EXTRACTIVE SPECTROPHOTOMETRIC … PROJECT WORK.pdf · 4.4.13 Caffeine Content in Bournvita 53...
Transcript of EXTRACTIVE SPECTROPHOTOMETRIC … PROJECT WORK.pdf · 4.4.13 Caffeine Content in Bournvita 53...
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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
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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
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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
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DEDICATION
This thesis is dedicated to God Almighty
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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
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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
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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.
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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
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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
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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
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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
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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
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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
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LIST OF FIGURES
4.1 Maximum Wavelength for 1ppm Caffeine Solution 45
4.2 Standard Calibration Curve of Caffeine 46
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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.
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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
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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.
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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.
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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.
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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.
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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
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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
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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
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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
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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.
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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
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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.
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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.
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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
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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
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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