffeine on Children

There is a common belief that caffeine consumption causes stunted growth in

children24. However, as with adults, nausea, urinary urgency, nervousness, or other

effects from an elevated caffeine intake via chocolate milk, sodas, cold medicines,

iced tea, coffee and other products that are widely used, may be reasons to limit the

amount that is been consumed by children.

33

2.4.5. Caffeine Intake during Pregnancy

There is a general agreement that pregnant women and those trying to conceive

should avoid consuming large quantities of caffeine. But after decades of

controversy and conflicting evidences, there is still no real consensus on how much

caffeine is safe during pregnancy. Women are generally advised to limit their

caffeine intake to less than 200mg per day, as those who consumed 200mg or more

of caffeine face the risk of miscarriage25. Besides miscarriage, caffeine can cause

blood vessels to constrict, thereby reducing blood flow to the placenta. The fact that

caffeine easily crosses the placenta, and reaches the baby (who then slowly

metabolizes it) could affect his developing cells. Some studies have suggested that

high levels of caffeine consumption by pregnant women may slightly reduce a

baby’s birth weight. One study has found a link between maternal caffeine

consumption equal to three cups of coffee per day and an increase risk of having a

son born with undescended testes. This happens when the testes don’t move from the

pelvis into the scrotum as they usually do in late pregnancy. Another research has

equally shown that babies whose mothers consumed more than 500mg of caffeine a

day had faster heartbeat and breathing rates and spent more time awake in the first

few days after birth25. Further studies have shown that it is pretty difficult for

pregnant women to absorb iron, due to the inhibiting effect of caffeine25.

2.4.6. Diuresis and Caffeine

Methylated xanthine tends to increase the glomerular filteration rate by improving

the general circulation or specifically the renal circulation. Caffeine increases the

34

number of functioning glomeruli and this finding especially in frogs led to the

suggestion that the xanthines raise filteration pressure by dilating the afferent

arterioles. Total renal blood flow is increased by caffeine and theobromine but it is

usually decreased by theophylline.

The direct actions of the drugs on kidney blood flow are augmented by their

stimulant action on heart that results in an increased cardiac output. Tolerance to

these actions develops with repeated dosage and may be already present in habitual

tea and coffee drinkers.

2.4.7. The Mutagenic Activity of Caffeine in Man

It has been suggested that caffeine may have mutagenic effect in man26. The melting

temperature of DNA18 is lowered by its interaction with caffeine. Experiment carried

out with Hela cells had proved to be the best available material for studying induced

chromosome breakage in human cells. Further studies which were extended to

leukocyte short-term cultures had proved essentially the same with those obtained

with Hela cells.

Hela cells were treated for 1 hour with varying concentrations of caffeine dissolved

in Eagle’s medium. Medium without caffeine was then added after the cells had

been washed twice and then incubated for another 24 hours. A chromosomal

analysis was then carried out, which interestingly revealed that caffeine is effective

in breaking chromosomes of human cells in culture. The amount of breakage is

almost linear with dose. However, at higher doses (10mg/mL) the effect seems to be

greater than linear. Since the production of breakage seems to be linearly dependent

35

on doses, it is possible that the much lower doses received by man in drinking coffee

or tea are mutagenic as well.

2.4.8. The Effect of Caffeine on Skeletal Muscle

Caffeine has been known as muscular stimulant. Caffeine as a potent cerebral

stimulant, superimposes volitional muscular activity over fatigue and thus increases

ones capacity for muscular work. Caffeine test on athletes performances has

however, showed that for 100m sprints, the administration of caffeine in the athletes

did not significantly improve their speed performance. However, in the high jump,

where muscular activity combines with mental precision in performance, it was

reported that 63% of the participants receiving caffeine improved their jump27.

2.4.9. Caffeine’s Effect on Those with Hepatitis/Liver Disease

Caffeine is metabolized through the liver. However, caffeine itself is not directly

harmful to the liver. Infact one study even suggested that coffee, but not other

caffeine containing drinks, may delay progression of liver disease to cirrhosis

(though not substantiated by other studies). However, in moderation one or two cups

of a caffeine bearing beverages per day may suppress the fatigue associated with

liver disease to some extent. Excessive intake of caffeine may put people with

chronic liver disease at increased risk of osteoporis and bone fractures. And in

people with cirrhosis, the metabolism of caffeine is slowed, resulting in higher

concentrations of caffeine in the blood. Thus, people with cirrhosis should limit their

36

caffeine intake to one cup of coffee or tea per day. Infact it is best for all people with

liver disease to consume caffeine in moderation29.

2.4.10. Parkinson’s Disease

Several large studies have shown that caffeine intake is associated with a reduced

risk of developing Parkinson’s disease (PD) in men, but studies in women have been

inconclusive30. The mechanism by which caffeine affects PD remains a mystery. In

animal models researchers have shown that caffeine can prevent the loss of

dopamine-producing nerve cells seen in Parkinson’s disease, but researchers still do

not know how this occurs31.

2.4.11. Effects of Caffeine on Memory and Learning

An array of studies found that caffeine could have nootropic effects, including

certain changes in memory and learning. However, it is still not definitely clear

whether the effect is negative or positive.

Researchers have found that long-term consumption of low dose caffeine (0.3g/dm3)

slowed hippocampus-dependent learning and impaired long-term memory. Caffeine

consumption for 4 weeks also significantly reduced hippocampal neurogenesis

compared to controls during the excitement. The conclusion was that long-term

consumption of caffeine could inhibit hippocampus-dependent learning and memory

partially through inhibition of hippocampal neurogenesis32.

37

In one study, caffeine was added to rat neurons in vitro. The dendritic spines (a part

of the brain cell used in forming connections between neurons) taken from the

hippocampus (a part of the brain associated with the memory tasks33.

Another study showed that subjects – after receiving 100 milligrams of caffeine-had

increased activity in brain regions located in the frontal lobe, where a part of the

working memory is located, and the anterior cingulum, a part of the brain that

controls attention. The caffeinated subjects also performed better on the memory

tasks34.

However a different study showed that caffeine could impair short-term memory and

increase the likelihood of the tip of the tongue phenomenon. The study allowed the

researchers to suggest that caffeine could aid short-term memory when the

information to be recalled is related to the current train of thought, but also to

hypothesize that caffeine hinders short-term memory when the train of thought is

unrelated35. In essence, focused thought coupled with caffeine consumption increase

mental performance.

2.4.12. World Consumption of Caffeine

Although coffee and other caffeine containing beverages were introduced in Europe

only a few hundred years ago, consumption of these beverages now occupies a

significant place in their national cultures. Caffeine is present in a number of dietary

sources consumed worldwide i.e. tea, coffee, cocoa beverages, chocolate bars, soft

drinks and more recently energy drinks36.

38

Estimate of Caffeine Consumption Worldwide are:

United States (196-423mg) per day

UK (359-621).

Caffeine consumption from all sources can be estimated to around 70 to 76

mg/person/day worldwide.

In 60-70kg individual, daily consumption is estimated at 2.4 to 40mg/kg i.e. 170-

300mg. While in 7-10 years old children, the daily consumption of caffeine ranges

from 0.5 to 1.8mg/kg36.

2.4.13. Therapeutic Effect of Caffeine

A lot of commercial preparations of caffeine are available and they are generally

prescribed for various therapeutic uses37

(i) Vigilance Stimulation: Caffeine is generally used in the form of coffee or tea,

to ward off tiredness or fatigue.

(ii) Bronchodilation: It is used in the treatment of chronic asthma and other

obstructive chronic lung diseases. Theophylline was formerly one of the drugs

mostly used in the treatment of asthma. Stimulation of respiration, treatment of

apnea of premature neonates, has been associated with caffeine and

theophylline by exploiting their stimulant effect that can reduce frequency and

duration of apnea.

Generally caffeine has found relevance in the preparation of pain-relieving drugs

especially for headache originating from eye strain38. In recent times, coffee has

been found to be useful in the treatment of constipation and may protect against the

39

pain of gallstones. Reduced risk of Parkinson’s disease development has been

identified with coffee drinkers.

Although coffee is relatively safe and will have no adverse health effects on the

majority of the population, there are probably rare cases of people who should avoid

caffeine because of its stimulating effects; especially those people with irregular

heartbeat or other heart conditions38.

2.4.14. Adverse Effect of Caffeine

Although Food and Drug Administration has classified caffeine, this does not mean

that caffeine is an innocuous compound. Some undesirable side effects of caffeine

administration are: Insomnia, restlessness, agitation, headaches and tremors. In

individuals who consume large amounts of caffeine daily (in excess of 1000mg/day

i.e. 10 cups of coffee), a syndrome referred to as caffenism may develop39. Sufferers

of caffenism are highly agitated over trivial matters; nervous twitches punctuate

their muscle movement, their breathing is short and rapid and they generally

experience visual hallucination. Consumption of caffeine has also been linked to an

increased frequency and severity of premenstrual syndrome (PMS). This interaction

is dose dependent with PMS increasing by as much as 5-fold for those women

consuming 8 to 10 cups of coffee daily. Finally, recent controversial therapeutic uses

of caffeine are closely linked to: Its efficacy in the eradication of skin funguses,

improvement in sperm mobility, to reinforce the toxic effect of chemicals used in

cancer therapy and to facilitate the production of seizures during electroconvulsive

therapy40.

40

2.4.15. Production of Caffeine

Caffeine extracted from coffee and tea during the decaffeination process is sold or

used as an additive. Being readily available as a byproduct of decaffeination,

caffeine is not usually synthesized41. If desired, it may be synthesized from dimethyl

urea and malonic acid42.

2.4.15.1. Decaffeination

Pure caffeine is a white powder, and can be extracted from a variety of natural

sources. Caffeine extraction is an important industrial process and can be performed

using a number of different solvents. Benzene, chloroform, trichloroethylene and

dichloromethane have all been used over the years but for reasons of safety,

environmental impact, cost and flavour, they have been super superseded by the

following main methods:

Water Extraction; coffee beans are soaked in water. The water, which contains not

only caffeine but also many other compounds that contribute to the flavour of coffee,

is then passed through activated charcoal, which removes the caffeine. The water

can then be put back with the beans and evaporated dry, leaving decaffeinated coffee

with a good flavour43. Coffee manufacturers recover the caffeine and resell it for use

in soft drinks and over-the-counter caffeine tablet

2.4.15.2. Supercritical Carbon Dioxide Extraction

Supercritical carbon dioxide is an excellent nonpolar solvent for caffeine (as well as

many other organic compounds), and is safer than the organic solvents that are used

41

for caffeine extraction. The extraction process is simple: CO2 is forced through the

green coffee beans at temperatures above 31.1oC and pressures above 73atm. Under

these conditions, CO2 is in a “supercritical” state: it has gas like properties which

allow it to penetrate deep into the beans but also liquid-like properties which

dissolve 97-99% of the caffeine. The caffeine-laden CO2 is then sprayed with high-

pressure water to remove the caffeine. The caffeine can then be isolated by charcoal

absorption (as above) or by distillation, recrystallization, or reverse osmosis43.

2.4.15.3. Extraction by Nonhazardous Organic Solvents

Organic solvents such as ethyl acetate present much less health and environmental

hazard than previously used chlorinated and aromatic solvents. The hydrolysis

products of ethyl acetate are ethanol and acetic acid, both nonhazardous in small

quantities. Another method is to use triglyceride oils obtained from spent coffee

grounds44.

2.4.16. Stereochemistry

The nitrogen atoms are all essentially planar (in SP 2 orbital hybridization). Even

though some are often drawn with three single bonds, the lone pairs on these atoms

are involved in resonance with adjacent double bonded carbon atoms, resulting in

the caffeine molecule having aromatic character45.

42

2.4.17. Religious View on Caffeine

Mormons and Christian Scientists46 do not consume caffeine. Followers of both

religions believe that God wishes them to be free of any addiction. However, this

seems to be a contradiction as many religious people still consume coffee and other

caffeine-bearing products to enable them stay awake over the night for prayers and

other spiritual activities.

2.4.18. Spectrophotometric Determination of Caffeine

The characteristic absorption of caffeine at 272nm is utilized to measure

quantitatively its presence in coffee and crude coffee47. Interfering impurities found

in these samples are removed by treatment with heavy magnesium oxide and zinc

ferrocyanide, plus in some cases permanganate oxidation. Rapidity and specificity

for caffeine are outstanding characteristics of the method. Results obtained by the

new method compare favourably with the Bailey-Andrew Procedure that is used as a

reference for comparison47. Detailed precision studies showing the new method to be

equal to or better than the Bailey-Andrew method are reported47. Decaffeinated

coffee will require a modification of the methods herein described which would

probably involve a solvent extraction step. The operation of a coffee decaffeinating

plant requires a great number of analytical caffeine determinations. For a number of

years, the A.O.A.C. official Power-Chestnut (g) method was used in the laboratory.

This method was satisfactory, but excessively time consuming for control

operations. In 1943, the Bailey-Andrew method(s), then official only for tea, was

successfully adapted for use here by increasing the specified amount of magnesium

43

oxide to 25grams and by making suitable changes in sample weight to permit its

application to decaffeinated products and solutions as well as to green or roasted

coffee. This method has since been made official for caffeine in coffee. The

procedure although more rapid than the Power-Chestnut, still requires an elapsed

time of at least 7 hours per determination. Even when greater number of this analysis

was being performed, it was never possible in the laboratory to perform the many

manipulations in much less than 1 man hour per sample.

In 1946, an investigation into the possibility of measuring concentration of caffeine

by utilizing its known absorption characteristics in the ultraviolet range was begun.

Absorption of light by caffeine was reported as early as 1905 by Hartley47. In 1919,

Henri reported that the absorption spectrum of caffeine had been studied47.

Castile and Rupport in 1928 reported the quantitative measurements on caffeine

indicating absorption maximum at 271 and 275nm at differing pH47. Adherence to

Beer’s law was generally indicated. Molecular extinction coefficients ranging from

8000 to 11530 were reported by these workers.

44

CHAPTER THREE

3.0. EXPERIMENTAL

3.1.0. Materials

All chemicals used were of analytical grade and were used as supplied unless

otherwise stated. A list of reagents employed during extraction of caffeine and its

spectrophotometric analysis are given in Table 1. The rats used were classified into

groups and these are summarized in Table 3.3

Table 1. Summary of Reagents Used For the Extraction and Spectrophotometric

Analysis.

Reagents. Source.

Pure Caffeine

Chloroform

Potassium Permanganate

Sodium Hydroxide

Magnesium Oxide

Potassium Thiocyanate (KSCN)

Sodium Tetraoxosulphate (VI) (Na2SO4)

Phosphoric Acid (H3PO4)

Hydrochloric Acid

Potassium Dichromate

Ammonium Hydroxide

Ethanol

Tannic Acid

SIGMA ALDRICH

«

«

«

«

«

«

«

«

«

«

«

«

45

Table 2. Summary of Analyzed Materials

Name Of Material Name Of Producer Source of Purchase

Nescafe

Bournvita

Milo

Coca Cola

Fanta

Power Horse

Red Bull

Guinness Small Stout

Gulder Beer

Chelsea London Dry Gin

Bacchus Tonic Wine

Cola-Acuminata

Cola-Nitida

Bitter Cola

Nestle Nig. Plc.

Cadbury Nig. Plc

Nestle Nig. Plc

Coca-Cola Company Plc

Coca-Cola Company Plc

GMbH LinZ, Austria

Arizonal Trading Company lagos

Guinness Nig. Plc

Nigerian Breweries

Nigerian Distillers Ltd. Ogun State

Nigerian Distillers Ltd. Ogun State

Sold Freely

Sold Freely

Sold Freely

ESCO Supermarket

ESCO Supermarket

ESCO Supermarket

ESCO Supermarket

ESCO Supermarket

ESCO Supermarket

ESCO Supermarket

ESCO Supermarket

ESCO Supermarket

ESCO Supermarket

ESCO Supermarket

Ogbogonogo Market, Asaba Delta State.

Ogbogonogo Market, Asaba Delta State

Ogbogonogo market, Asaba Delta State

46

Table 3. Experimental Rats

Groups Type of Rat Source

A1

A2

A3

A4

B1

B2

B3

B4

C1

C2

C3

C4

D1

D2

D3

D4

E1

E2

E3

E4

WISTAR ALBINO RATS

DEPARTMENT OF

VETERINARY

MEDICINE, UNIVERSITY

OF NIGERIA, NSUKKA

47

3.1.1 Instruments/Apparatus

All weighings were carried out using Mettler Toledo Analytical balance. A Fischer

Scientific centrifuge was utilized during the extraction of caffeine from kola nuts.

The melting points of the synthesized and pure caffeine were determined using John

Fischer melting point apparatus. The temperature was recorded from the in-built

thermometer connected to the apparatus.

Electronic spectra of the analyzed products, pure caffeine and synthesized caffeine

were obtained with a Perkin Elmer model Lambada EZ301 spectrophotometer

equipped with computer and printer.

Other apparatus employed involve glassware’s of various types, separation flask,

pipette, retort stands, gloves, filter paper (7cm), masking tape, rubber tubing’s and

Bunsen burner.

3.2.0. METHODS

3.2.1. UV- Spectra of Caffeine.

A Perkin Elmer, model Lambada EZ301 spectrophotometer was used. The electronic

absorption spectra in the UV-visible range was recorded between 110 to 396nm,

using chloroform as solvent.

3.2.2. Preparation of Calibration Curve from the Pure Caffeine.

Caffeine solutions containing 0.10, 0.25, 0.50, 1.00, 1.50 and 2.00mg of caffeine

dissolved in 100cm3 chloroform were prepared from stock solution of 100mg

caffeine in 100cm3 chloroform. Absorbance of these solutions was recorded at

276nm, since this had earlier been established to be the max of caffeine.

48

3.2.3. Caffeine Determination in Alcoholic/Non-alcoholic Beverages Using

Association of Official Analytical Chemists (AOAC Method 962-13)

Procedure:

10cm3 of test portion was pipetted into 125mL separation flask. This was

thoroughly mixed with 5cm3 of 1.5% KMnO4 solution, allowed to stand for five

minutes, and 10cm3 of reducing solution (5g NaSO4 + 5g KCSN dissolved in

100cm3 distilled water) was added and thoroughly stirred. 1cm3 of 15% H3PO4

solution was thereafter added, followed by addition of 1cm3 25% NaOH solution.

The solution was thoroughly mixed. This was followed by the addition of 50cm3

chloroform to effect caffeine extraction. Two layers were formed. The lower layer

was drained through 7cm filter paper into 100cm3 glass-stoppered volumetric flask.

Further 2-3cm3 chloroform was added to the separation flask and drained again to

rinse separator stem. The paper was again washed with 2-3cm3 chloroform, the

drained solution was again subjected to re-extraction with 40cm3 chloroform, and

paper and stem were thoroughly washed as before. Finally the solution in the

volumetric flask was finally diluted to mark with chloroform. From this solution, the

absorbance of the solution was obtained at the max of caffeine against chloroform

blank. The corresponding concentration of caffeine was extrapolated from the

calibration curve

3.2.4. Extraction of Caffeine From Kola nuts

Procedure:

(a) One (1) gram of kola nut (Cola acuminata) i.e. Urhobo or Ibo type of Kola nut

was crushed in a mortar. This was further blended for 30minutes into a paste.

49

Chloroform was added to extract the caffeine. The chloroform layer was then

separated from the water layer by centrifugation. The centrifuge speed was set at 300

rpm. The chloroform layer was filtered and the filtrate evaporated to dryness. The

residue after evaporation was recrystallised as white powder (Mp 230oC). However,

to determine the concentration of caffeine in the one gram of kola nut, a sample of

the filtrate in 1cm quartz cell against the chloroform blank was used for the UV

spectrum. The caffeine content was determined from the calibration curve, earlier

prepared from the pure caffeine.

(b) Cola Nitida (gworo) and Bitter Kola.

The above experimental procedure was used in extracting caffeine from Cola

Nitida and Bitter Kola.

3.2.5. Coffee (Product of Nestle (Nig.) Plc.)

Caffeine was extracted from 1g of coffee using 100cm3 chloroform in a separating

funnel. The UV spectrum was obtained using a small portion of the extract. The

same experimental procedure was adapted in extracting caffeine from Bournvita

(Product of Cadbury Nig.Ltd.) and Milo (Product of Nestle Nig.Plc.)

3.2.6. Determination of Caffeine from Soft Drinks.

Experimental procedure for the determination of caffeine content in non-alcoholic

beverages was adopted in the determination of caffeine contents in coca-cola, Fanta

50

(Products of Coca-cola Bottling Company Plc.), Power Horse (product of GmbH

Linz, Austria), Red Bull (product of Arizona Trading Company, Lagos).

3.2.7. Determination of Caffeine in Alcoholic Beverages.

Experimental procedure in 3.2.3 was used in the determination of caffeine content in

Guinness Small Stout (product of Guinness Nig. Plc.), Gulder (product of Nigerian

Breweries, Iganmu Lagos), Chelsea London Dry Gin (product of Nigeria Distillers

Ltd, Sango Ota, Ogun State), and Bacchus Tonic Wine (Nigerian Distillers Ltd,

Sango Ota, Ogun State).

3.2.8. Determination of Caffeine Content in the Livers, Hearts and Kidneys of

Wistar Albino Male Rats)

In this study, twenty (20) wistar Albino Male Rats obtained from the Faculty of

Veterinary Medicine, University of Nigeria Nsukka, were used. These were divided

into five (5) groups: A, B, C, D, and E of Four rats each. The group D was

designated as the control group, and this was fed orally with normal animal feed

from Ewu Flour Mill. The B groups was made up of four rats, and were fed orally

with animal feed that was mixed with 1mg of pure caffeine per 1kg body weight,

while group C is made up of four rats fed with normal animal feed that was mixed

with 2mg of pure caffeine per Kg body weight. Group A consist of four (4) rats, fed

with normal animal feed but mixed with energy drink (Power Horse) that contain

1mg of pure caffeine per 1kg body weight. The last group (E) also contains four rats

fed orally with the normal animal feed but mixed with energy drink (Power Horse)

that contains 2mg of pure caffeine per kg body weight.

51

See Table below for the summary of the feeding pattern and the body weight of the

experimental rats.

Table 4: Feeding Pattern and Weights of Experimental Rats

Groups

Weight Before the

Experiment.

FEEDING PATTERN

A1 107.5 2g NORMAL ANIMAL FEED MIXED WITH

ENERGY DRINK (POWER HORSE) THAT

CONTAINS 1mg OF CAFFEINE A2 99.7

A3 88.80

A4 102.4

B1 100.80 2g NORMAL ANIMAL FEED MIXED WITH

1mg PURE CAFFEINE PER 1kg BODY

WEIGHT

B2 101.20

B3 102.00

B4 103.00

C1 102.04 2g NORMAL ANIMAL FEED MIXED WITH

2mg OF PURE CAFFEINE PER 1kg BODY

WEIGHT

C2 96.20

C3 115.60

C4 103.22

D1 105.2 NORMAL ANIMAL FEED OF 2g PER DAY

D2 112.2

D3 103.0

D4 85.6

E1 101.02 2g NORMAL ANIMAL FEED MIXED WITH

ENERGY DRINK (POWER HORSE) THAT

CONTAINS 2mg OF CAFFEINE

E2 92.72

E3 109.21

E4 99.05

Procedure:

The initial weights of rats in the various groups were taken and recorded as shown in

the Table above. The final weights of rat were taken after the 14th day when feed and

52

caffeine have been administered as indicated in Table 4. The choice of a single sex

(i.e. all male) was to prevent copulation that could cause pregnancy during the

period of the study. In this particular study, it is terminal rather than sequential.

These rats were acclimatized for three days before they were randomly assigned to

five groups of four rats each. The rats were housed in steel cages at room

temperature of 25oC and exposed to a normal daylight/dark cycle under humid

tropical condition. Apart from the rations administered to these rats, enough drinking

water was supplied throughout the duration of the experiment. All the rats received

humane care in accordance with the guidelines of National Institute of Health U.S.A.

for ethical treatment of laboratory animals. Hence after the 14th day of extensive

feeding, monitoring, the rats were all euthanized. The organs of interest viz: Heart,

Kidney and Liver were neatly harvested, properly labeled and stored in a

refrigerator. Thereafter the individual organs (Liver, Kidney and Heart) of the

individual rats were blended seperately into paste. This was followed by carrying out

the extraction of caffeine by the addition of chloroform and centrifuging at 300 rpm.

The resultant two distinct layers were separated. The solution in the 100cm3

volumetric flask was re-extracted with 40cm3 chloroform and made to mark. From

the solution, its absorbance at max was taken and the corresponding concentration

extrapolated from the standard calibration curve. However, before the absorbance

was taken, the extract was subjected to a confirmatory test using Murexide and

Tannic acid test respectively to establish the presence of caffeine.

Furthermore, a TWO FACTOR ANOVA STATISTICAL analysis method was used

to validate analyzed body organs.

53

3.2.9. Melting Point Determination

John Fischer melting point apparatus was used for this determination. It involves

placing a minute quantity in a sample plate container, which gets heated up once the

electrical source, is switched on. Over the plate is a viewing lens, through which the

sample is viewed as the temperature rises. As soon as the sample is viewed to melt

or decompose, the temperature is recorded from the inbuilt thermometer connected

to the apparatus.

3.2.10. Murexide Confirmatory Test For Caffeine

1cm3 of hydrochloric acid was added to 5ppm, and 1ppm pure caffeine in a

porcelein dish. This was followed by addition of 50mg of potassium dichlorate and

content was evaporated to dryness. The dish was inverted over a vessel containing

10% Ammonium hydroxide

3.2.11. Tannic Acid Confirmatory Test For Caffeine

10% Ethanolic acid solution of tannic acid was added dropwise to 5ppm and 1ppm

solutions of caffeine. However, the test was replicated for kola nut extract, kidney,

liver and heart.

54

CHAPTER FOUR

4.0. Results & Discussion

4.1. Physical Properties

4.1.1. Melting Point of Pure and Extracted Caffeine

Table 5

S/N Type of Test Unit Pure Caffeine (Imported) Extracted Caffeine

1 Melting Point OC

235

233

Table 5 above is the summary of melting point of caffeine.

55

4.1.2. Confirmatory Test for Caffeine

4.1.2.1. Murexide Test

Table 6 Summary of Murexide Confirmatory Test

S/N Sample Test Colour Change Remark

1

1

Caffeine Standard

5.0ppm

Murexide Deep purple that disappears

with NaOH

Caffeine present

2

2

Caffeine Standard

1ppm

Murexide Deep purple that disappears

with NaOH

Caffeine present

3

3

Extract from Kola nut Murexide Purple that disappears with

NaOH

Caffeine present

4

4

Extract from Kidney Murexide Pale colour that disappears

with NaOH

Trace amount of caffeine

5

5

Extract from Liver Murexide Pale colour that disappears

with NaOH

Trace amount of caffeine

5

6

Extract from Heart Murexide Pale colour that disappears

with NaOH

Trace amount of caffeine

56

4.1.2.2. Tannic Acid Test for Caffeine

Table 7: Summary of Tannic Acid Confirmatory Test

S/N Sample Test Colour Change Remark

1 Caffeine Standard 5.0ppm Tannic White Insoluble Precipitate Caffeine present

2 Caffeine Standard 1ppm Tannic White Insoluble Precipitate Caffeine present

3 Extract from Kola nut Tannic White Insoluble Precipitate Caffeine present

4 Extract from Kidney Tannic White Insoluble Precipitate Caffeine present

5 Extract from Liver Tannic White Insoluble Precipitate Caffeine present

6 Extract from Heart Tannic White Insoluble Precipitate Caffeine present

57

4.2. Maximum Wavelength for 1ppm Caffeine Solution

Figure 1: maximum wavelength for 1ppm caffeine solution

Fig 1 above shows the maximum wavelength obtained from scanning 1ppm caffeine

in chloroform solution. The caffeine at 1ppm has max centered at 276nm. This was

in close conformity with what was reported by earlier work46. At concentration of

10-3 to 10-6 M, at room temperature, Beer-Lambert’s Law was obeyed and this was

further confirmed by the constancy of Є (molar absorbtivity value) calculated to be

1922.3 from the calibration curve, as shown below.

58

59

4.3. Standard Calibration Curve for Caffeine

Figure 2: Standard Calibration Curve for Caffeine

From Fig 2 above a straight-line curve that passes through the origin was obtained.

60

4.3.1. Estimation of Molar Absorptivity (Є) From the Values of Absorbance

and Concentration Used In the Calibration of the Standard Calibration Curve

Table 8: Summary of Estimate of Molar Absorptivity (Є) Values

S/N Absorbance Quantity of Caffeine dissolved

in 100cm3 Chloroform

Actual Concentration

of Caffeine

Molar Absorbtivity

(Є)

1 0.009 0.10mg 5.1496 x 10-6M 1747.71

2 0.23 0.25mg 1.2874 x 10-5M 1786.55

3 0.049 0.5mg 2.5748 x 10-5M 1903.06

4 0.099 1.0mg 5.1496 x 10-5M 1922.48

5 0.149 1.5mg 7.7244 x 10-5M 1928.95

6 0.198 2.0mg 1.0299 x 10-4M 1922.5

4.3.2. Determination of Actual Concentration of Caffeine

Molecular Weight of Caffeine = 194.19

1Molar solution of caffeine will require 194.19g dissolved in 1dm3 solvent

194.19mg dissolved in 100cm3 chloroform =0.001M solution

100mg dissolved in 100cm3 chloroform = 5.1496 x 10-3

Hence,

0.1mg of caffeine dissolved in 100cm3 chloroform = 5.1496 x 10-3

= 5.1496 x 10-6

0.25mg 5.1496 x 10-3 ÷ 400 = 2.2874 x 10-5

─ 0.5mg 5.1496 x 10-3 ÷ 200 = 2.5748 x 10-5M

61

1.0mg 5.1496 x 10-3 ÷ 100 = 5.1496 x 10-5M

1.5mg 5.1496 x 10-3 ÷ 66.6667 = 7.7244M

2.0mg 5.1496 x 10-3 ÷ 50 = 1.0299 x 10-4M

4.3.3. Determination of Molar Absorptivity (Є)

The Beer – Lambert law is

A = Єbc ---------------- (1)

Where,

A = Absorbance

Є = Molar Absorptivity

b = Length of cell = 1cm

c = Concentration of solution

From (1) above

Є = A ÷ bc

For 0.1mg at concentration of 5.1496 x 10-6M

Є = 0.009 ÷ 1 x 5.1496 x 10-6 = 1747.71

For 0.25mg,

Є = 0.023 ÷ 1 x 5.2874 x 10-5 = 1786.55

For 0.5mg,

Є = 0.049 ÷ 1 x 2.5748 x 10-5 = 1903.06

For 1.0mg,

62

Є = 0.099 ÷ 1 x 7.7244 x 10-5 = 1922.48

For 1.5mg,

Є = 0.149 ÷ 1 x 7.7244 x 10-5 = 1928.95

For 2.0mg,

Є = 0.198 ÷ 1 x 1.0299 x 10-4 = 1922.50

4.4. Determination of Caffeine Content in Some Food Products

4.4.1. Caffeine Content in Red Bull

Absorbance at max 276 = 0.022 x 150

= 3.3

Regression Factor = 10.40

Concentration of caffeine in 100cm3 of Red Bull

= 10.40 x 3.3

= 34.617mg per 100cm3

1cm3 of Red Bull = 34.617 ÷ 100

= 0.34617mg

250cm3 = 0.34617 x 250cm3

= 86.5

= 87mg

4.4.2. Caffeine Content in Power Horse

Absorbance at max 276 = 0.025 x 100

63

= 2.5

Regression Factor = 10.4090

Concentration of Caffeine in 100cm3 of Power Horse

= 10.4090 x 2.5

= 26.0225mg per 100cm3 chloroform.

1cm3 of Power Horse = 26.0225 ÷ 100

= 0.260225mg

330cm3 = 0.260225 x 330cm3

= 85.7

= 86mg

4.4.3. Caffeine Content in Coca – Cola

Absorbance at max 276 = 0.099 x 10

= 0.99

Regression Factor = 10.4090

Concentration of Caffeine in 100cm3 of coca – cola

= 10.4090 x 0.99

= 10.30491

1cm3 of coca – cola will contain = 10.30491 ÷ 100

= 0.1030491

In 350cm3 of coca – cola = 0.1030491 x 350cm3

= 36.07mg

64

4.4.4. Caffeine Content in Fanta

Absorbance at max 276nm = 0.028 x 10

= 0.28

Regression Factor = 10.4090

Concentration of Caffeine in 100cm3 of Fanta

= 10.4090 x 0.28

= 2.91452

1cm3 of Fanta will contain = 2.91452 ÷ 100

= 0.0291452

In 350cm3 of Fanta will contain = 0.0291452 x 350cm3

= 10.2mg

4.4.5. Caffeine content in Guinness Stout

Absorbance at max 276nm = 0.001

Regression Factor = 10.4090

Concentration of Caffeine in 100cm3 of Guinness stout

= 10.4090 x 0.001

= 0.010409

1cm3 of Guinness stout will contain = 0.010409 ÷ 100

= 0.00020818mg caffeine

In 325cm3 of Guinness stout = 0.00020818 x 325cm3

= 0.068mg

65

4.4.6. Caffeine Content in Bacchus Tonic Wine

Absorbance at max 276nm = 0.028 x 10

= 0.28

Regression Factor = 10.4090

Concentration of Caffeine in 100cm3 Bacchus Tonic Wine

= 10.4090 x 0.28

= 2.91452mg

1cm3 of Bacchus Tonic Wine will contain = 2.91452 ÷ 100

= 0.0291452

In 375cm3 of Bacchus Tonic Wine = 0.0291452 x 375cm3

= 11mg

4.4.7. Caffeine Content in Gulder Beer

Absorbance at max 276nm = 0.001

Regression Factor = 10.4090

Concentration of Caffeine in 100cm3 of Gulder Beer

= 10.4090 x 0.001

= 0.010409mg

1cm3 of Gulder Beer will contain = 0.010409 ÷ 100

= 0.00010409

In 600cm3 of Gulder Beer = 0.00010409 x 600cm3

= 0.063mg

66

4.4.8. Caffeine Content in Chelsea London Dry Gin

Absorbance at max 276nm = 0.001

Regression Factor = 10.4090

Concentration of Caffeine in 100cm3 of Chelsea London Dry Gin

= 10.4090 x 0.001

= 0.010409mg

1cm3 of Chelsea London Dry Gin will contain = 0.010409 ÷ 100

= 0.00010409

In 375cm3 of Chelsea London Dry Gin = 0.00010409 x 375cm3

= 0.04mg

4.4.9. Caffeine Content in Kola Nut (Cola Nitida)

Maximum Absorbance Recorded for 1g of Kola nut dissolved in 100cm3 chloroform =

0.55

Concentration of caffeine in 1g Kola nut

= 0.55 x 10.4090 (Regression Factor)

= 5.73mg

4.4.10. Caffeine Content in Kola Nut (Cola Acuminata)

Maximum Absorbance Recorded for 1g of Kola nut dissolved in 100cm3 chloroform =

0.57

Concentration of caffeine in 1g Kola nut

67

= 0.57 x 10.4090 (Regression Factor)

= 5.93mg per 1g kola nut

4.4.11. Caffeine Content in Kola Nut (Bitter Kola)

Maximum Absorbance Recorded at max 276 = 0.074

Concentration of caffeine in 1g bitter kola

= 0.074 x 10.4090

= 0.77mg in 1g bitter kola

4.4.12. Caffeine Content in Nescafe Instant Coffee

Maximum Absorbance at max 276nm = 0.182

Regression Factor = 10.4090

Concentration of Caffeine in 1g of Nescafe Instant Coffee

= 10.4090 x 0.182

= 1.89mg per 1g coffee

50g Nescafe Instant Coffee = 94.72mg

4.4.13. Caffeine Content in Bournvita

Maximum Absorbance at max 276nm = 0.0019

Regression Factor = 10.4090

Concentration of Caffeine in 1g of Bournvita

= 10.4090 x 0.0019

= 0.02mg per 1g Bournvita

68

450g Bournvita will contain = 9.0mg

4.4.14. Caffeine Content in Milo

Maximum Absorbance at max 276nm = 0.0029

Regression Factor = 10.4090

Concentration of Caffeine in 1g of Milo

= 10.4090 x 0.0029

= 0.0302mg per 1g Milo

450g Milo will contain = 13.59mg

69

4.4.15. Summary of Caffeine Content of Analyzed Food Products

Table 9: Concentration of Caffeine in Some Food Products

S/N SAMPLE MANUFACTURER WT/VOL. OF SAMPLE (g/cm3)

CONCENTRATION OF CAFFEINE

1 Cola Nitida (gworo) Sold Freely 1g 5.73mg

2 Cola Acuminata (Native Kola nut)

Sold Freely 1g 5.93mg

3 Bitter kola Sold Freely 1g 0.77mg

4 Nescafe Nestle Nig. Plc 1g 1.89mg

5 Bournvita Cadbury Nig. Plc 1g 0.02mg

6 Milo Nestle Nig. Plc 1g 0.03mg

7 Coca-Cola Coca-Cola Company Plc 1cm3 0.1mg

8 Fanta Coca-Cola Company Plc 1cm3 0.03mg

9 Power Horse Power Horse Energy Drink GmbH Linz, Austria

1cm3 0.26mg

10 Red Bull Arizonal Trading Company Lagos

1cm3 0.35mg

11 Guinness Small Stout Guinness Nig. Plc 1cm3 2.08x10-4

12 Gulder Nigerian Breweries Iganmu, Lagos

1cm3 1.04x10-4

13 Chelsea London Dry Gin Nigerian Distillers Ltd, Sango Ota, Ogun State

1cm3 1.04x10-4

14 Bacchus Tonic Wine Nigerian Distillers Ltd, Sango Ota, Ogun State

1cm3 2.91x10-2

70

4.5.0 Determination of Caffeine Content in the Livers, Kidneys and Hearts of

Experimental Rats

4.5.1.A. Caffeine Content in the Heart of Group A Rats

Caffeine Concentration in Rat A1

Maximum Absorbance = 0.0328

Regression Factor = 10.0024

Caffeine Content = 10.0024 x 0.0328

= 0.3281

1g Heart = 0.3281 ÷ 0.5024

= 0.066mg

4.5.1.B Caffeine Content in the Kidney of Group A Rats

Caffeine Concentration in Rat A1

Maximum Absorbance = 0.0266

Regression Factor = 10.0024

Caffeine Content = 10.0024 x 0.0266

= 0.26606

For 1g of kidney = 0.26606 ÷ 1.0087

= 0.264mg

71

4.5.1.C Caffeine Content in the Liver of Group A Rats

Caffeine Content of Rat A1

Maximum Absorbance = 0.375

Regression Factor = 10.0024

Caffeine Content = 10.0024 x 0.375

= 3.7509

For 1g of Liver = 3.7509 ÷ 3.8003

= 0.98mg

72

Table 10: Initial and Final Weights of Experimental Rats

Groups Weight Before the

Experiment.

Wt. after 14th day of

Feeding & Monitoring

A1 105.2 108.1

A2 112.2 119.63

A3 103.0 105.04

A4 85.6 89.04

B1 100.80 99.01

B2 101.20 99.26

B3 102.00 101.23

B4 103.00 101.46

C1 102.04 99.41

C2 96.20 95.20

C3 115.60 112.11

C4 103.22 102.09

D1 107.5 123.49

D2 99.7 DIED

D3 88.80 96.10

D4 102.4 112.22

E1 101.02 107.15

E2 92.72 97.42

E3 109.21 135.53

E4 99.05 87.6

73

TABLE 11 Distribution of Caffeine in the organs of experimental Rats.

Weight of organs and rats (g)

Concentration of

caffeine in the organs (mg) Heart Kidney Liver Wt.of rat Heart Kidney Liver A1 0.5024 1.0087 3.8003 108.1 0.066 0.264 0.987 A2 0.5315 1.1404 4.769 119.63 0.097 0.289 1.124 A3 0.474 0.8785 3.2475 105.04 0.053 0.289 0.876 A4 0.4152 0.7013 2.8352 89.04 0.049 0.316 1.397 B1 0.5213 0.9099 3.6548 99.01 0.089 0.507 1.613 B2 0.471 1.0025 4.3057 99.26 0.091 0.513 1.527 B3 0.6874 1.0031 4.3067 101.23 0.092 0.517 1.688 B4 0.6877 1.0034 4.3065 101.46 0.095 0.52 1.687 C1 0.3889 0.8021 3.0754 99.41 0.022 0.416 1.518 C2 0.5359 0.8644 3.6344 95.20 0.025 0.416 1.427 C3 0.5803 0.9821 3.6825 112.11 0.037 0.417 1.633 C4 0.4653 0.8757 3.5867 102.09 0.031 0.419 1.517 D1 0.5494 0.8463 3.9385 123.49 0.002 0.003 0.002 D2 0.4199 0.9873 3.9667 96.1 0.002 0.002 0.003 D3 0.5151 0.9767 4.384 112.22 0.002 0.003 0.003 D4 E1 0.4738 0.9847 3.8638 107.15 0.068 0.628 2.897 E2 0.461 0.8385 4.4216 97.42 0.053 0.513 2.913 E3 0.5953 1.0399 3.9384 135.53 0.087 0.714 2.88 E4 0.4095 0.7544 2.8441 87.6 0.056 0.509 2.616

74

Statistical Analysis of the Organs Analyzed

(Anova : TWO Factor) statistical analysis of analyzed body organ is presented in

Table 12 below.

Table 12 ANOVA TWO FACTOR STATISTICAL ANALYSIS

SUMMARY Count Sum Average Variance Row 1 3 5.3114 1.7705 3.1543 Row 2 3 6.4409 2.1470 5.2490 Row 3 3 4.6 1.5333 2.2447 Row 4 3 3.9517 1.3172 1.7486 Row 5 3 5.086 1.6953 2.9174 Row 6 3 5.7792 1.9264 4.3164 Row 7 3 5.9972 1.9991 4.0188 Row 8 3 5.9776 1.9925 4.0440 Row 9 3 4.2664 1.4221 2.0927 Row 10 3 5.0347 1.6782 2.8969 Row 11 3 5.2449 1.7483 2.8462 Row 12 3 4.9277 1.6426 2.8768 Row 13 3 5.3342 1.7781 3.5226 Row 14 3 5.3739 1.7913 3.6298 Row 15 3 5.8758 1.9586 4.4652 Row 16 3 5.3223 1.7741 3.3404 Row 17 3 5.7211 1.9070 4.7779 Row 18 3 5.5736 1.8579 3.2959 Row 19 3 4.008 1.3360 1.7355 Column 1 19 9.6649 0.5087 0.0068 Column 2 19 17.5999 0.9263 0.0119 Column 3 19 72.5618 3.8190 0.2934

ANOVA

Source of variation SS Df MS F P-value F crit. Row 2.7769 18 0.1543 1.9549 0.0428 1.8986 Columns 123.5052 2 61.7526 782.5112 0.0000 3.2594 Error 2.8410 36 0.0789 Total 129.123 56

75

4.6. Discussion

The study was necessitated by the observed indiscriminate consumption of

caffeinated products in recent times without minding the possible health hazards

from its use over time even at a low concentration

The analysis presented in Table 2 has instructively shown that both Kola Acuminata

and Nitida contain a high caffeine contents as consumption of 50g of kolanuts would

amount to an intake of 281mg of caffeine. As for energy drinks, especially Power

Horse that contains 0.25mg per cm3 it would mean that consumption of 330cm3 can

of Power Horse would translate to an intake of 82mg. Another that presented a high

value of caffeine is Nescafe. At 1.84mg per g, a consumption of 100g of the product

would amount to total intake of 184mg of caffeine. Other products assayed did not

indicate significant caffeine presence except coca-cola, which registered 0.1mg per

cm3 of product. This shows that a consumption of a bottle of coca-cola will translate

to 35mg of caffeine intake.

From Table 10 (Analysis of Organs), it is crystal clear that there is a significant

presence of caffeine in the various organs assayed. However, the degree of presence

is directly proportional to the amount of caffeine intake either directly or as

ingredient of beverages. This is exemplified through the statistical analysis

(ANOVA) that succinctly revealed significant variations in the caffeine contents

deposited in the livers, kidneys and hearts of rats as a result of treatment with pure

caffeine and caffeine bearing products especially when F calculated is higher than F

tabulated for both rows and columns.

76

The highest caffeine concentration was recorded in the liver of the rats; this was

followed by the caffeine concentration in the kidneys, while the hearts had the least

deposit of caffeine concentration.

A noticeable trend, particularly of how the amount of caffeine consumed affects the

body weight was observed. In the control group, the rats averagely gained 11.08%

weight, while those in-group A & E gained 3.87% and 6.07% weight respectively.

However for rats in-group B & C, fed with pure caffeine in their diet, they recorded

a weight loss of 1.48% and 1.98% respectively. This no doubt corroborates earlier

reported work that caffeine is a useful part of a weight control programme25. It has

been further reported that caffeine when ingested with a meal, increases the rate at

which the food is converted into useable energy. Also when taken in between meals,

it causes fats to be transferred from deposits in the cells to the blood stream. As free

fatty acids they can be used as energy by most of the organs of the body. Caffeine

also raises the activity level of the body, which thus causes energy derived from

food to be used up rapidly rather than being stored as fat. Furthermore, caffeine is

known to stimulate the temperature regulating centres of the body, which in turn

produces an increase in body temperature. To sustain these changes, energy that

might otherwise be deposited as fat is used. Thus even when the body is at rest, a

greater amount of food is burned. However, the added weight exhibited by rats in

groups A and E even when fed with equivalent amounts of caffeine as in groups B &

C, may be attributable to the presence of other additives in the energy drink which

must have helped to moderate the direct impact of caffeine on the various body

organs.

77

4.7. Recommendation

Considering the analysis result as it relates to caffeine presence in some selected

food products and its consequent deposits in the body organs: heart, kidney and

liver, it is evidently clear that severe health challenges await consumers of

caffeinated products even at low concentrations, if they are not timeously sensitized

to dangers inherent in such consumptions. There is no doubt, unregulated and

unguided consumption will promote ailments that are detrimental to the wellbeing of

the consumers. The popularity accorded the consumption of highly caffeine enriched

energy drinks in recent times has become worrisome. Therefore it has become

imperative, that the underlisted recommendations though not exhaustive are being

proposed to serve as the necessary guide for those people that are deeply enmeshed

in the consumption of caffeine bearing products.

(1) Hypertensive Patients – People who have been diagnosed to be hypertensive

should abstain from the consumption of products that are highly rich in caffeine.

These products such as kola nut (Kola acuminata and nitida), coffee and energy

drinks are capable of aggravating hypertensive situations, so consumption of these

products should be avoided or taken in moderation.

(2) Stunted Growth – This was demonstrated by the rats in groups B and C. Stunted

growth due to intake of caffeine, may occur in children of ages between 1 – 18

years. Therefore it is advisable that children should avoid excessive intake of

caffeine-bearing products.

78

(3) Abortion in Pregnant Mothers – Excessive intake of caffeine beyond 150mg per

day could cause abortion in pregnant mothers. Pregnant mothers when consuming

caffeine-rich products should exercise utmost caution.

(4) The Food and Drug Regulatory Agencies e.g. NAFDAC should ensure that the

quantity of caffeine that goes into drinks, foods, and drugs production should not

exceed permissible limits that will engender health crisis.

(5) There is no gainsaying the fact, that anything taken in excess would always cause

discomfort to our well-being. Even the water we take, if not taken in moderation

could be injurious to the body. Therefore it is incubent on all to exercise utmost

caution when taking caffeine-bearing products.

4.8. Conclusion

Extractive Spectrophotometric determination of caffeine has been successfully

carried out at 276nm. Beer-Lambert’s law is obeyed between the concentration

ranges of 4 x 10-6 to 8 x 10-6M. Caffeine content of some food products and their

consequent deposit on vital body organs has been determined

spectrophotometrically. The study has brought to the fore, that whenever a quantity

of caffeine-bearing product is consumed, probably due to incomplete metabolism, it

causes certain amounts of caffeine to be deposited in the livers, kidneys and hearts

of humans as indicated in the experimental rats. Therefore, it is advisable to exercise

utmost caution in the consumption of caffeine-bearing products so as to minimize

the overloading of livers, kidneys and hearts with caffeine. There is no doubt,

prolonged accumulation of caffeine on these organs could provoke the onset of liver

cirrhosis, kidney dysfunction and heart congestion as the case may be. Finally, even

79

though caffeine has been proved to be a weight loss aid, serious consideration should

be given to the caffeine’s side effects before advocating its use for this purpose.

80

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87

Appendices

Appendix 1 ……….Concentration of Caffeine in (Cola-Nitida)

Appendix 2 ……….Concentration of Caffeine in Cola Acuminata.

Appendix 3 ……….Concentration of Caffeine in Bitter Kola.

Appendix 4 ……….Concentration of Caffeine in Nescafe.

Appendix 5 ……….Concentration of Caffeine in Bournvita.

Appendix 6 ……….Concentration of Caffeine in Milo.

Appendix 7 ……….Concentration of Caffeine in Coca-cola.

Appendix 8 .………Concentration of Caffeine in Fanta

Appendix 9 ……….Concentration of Caffeine in Power Horse

Appendix 10 ……….Concentration of Caffeine in Red Bull.

Appendix 11 ……….Concentration of Caffeine in Guinness Small Stout.

Appendix 12 ……….Concentration of Caffeine in Gulder Beer.

Appendix 13 ……….Concentration of Caffeine in Chelsea London Dry Gin.

Appendix 14 ……….Concentration of Caffeine in Bacchus Tonic Wine.

Appendix 15 ……….Caffeine Concentration in the Heart of Rat A1

Appendix 16 ……….Caffeine Concentration in the Heart of Rat A2

Appendix 17 ……….Caffeine Concentration in the Heart of Rat A3

Appendix 18 ……….Caffeine Concentration in the Heart of Rat A4

Appendix 19 ……….Caffeine Concentration in the Kidney of Rat A1

Appendix 20 ……….Caffeine Concentration in the Kidney of Rat A2

Appendix 21 ……….Caffeine Concentration in the Kidney of Rat A3

Appendix 22 ……….Caffeine Concentration in the Kidney of Rat A4

88

Appendix 23 ……….Caffeine Concentration in the Liver of Rat A1

Appendix 24 ……….Caffeine Concentration in the Liver of Rat A2

Appendix 25 ……….Caffeine Concentration in the Liver of Rat A3

Appendix 26 ……….Caffeine Concentration in the Liver of Rat A4

Appendix 27 ……….Caffeine Concentration in the Heart of Rat B1

Appendix 28 ……….Caffeine Concentration in the Heart of Rat B2

Appendix 29 ……….Caffeine Concentration in the Heart of Rat B3

Appendix 30 ……….Caffeine Concentration in the Heart of Rat B4

Appendix 31 ……….Caffeine Concentration in the Kidney of Rat B1

Appendix 32 ……….Caffeine Concentration in the Kidney of Rat B2

Appendix 33 ……….Caffeine Concentration in the Kidney of Rat B3

Appendix 34 ……….Caffeine Concentration in the Kidney of Rat B4

Appendix 35 ……….Caffeine Concentration in the Liver of Rat B1

Appendix 36 ……….Caffeine Concentration in the Liver of Rat B2

Appendix 37 ……….Caffeine Concentration in the Liver of Rat B3

Appendix 38 ……….Caffeine Concentration in the Liver of Rat B4

Appendix 39 ……….Caffeine Concentration in the Heart of Rat C1

Appendix 40 ……….Caffeine Concentration in the Heart of Rat C2

Appendix 41 ……….Caffeine Concentration in the Heart of Rat C3

Appendix 42 ……….Caffeine Concentration in the Heart of Rat C4

Appendix 43 ……….Caffeine Concentration in the Kidney of Rat C1

Appendix 44 ……….Caffeine Concentration in the Kidney of Rat C2

Appendix 45 ……….Caffeine Concentration in the Kidney of Rat C3

89

Appendix 46 ……….Caffeine Concentration in the Kidney of Rat C4

Appendix 47 ……….Caffeine Concentration in the Liver of Rat C1

Appendix 48 ……….Caffeine Concentration in the Liver of Rat C2

Appendix 49 ……….Caffeine Concentration in the Liver of Rat C3

Appendix 50 ……….Caffeine Concentration in the Liver of Rat C4

Appendix 51 ……….Caffeine Concentration in the Heart of Rat E1

Appendix 52 ……….Caffeine Concentration in the Heart of Rat E2

Appendix 53 ……….Caffeine Concentration in the Heart of Rat E3

Appendix 54 ……….Caffeine Concentration in the Heart of Rat E4

Appendix 55 ……….Caffeine Concentration in the Kidney of Rat E1

Appendix 56 ……….Caffeine Concentration in the Kidney of Rat E2

Appendix 57 ……….Caffeine Concentration in the Kidney of Rat E3

Appendix 58 ……….Caffeine Concentration in the Kidney of Rat E4

Appendix 59 ……….Caffeine Concentration in the Liver of Rat E1

Appendix 60 ……….Caffeine Concentration in the Liver of Rat E2

Appendix 61 ……….Caffeine Concentration in the Liver of Rat E3

Appendix 62 ……….Caffeine Concentration in the Liver of Rat E4