FACULTY OF PHYSICAL SCIENCE - University of Nigeria, Nsukka · 2017-02-22 · 2 tittle page...

PHYTOCHEMICAL SCREENING, ANTIOXIDANT

CAPACITY AND ANTIMALARIAL ACTIVITIES OF

DEPARTMENT OF PURE AND INDUSTRIAL



UNRIPE COCONUT FLUID.


CHEMISTRY

ONYEYILIM EBUKA LEONARDPG/M.Sc/14/68312

FACULTY OF PHYSICAL SCIENCE

Onah Ifeanyi

Digitally Signed by: Content manager’s

DN : CN = Webmaster’s name

1




ONYEYILIM EBUKA LEONARD

FACULTY OF PHYSICAL SCIENCE

: Content manager’s Name

Webmaster’s name

2

TITTLE PAGE

PHYTOCHEMICAL SCREENING, ANTIOXIDANT CAPACITY AND

ANTIMALARIAL ACTIVITIES OF UNRIPE COCONUT FLUID.

BY


PG/M.Sc/14/68312

DEPARTMENT OF PURE AND INDUSTRIAL CHEMISTRY

UNIVERSITY OF NIGERIA, NSUKKA

JANUARY, 2016

DEPARTMENT OF PURE AND INDUSTRIAL CHEMISTRY FACULTY OF PHYSICAL SCIENCES

3

UNIVERSITY OF NIGERIA, NSUKKA RESEARCH PROJECT (CHM592)

TOPIC:

PHYTOCHEMICAL SCREENING, ANTIOXIDANT CAPACITY AND

ANTIMALARIAL ACTIVITIES OF UNRIPE COCONUT FLUID

A RESEARCH PROJECT SUBMITTED IN PARTIAL FULFILLMENT OF

THE REQUIREMENT FOR THE AWARD OF THE DEGREE OF MASTER

OF SCIENCE (MSc) IN ORGANIC CHEMISTRY

BY


PG/M.Sc/14/68312

SUPERVISOR: DR B.E EZEMA

JANUARY, 2016

4

CERTIFICATION

Mr. Onyeyilim, Ebuka Leonard, a postgraduate student in the Department of Pure and Industrial

Chemistry with registration number PG/M.Sc/14/68312, has satisfactorily completed the

requirements for research for the degree of Master of Science in Pure and Industrial Chemistry.

The work embodied in this thesis is original and has not been submitted in part or full for any other

diploma or degree in this or any other university.

……………………. ………………………….

Dr B.E. Ezema Dr E.A. Ochonogor

Supervisor Head of Department

……………………

External Examiner

Date ……………………

5

DEDICATION

This work is dedicated to Almighty God for His unwavering faithfulness throughout my MSc

programme.

6

ACKNOWLEDGEMENT

I wish to thank God for the gift of life, grace and resources in completing this work. I express my

profound gratitude to my supervisor, Dr B.E. Ezema, whose efforts, encouragement, concern and

spirit of understanding made this work possible.

I am also grateful to the head of department of Pure and Industrial Chemistry,University of

Nigeria, Nsukka, Dr. A. E. Ochonogor, and other staff in the Department. I am indebted to my

parents, Mr. and Mrs. Onyeyilim for their moral and financial support from my primary education

till date. I appreciate a lot.

Ultimately, I remain grateful to my siblings and friends who have been there for me throughout my

programme. May God bless you all!

Onyeyilim, Ebuka Leonard.

7

ABSTRACT

The phytochemical, antioxidant, antimicrobial and antimalarial activities of the fluid inside an

unripe coconut fluid were investigated and at the end of the day, the phytochemicals were found to

be terpenoids, flavonoids, alkaloids, tannins and saponins and the percentage composition of these

classes of compounds were also determined. This component of an unripe coconut had fluid which

is very effective in destroying malaria parasites in the same way the strength showed on the

organism by the control drug which is Atesurnate. The antioxidant capacity of the fluid was also

determined vis-à-vis and they were interestingly good results.

8

TABLE OF CONTENTS

Title page i

Certification iii

Dedication iv

Acknowledgement v

Abstract vi

Table of Contents vii

CHAPTER ONE

1.0 Introduction 1

1.1 Natural products and medicinal chemistry 1

1.2 Statement of problem 2

1.3 Objectives of study 2

1.4 Justification of study 3

CHAPTER TWO

2.0 Review of Related Literature 4

2.1 Phytochemical analysis of unripe coconut fluid 4

2.1.1 Alkaloids 4

2.1.1.1 Classification of alkaloids 5

2.1.1.2 Properties 6

2.1.2 Tannins 6

2.1.2.1 Structure of tannins 7

2.1.2.2 Classification of tannins 8

2.1.3 Terpenoids 10

9

2.1.3.1 Structure of terpenoids 10

2.1.4 Flavanoids 18

2.1.4 Saponins 25

2.2 Phytohormones present coconut fluid 26

2.2.1 Auxin 26

2.2.2 Cytokinins 27

2.2.3 Gibberellins 28

2.3 Nutritional benefits of unripe coconut fluid 28

2.4 Natural products with antimalarial activities 30

2.5 Structures of some established antimalarial drugs 30

CHAPTER THREE

3.0 Experimental Section 32

3.1 Qualitative and quantitative phytochemical screening of unripe coconut

Fluid 32

3.1.1 Qualitative phytochemical screening test 32

3.1.1.1 Test for tepernoids 32

3.1.1.2 Test for flavonoids 32

3.1.1.3 Test for alkaloids 33

3.1.1.4 Test for tannins 33

3.1.1.5 Test for saponins 34

10

3.1.2 Quantitative phytochemical screening test 34

3.1.2.1 Terpenoids 34

3.1.2.2 Flavonoids 34

3.1.2.3 Alkaloids 34

3.1.2.4 Tannins 35

3.1.2.5 Saponins 35

3.2 Antimicrobial screening test 35

3.2.1 Minimum Inhibitory Concentration (MIC) testing 36

3.3 Acute toxicity screening test 37

3.4 Innoculation of the parasitaemia 37

3.4.1 Determination of malaria parasite (MP+) 38

3.5 Determination of antioxidant activities 38

3.5.1 Determination of Superoxide Dismutase (SOD) 38

3.5.2 Determination of vitamin C 39

3.5.3 Determination of vitamin E 40

3.6 Haematological test 40

3.6.1 Determination of total white blood cell count 40

3.6.2 Determination of total red blood cell count 41

3.6.3 Determination of haemoglobin concentration 41

3.7 Liver function test 41

3.7.1 Determination of the aspartate aminotransferase activity 42

11

3.7.2 Determination of the alanine transaminase activity 42

3.7.3 Determination of total bilirubin 43

3.8 The kidney function test activity 43

3.8.1 Determination of urea 44

3.8.2 Determination of uric acid 44

3.8.3 Determination of acid phosphatase 45

3.8.4 Determination of creatinine 45

3.9 Determination of trace elements 45

3.9.1 Serum chloride determination 45

3.9.2 Serum sodium determination 46

3.9.3 Serum potassium determination 46

3.9.4 Serum zinc determination 47

CHAPTER FOUR

4.0 Results and discussion 48

4.1 Results of phytochemical screening of unripe coconut fluid 49

4.2 Results of antimicrobial activity 50

4.3 Result of acute toxicity test (LD50) 51

4.4Result of percentage parasitaemia 52

4.5 Results of antioxidant activities 53

4.6 Results of haematological test 56

4.7 Results of liver function test activities 59

4.8 Results of kidney function test activities 61

12

4.9 Results of trace element determination 63

CHAPTER FIVE 66

5.0 Conclusion 67

References

13

LIST OF TABLES

Table 1: Qualitative phytochemical screening of unripe coconut fluid 48

Table 2: Quantitative Phytochemical Screening of unripe coconut fluid 49

Table 3: Antimicrobial Sensitivity Testing of the unripe coconut fluid 50

Table 4: Inhibition Zones Diameter (mm) 50

Table 5: Acute toxicity result table 51

Table 6: 4days after inoculation of malaria parasite 52

Table 7: 7days treatment of malaria parasite 52

Table 8: Determination of Superoxide Dismutase (SOD) 54

Table 9: Determination of vitamin E 55

Table 10: Determination of vitamin C 55

Table 11: Determination of Total White Blood cell count 56

Table 12: Determination of Total Red Blood cell Count 57

Table 13: Determination of Haemoglobin Concentration 58

Table 14: Aspartate Aminotransferase (AST) Activity 59

Table 15: Alanine Aminotransferase (ALT) Activity 59

Table 16: Determination of total bilirubin 60

Table 17: Determination of Creatinine 61

Table 18: Determination of Urea 61

Table 19: Determination of Uric acid 62

Table 20: Determination of Acid phosphate 62

14

Table 21: Serum Potassium determination 63

Table 22: Serum Sodium determination 64

Table 23: Serum Chloride determination 64

Table 24: Serum Zinc determination 65

15

LIST OF FIGURES

Fig 1: UV-Visible spectral of unripe coconut fluid

Fig 2: FTIR spectral of unripe coconut fluid

16

ABBREVIATIONS

EDTA Ethylene diamine tetra acetic acid

MIC Minimum inhibitory concentration

UC Untreated control

SC Standard control

NC Normal control

NST No sign of toxicity

ND No death

GV Gential violet

HB Haemoglobin

ALT Alanine transaminase

AST Aspartate aminoferase

ACP Acid phosphatase

SOD Superoxide dismutase

SDA Saboround dextrose agar

17

CHAPTER ONE

1.0 INTRODUCTION

1.1 NATURAL PRODUCTS AND MEDICINAL CHEMISTRY

A natural product is a chemical compound or a substance produced by a living organism

that is found in nature1.Within the field of organic chemistry, the definition of natural products is

usually restricted to mean purified organic compounds isolated from natural sources.2 Natural

products sometimes have pharmacological or biological activity that can be of therapeutic benefit

in treating diseases. As such, natural products are active components not only for most traditional

medicines but also for many modern medicines.3

A significant number of anti-infectives are based on natural products. The first antibiotic to

be discovered is Penicillin [1].

R

O

NH

NO

S

CH3

CH3

H

OOH

1

A large number of currently prescribed drugs have been directly derived from natural products. For

example the bark of the Willow tree has been known from antiquity to have pain relieving

properties4; this is due to the presence of the natural product salicin [2] which in turn may be

hydrolyzed to salicylic acid [3], a synthetic derivative acetylsalicylic acid [4] better known as

aspirin widely usedas a pain reliever was obtained when salicylic acid is treated with acetic

anhydride (scheme 1)

18

O O

OH

OH OH

OH

OH

O

OH

OOH OCH3

O

O

CH3

hydrolysis acetic anhydride

conc H2SO

4

2 3 4

Scheme 1

Unripe coconut fluid is one of the world’s most versatile natural products with increasing

scientific evidence that support its role in health and medicinal application. Unripe coconut fluid is

the liquid endosperm of immature coconut. The drink is extracted from immature coconut, which

implies it has not yet accumulated fat and has low sugar content5. The sugar and protein contents

increase as the coconut matures. It is recommended for weight loss phases because it increases the

rate of metabolism. Unripe coconut fluid is a hydrating drink, a natural isotonic drink, a universal

donor which can be used in blood transfusion because it has the same salt concentration as cells

and blood.6

1.2 STATEMENT OF THE PROBLEM

Despite the use of the various parts of coconut palm in herbal medicine, reports on

antimalarial activities, antioxidant capacity and phytochemical screening of unripe coconut water

are rather scanty. Hence it is imperative to examine its antimalarial activities, its antioxidant

capacity in blood serum and the phytochemicals in unripe coconut water.

1.3 OBJECTIVES OF THE STUDY

The specific objectives of this study are:

i) to determine the antimalarial activities of the unripe coconut water

ii) qualitative and quantitative phytochemical screening of unripe coconut water.

iii) to determine its antimicrobial activities

iv) to determine itsacute toxicity (LD50)

v) to determine hematological assay.

vi) to determine the antioxidant capacity of the unripe coconut water in the blood serum and

vii) to determine the effect of the unripe coconut water in the body organs (Liver and kidney).

19

1.4 JUSTIFICATION OF THE STUDY

The wide applications of unripe coconut water especially in medical fields can be justified

by its unique chemical composition of sugar, vitamins, minerals, amino acids, antioxidants,

phytochemicals and phytohormones4,7

. For example Vitamin E is an important antioxidant that

protects unsaturated oil from being destroyed in the body by oxygen. It is also a potent water-

soluble antioxidant in humans and most flavonoids have antioxidant properties8. This antioxidant

blocks some of the damage caused by free radicals and substances that damage DNA9. Some

alkaloids and terpenoids have antimalarial activities.10

20

CHAPTER TWO

LITERATUREREVIEW

2.1 PHYTOCHEMICAL ANALYSIS OF UNRIPE COCONUT FLUID

According to Kindersley11

, phytochemicals derived from coconut fluid can be used as therapeutic

agents. Dichter and Delanty12

discovered that they reduce the risk of cancer due to dietary fibers,

polyphenols, antioxidants and anti-inflammatory effects. Osman and co-workers 13

coined a term to

refer to foods rich in phytochemicals as therapeutic foods, which can provide health benefits

beyond those supplied by the traditional nutrients they contain.

2.1.1 ALKALOIDS

Long and co-workers14

reported Morphine [5], Cocaine[6], codeine [7] and Nicotine[8] as

alkaloids found inmany natural products,drugs and poisons.

O

NCH3

H

OH

OH

H

5 O

O

O

CH3

O

NCH3

6

O

NCH3

H

OH

O

H

CH3

7

N

N

CH3

8

According to Prudhomme15

, alkaloids are basic nitrogenous compounds. Hoehn and co-

workers16

in their research discovered that alkaloids can be found in coconut fluid and various

parts of plant.

21

2.1.1.1 CLASSIFICATION OF ALKALOIDS

According to Yang and Cordell 17

alkaloids are classified according to the chemical

structures in two broad divisions:-

i) Non-heterocyclic or a typical alkaloids or biological amines. Example Ephedrine [9]

ii) Heterocyclic alkaloids. Examples pyridine [10], pyrrole [11], quinolizidine [12], histidine

[13], tryptophan [14], proline [15], quinolone [16], indole [17], isoquinoline [18], purine

[19], aporphine [20] and piperidine [21].

NH

CH3

CH3

OH

9

N

10

NH

11

N

12

O

NH2

NH

N

R

13

O

NH2NH

R

14

O

NH

R

15

N

16

NH

17

N

18

N

N NH

N

19

N CH3

20

NH

21

22

2.1.1.2 PROPERTIES

Sańchez and co-workers18

discovered that alkaloids can form salts when reacted with acids. They

further explained in their work that the knowledge of the solubility of alkaloids and their salts is of

considerable pharmaceutical importance. Akinaga and co-workers19

provided a method for the

isolation of alkaloids from coconut fluid and their separation from the non-alkaloidal by exploring

differences in solubilities between alkaloids and their salts.

Oguntoye and Durowade20

reported the methods for identifying alkaloids in coconut fruits and

other natural products.

2.1.2 TANNINS

Preliminary phytochemical screening was carried out on the various extracts of ripe coconut fruit

byCook and Samman21

, and itshowed the presence of tannins, alkaloids, flavonoids and

carbohydrates. They observed that tannins appeared as light yellow or white amorphous powders

or shiny, nearly colourless, loose masses with a characteristic strange smell and astringent taste. In

medicine, the tannin-containing plant extracts are used as astringents, against diarrhea, as

diuretics,against stomach and duodenal tumors and as anti-inflammatory, antiseptic, and

haemostatic pharmaceuticals.21,19

Hemingway and Karchesy22

reported on thetannin derivatives extracted from the bark of mango

tree. These derivatives are used in the dyestuff industry as caustics forcationic dyes (tannin dyes),

and also in the production ofinks.

23

2.1.2.1 STRUCTURE OF TANNINS

According to Trease and Evan23

tannins are polyphenols with basic unit structure or monomer

ofgallic acid [22]and flavones [23] unit. The same authors23

further stated that tannin molecules

require at least 12 hydroxyl- and at least 5 phenyl groups to function as protein binders.

OH

OHOH

OHO

22

O

O

23

They also reported of the following derivatives of tannins found in Caesalpinia Spinosa plant:

catechin [24], meta-digalloyl [25], chebuloyl [26], elaeocarpusoyl [27], flavogallonyl [28],

sanguisorboyl [29] and dehyrochebuloyl [30]

O

OH

OH

OH

R

OH

OH

24

O O

RO

R

OH

OH

OH

OH

OH

25

OHO

R

OHOH

OH

O

26

24

OH

OH

R

O OH OH

OH

O

O

O

O

OH

OH OHOH OH

27

OH

OH

OH

O

R

O

R

OH

OH

28

OOH

OH OH OH OH OH OH

OH

O

OHO

O

R R

29

O

OH

O

OH

OH

R

O

O OH

OR

30

2.1.2.2 CLASSIFICATION OF TANNINS

According to Obidoa and co-workers24

tannins are classified based on specific

structuralcharacteristics and chemical properties. Based on the researches carried out on coconut

fruit the researchers reported many types of tanninsfractionated hydrolytically into their

components. As an example treatment with hot water led to the classification of such tannins as

‘hydrolysable tannins while non-hydrolysable, oligomeric and polymeric pro-anthocyanidins were

classified as condensed tannins.

Haslam25

divided tannins into four major groups based on their structural characteristics.

Namely:

25

i) Gallotannins:these are all those tannins in which galloyl units or their meta-depsidic

derivatives are bound to diverse polyol-,catechin-, or triterpenoid units with [31] as an

example.

ii) Ellagitannins: Ellagitannins form by far the largest group of known tannins.They are

formed from the gallotannins by the oxidative coupling of, at least, two galloyl units with

in [32]as an example.

iii) Compex tannins: The structures of the complex tannins are built up from a gallotannin

unit or an ellagitannin unit, and a catechin unit with[33]as an example.

iv) Condensed tannins: Condensed tannins are oligomeric and polymeric proanthocyanidins

consisting of coupled flavan-3-ol (catechin) units with[34] as an example.

OO

O

O

O

OHOHOH

R

OR

OR

R

31

OH

OH

OH

O

OH

OH

OH

O

O O

O

OR

OR

O R

32

O

O

O

O

O

OR

R

OOH

OH

OH

OH OH

OR

33

O

OH

OH

O

OH

OH

OH

OH

OH

H

catechin moiety

OH

catechin moiety

(OH)

34

Porter26

in the evaluation of total phenolic contents of coconut fluid reported tannins as

polyphenolic secondary metabolites of higher plants.

26

2.1.3 TERPENOIDS

According to Antherden27

,terpenoids are large and diverse class of naturally occurring organic

chemicals similar to terpenes, derived from five-carbon isoprene units assembled and modified in

thousands of ways. Osagie28

observed that terpenoids can be found in coconut fluid and all classes

of living things, and are the largest group of natural products.Akinleyeand Fabunmi29

reported that

about60% of known natural products are terpenoids after quantitative screening of coconut fluid

and other natural systems.

2.1.3.1 STRUCTURE OF TERPENOIDS

According to Sofowora30 terpenesderived biosynthetically from units of isoprene [35], has a

molecular formula C5H8. The basic molecular formula of terpenes are multiples of that that

isoprene units (C5H8)n where n is the number of linked isoprene units.

CH2

CH2

CH3

35

In another development, Brian and co-workers31

stated that isoprene units may be linked ‘’head to

tail’’ to form linear chains or they may be arranged to form rings. According to Mitscher and co-

workers32

isoprene itself does not undergo the building process, but rather activated forms,

isopentyl pyrophosphate [36] and dimethylallyl pyrophosphate [37]are the components in the

biosynthetic pathway.

27

O

P

O

P

O-

O

O-

O

O-

CH3

CH3

36

O

P

O

P

O-

O

O-

O

O-

CH3

CH3

37

Hemiterpenes: Narayana and co-workers33

reported Prenolor 3-methyl-2-buten-1-ol[38]as one of

the simplest hemiterpene.

CH3 OH

CH3

38

Okwu34

discovered that prenol [38] occurs naturally in coconut water, cranberry, bilberry,

currants, grapes, raspberry, blackberry, tomato, white bread, hop oil, coffee, arctic bramble,

cloudberry and passion fruit. The same author34

further stated that prenol is a building block of

isoprenoid alcohols which have the general formula: (C5H8)nH2O

The simplest isoprenoid alcohol is geraniol[39] (n = 2): higher oligomers include

farnesol[40] (n = 3) and geranylgeraniol[41] (n = 4).

CH3 OH

CH3 CH3

39

CH3

CH3 CH3

OH

CH3

40

CH3

CH3 CH3 CH3

OH

CH3

41

28

Stray35

synthesized [38] by reacting formaldehyde[43] with isobutene[42], followed by the

isomerization of the resulting isoprenol[44] (3-methyl-3-buten-1-ol).

CH3 CH2

CH3

H

O

H CH2

CH3

OH

+

42 43 44CH3

CH3

OH

38

Isomerization

Monoterpenes:According to Pascaline,et al36

, monoterpenes are a class of terpenes that consist of

two isoprene units and have the molecular formula C10H16as seen in myrcene [45].

CH2

CH2

CH3CH3

45

According to Kocheet al37

geranyl pyrophosphate can also undergo two sequential cyclization

reactions to form bicyclic monoterpenes, such as pinene[48] which is the primary constituent of

pine resin.

CH3

CH348

29

Pascaline, et al38

reported the two structural isomers of pinene: α-pinene and β-pinene. These two

isomers of pinene constitute the major components of turpentine.

Sesquiterpenes: According to Singh, et al39

sesquiterpenes are a class of terpenes that consist of

three isoprene units and have the generalempirical formula C15H24. Kartnig40

reported

cadinenes[49] a sesquiterpenes with two fused six-membered rings which can be modified

biochemically to produce related sesquiterpenoids.

CH3CH3

CH3

CH3

49

Ghasemzadehand co-workers41

reported that there are more cyclic sesquiterpenes than cyclic

monoterpenes because of the increased chain length and addditional double bonds.The same

authors41

also reported Zingiberene [50] a monocyclicsesquiterpene which is the predominant

constituent of the oil of ginger (Zingiber officinale), from which it gets its name. They further

stated that 50 gives ginger its distinct flavour.

CH3

CH3

CH3

CH3

50

30

Edward42

reported another sesquiterpene Humulene[51] derived from farnesyl

diphosphate(FPP).

CH3

CH3

CH3

51

Diterpene: Edward42

further reported of derived diterpene farnesyl from geranyl pyrophosphate.

Martin and co-workers43

gave the following notable examples of ditepernes found in some natural

products: Abietic acid [52], cafestol [53] a diterpene present in coffee, cambrene A [54], ferruginol

[55], forskolin [56], kahweol [57], labdane [58], lagochilin [59], sclarene [60], stemarene [61],

steviol [62], texadiene [63], cassaic acid [64], abietadiene [65] and trisporic acid [66].

CH3

CH3

OOH

H

H

CH3

CH3

52

O

OH

CH3

OH

H

H

H

53

CH3

CH3

CH2

CH3

CH3

54

CH3 CH3

CH3

CH3OH

CH3

CH3

55

CH3 CH3

O

CH3 O

O

OH

CH2

CH3

H

OH

56

O

OH

OHCH3

CH3

57

31

CH3

CH3

CH3

CH3CH3

CH3CH3

58

CH3 CH3

CH2

CH2

CH2

60

CH3

CH3

H

CH3CH3

61

CH2

CH3

CH3

CH3

CH3

O

OH

62

CH3

CH3

CH3

CH3

H

63CH3 CH3

CH3

O

COOH

CH3H

OH

64

CH3

CH3

CH3 CH3

H

CH3

65

CH3

CH2

CH3

CH2

CH3CH3

HOOC

66

Sesterterpenes:Pichersky44

reported a raresesterterpenoidgeranylfarnesol[67].

CH3 CH3CH3

CH3 OH

3

67

Triterpenoid:Günata and coworker45

assembledtriterpenes from a five-carbon isoprene unit

through the cytosolic mevalonate pathway to make a thirty-carbon compound.Sandhya and

Rajamohan46

gave some notable examples of triterpenoids found in coconut oil and most natural

32

products. They include:Betullinic acid [68], sterol [69], cholesterol [70], campesterol [71],

stigmasterol [72], stigmastanol [73]

OH

CH3CH3

H

H

H

CH2

CH3

O

OH

CH3 CH3

H

68

OH

69

OH

CH3

CH3CH3

H

HH

H

CH3

CH3

70

OH

CH3

CH3CH3

H

HH

H

CH3

CH3 CH3

71

OH

H

HH

H

CH3

CH3

CH3

CH3CH3

CH3

72OH

H

HH

H

CH3

CH3

CH3

CH3

CH3

CH3

73

Tetraterpenes: Thimmappa and coworkers47

identified carotenoids as the major class of

tetraterpenes. Classification of carotenoids was earlier reported by Zelena and coworkers50

. They

classified carotenoids as oxygenated and unoxygenated carotenoids. With molecules containing

oxygen, such as lutein [74], zeaxanthin [75], and crptoxanthin [76] are known as xanthophylls.

33

CH3CH3

CH3CH3

CH3CH3

CH3 CH3

OH

OH

CH3

74

CH3CH3

CH3

CH3

CH3CH3

CH3 CH3

OH

OH

CH3

CH3

75

CH3CH3

CH3CH3

OH

CH3

CH3

CH3

CH3

CH3

CH3

76

The unoxygenated (oxygen free) carotenoids such as α-carotene, β-carotene, and lycopene, are

known as carotenes [77].

CH3CH3

CH3

CH3

CH3

CH3

CH3CH3

CH3CH3

H

77

Tripoli and coworkers49

discovered that people consuming diets rich in carotenoids from natural

foods, such as fruits and vegetables, are healthier and have lower mortality from a number of

chronic illnesses.Dewick50

stated that humans and animals are mostly incapable of synthesizing

34

carotenoids and must obtain them through their diet.Boutanaevand co-workers51

proposed that

carotenoids are used in ornamental traits.

Polyterpenes:According to Okwu34

polyterpenes consist of long chains of many isoprene

units[78].Natural rubber consists of polyisoprene[79] in which the double bonds are cis34,52

.

CH3CH3

RR

n

CH2 CH2

CH3

n 78 79

polymerization

Babu, etal53

reported gytta-percha [80] a polyisoprene with trans double bond.

CH3

CH3 CH3

CH3

CH3 CH3

n80

2.1.4 FLAVANOIDS

According to Miller54

Flavonoids are water soluble polyphenolic molecules containing 15 carbon

atoms.Saslowsky and Winkel-Shirley55

stated that flavonoids belong to the polyphenol family.

Fernandez and coworkes56

discovered that flavonoids occur in coconut fruit and vegetablesin the

form of glycosides and sometimes as acylglycosides. Rijke and Niessen57

visualized flavonoids as

two benzene rings which are joined together with a short three carbon chain.

Chalcone:Jiang58

and co-workersreportedBenzylideneacetophenone[81] as the parent member

of the chalcone series.

35

O

81

Flavones: David59

reported 2-phenylchromen-4-one (2-phenyl-1-benzopyran-4-one) [82] as

the backbone of this class of flavonoids.

O

O

82

Si and co-workers60

reported natural some flavones apigenin (4,5,7- trihydroxyflavone) [83],

liteolin, (3,4,5,7 tetrahydroxyflavone) [84] , tangeritin (4,5,6,7,8- pentamethoxyflavone) [85],

chrysin (5,7-dihydroxyflavone) [86], baicalein(5,6,7-trihydroxyflavone) [87],

scutellarein(5,6,7,4'-tetrahydroxyflavone) [88], wogonin (5,7-dihydroxy-8-methoxyflavone)

[89] and 6-hydroxyflavone [90] found in most fruits and vegetables.

O

OOH

OH

OH

83

O

OOH

OH

OHOH

84

36

O

OO

O

O

O

O

CH3

CH3

CH3

CH3 CH3

85

O

OOH

OH

86

O

O

OH

OH

OH

87

O

O

OH

OH

OH

OH

88

O

O

OH

OH

O

CH3

89

O

O

OH

90

Larget and co-workers61

reported of the Synthesis of some synthetic flavonediosmin[91],

flavoxate[92], and 7,8-dihydroxyflavone[93].

O

OHOH

CH3

OH

O

O O

OH OH

OH

O O

OOH

OH

O

CH3

91

37

N

O

OOH

O O

92

O

O

OH

OH

93

Flavanols: Tohgeand coworkers62

reported 2-phenyl-3,4-dihydro-2H-chromen-3-ol as the parent

skeleton of flavanols. Ruidavets63

reported the formation ofcatechin[94], epicatechin gallate[95],

gallocatechin [96], epigallocatechin[97] and theaflavins[98] by condensation of flavan-3-ol.

OOH

OH

OH

OH

94

OOH

OH

OH

OH

O

OOH

OH

OH95

OOH

OH

OH

OH

OH

96

OOH

OH

OH

OH

O

O

OH

OH

OH

OH

97

38

OOH

OH

OH

OH

O

OH

OOH

OH OH

98

Flavanones:Nibbs and Scheidt64

reported numerous methods for the enantioselective chemical and

biochemical synthesis of the following flavanones, namelybutin [99], eriodictyol [100], hesperetin

[101], homoeriodictyol [102], isosakuranetin[103], naringenin [104], pinocembrin [105] and

sterubin[106]

OOH

O

OH

OH

99

OOH

O

OH

OH

OH

100

OOH

O

O

OH

OH

CH3

101

OOH

OOH

O

OH

CH3

102

39

OOH

OOH

O CH3

103

OOH

OOH

OH

104

OOH

OOH

105

O

OOH

OH

OH

OCH3

106

Anthocyanins: Winkel-Shirley65

synthesizedanthocyanins via the phenylpropanoid pathway.

Mazza and Francis66

reported the occurrence of anthocyanins in nearly all tissues of higher plants,

including leaves, stems, roots, flowers and fruits of coconut.

According to Wroslstad and co-workers67

anthocyanins[107] are generally degraded at higher pH

and can also be used as pH indicators because of their colour changes with pH. The same authers67

classified anthocyanins according to their substitutions on anthocyanins.

OR7

R6

R5

R3

R3

R4

R5

107

40

Isoflavonoids: Accordinng to Bitisand co-workers68

many isoflavonoid compounds have

biological effects via the estrogen receptor. The same authers68

reported 3-phenylchromen-4-one

108]as the main representative of this class of flavonoids.

O

O

108

Recently, Salucciand co-workers69

identified some natural isoflavonoids as toxins, including

biliatresone which were reported to have the capacity to causebiliary atresia when infants are

exposed to the plant product. Williams and co-workers70

reported Isoflavones[108], Isoflavonones

[109]Isoflavans [110] and Pterocarpans[111] as structural groups similar to isoflavonoids.

OCH3

O OH

109

O

110

OH

O

O

CH3 CH3

H

H

111

Skerget and co-workers71

reportedIsoflavonoidsapigenin and luteolinfrom the flavonoid

biosynthesis pathway via liquiritigenin[112] or naringenin[113]

41

OH

O

OH

112

OH

O

OH

OH

113

2.1.5 SAPONINS

According to Francis and co-workers72

Saponins[114] are phytochemicals which can be

found in coconut fluid, most vegetables, beans and herbs among others.

CH3

CH3

CH3 CH3

CH3

CH3

OSugar

OH

CH3

OSugar

114

Sun and co-workers73

reported peas, soybeans, and some herbs with names indicating

foaming properties such as soapwort, saoproot, soapbark and soapberry as the best known sources

of saponins.

Skene and co-workers74

described saponinsare glycosides with foaming characteristics. The

foaming ability of saponins is caused by the combination of a hydrophobic (fat-soluble) sapogenin

and a hydrophilic (water-soluble) sugar part75

. Saponins have a bitter taste76

. Some saponins are

toxic and are known as sapotoxin76

.

42

Jonathan and co-workers77

reported of the glycoside-free portions of the saponins

sapogenins. Some steroidal sapogenins can serve as a practical starting point for the semi

synthesis of particular steroid hormones78

.

Li and co-workers79

reported Yamogenin [115], a class of sapogenins, found in the

herbTrigonella foenum-graecumand other plants.

O

O

CH3

H

H H

H

OH

HCH3

CH3

CH3

115

2.2 PHYTOHORMONES PRESENT IN COCONUT FLUID

According to Kemdeand Zeevaart80

, phytohormones are a group of naturally occurring

organic compounds that play crucial roles in regulating plant growth in a wide range of

developmental processes.

2.2.1 AUXIN

Coconut fluid contains indole-3-acetic acid (IAA) [116]as reported by Tucker and

Roberts81. IAA is a weak acid (pKa = 4.75) that is synthesized in the meristematic regions

located at the shoot apex and subsequently transported to the root tip in plants.

NH

COOH

116

43

2.2.2 CYTOKININS

According Haberer and Kieber82 cytokinins found in coconut water support cell division,

and thus promote rapid growth. Letham83

reported various cytokinins contained in coconut

water.

The same auther83

reported two types of cytokinins:

i. Adenine-type cytokinins represented by kinetin[117]84

,6-benzylaminopurine[118]85

and

zeatin [119]86

and

ii. Phenylurea-type cytokinins like diphenylurea[120]84

and thidiazuron[121].85,

N

NNH

N

NH

O

117

N

NNH

N

NH

118

N

NNH

N

NHCH3

OH

119

NH NH

O

119

NH NH

O

SN

N120

The first cytokinin, kinetin was discovered by Miller et al.86.

44

2.2.3 GIBBERELLINS (GAs)

According to Chenand co-workers87gibberellins [112] are plant hormones that regulate

growth and influence various developmental processes. Gibberellinsare numbered

neither by their structural information nor by their functions, but rather in the order of their

identification. Yong and co-workers88 successfully detected and quantified some GA1 and

GA3 in coconut fluid.

OH

CH3

H

CH2

O

OC H

CO2H

122

2.3NUTRITIONAL BENEFITS OF COCONUT FLUID

According to Heo89and co-workers90

Coconut fluid contains vitamins B1, B2, B3, B5, B6, B7 and

B9, amino acids, carbohydrates, antioxidants, enzymes, health enhancing growth hormones, and

other important nutrients. Due its electrolyte (ionic mineral) content it is said to be similar to

human plasma and it has gained international acclaim as a natural sports drink for oral

rehydration91,92

. As such, it has proven superior to commercial sports drinks and unlike other

beverages, it is completely compatible with the human body, in so much so that it can be infused

directly into the bloodstream93,94

. In fact, doctors have used coconut fluid (unripe and mature)

successfully as an intravenous fluid for over sixty years95

. Published medical research works and

clinical observation have shown that coconut fluid (unripe and mature) can be used as follows:

45

i. makes an excellent oral rehydration sports beverage - replaces electrolytes from exercise,

heat stress and illness

ii. aid in exercise performance

iii. natural isotonic beverage – contains the same level of electrolytes found in human blood

iv. has 15 times the amount of potassium as most sports and energy drinks (264 mg vs 12.5 mg

/100 mL)

v. reduce problems for infants suffering from intestinal disturbances

vi. cardioprotective: helps regular blood pressure (due to high potassium); improves

circulation

vii. reduces swelling in hands and feet

viii. prevents abnormal blood clotting

ix. aid in kidney function including those with kidney stones; Nutritional support for those

with urinary tract/bladder problems

x. help balance blood sugar in diabetics

xi. improve digestion

xii. reported by some people to reverse cataracts

xiii. contain nutrients that feed friendly gut bacteria

xiv. help relieve constipation or diarrhea

xv. possesses anti-aging properties

xvi. nutritional support for healthy skin: restores strength and elasticity to skin; reduces age

spots; reduces wrinkles and sagging

xvii. regulate the functioning of the intestine which promotes smoother, more hydrated skin

46

xviii. enhances healing of wounds and lesions

xix. supports good vision and provides nutritional support in those who have a tendency

towards glaucoma

xx. contain potent antioxidants

2.4 NATURAL PRODUCTS WITH ANTIMALARIAL ACTIVITIES

Quinine, an alkaloid from the bark of Cinchona (Rubiaceae) plant was reported by Farnsworth96

.

Gen and Lin97

discovered the plant Artemisia annua Lwhich contains a sesquiterpene-lactone with

a potent antimalarial activity. Other medicinal plants showing antimalarial activities include:

Acanthospermum australe98

, Esenbeckia febrifuga99

,Lisianthus specious100

and Tachia

guianensis101

2.5 STRUCTURES OF SOME ESTABLISHED ANTIMALARIAL DRUGS

A new class of antimalarial drugs that are effective across various stages of the malaria

parasite’s life cycle has been developed by Li and co-workers102

. These antimalarial drugs include:

Artemisinin [123], primaquine [124], lapachol [125], atovaquone [126], proguanll [127],

artesunate [128], artemether [129] and lumefantrin [130].

O

O

O

CH3

H

CH3

H

CH3

H

O O

123

N

NHNH2

CH3

OCH3

124

47

O

O

OH

OCH3

CH3

125

O

O

OH

Cl 126

Cl

NH NH

NH

NH NHCH3

CH3

127

O

OOO

CH3

CH3

H

H H

CH3

OOH

O

O

128

O

OOO

CH3

CH3

H

H H

CH3

O

CH3

129

OH

N

CH3

CH3

ClCl

Cl

129

`

48

CHAPTER THREE

EXPERIMENTAL

3.0 MATERIALS AND METHOD

Samples of unripe coconut were collected from the University of Nigeria, Nsukka campus and

authenticated at the Department of Plant science and Biotechnology by a taxonomist. The

mesocarp was carefully removed to get the endocarp which habours the clear liquid of unripe

coconut fluid. This clear liquid was extracted from the endocarp with the aid of a syringe into a

clean container.

3.1. QUALITATIVE AND QUANTITATIVE PHYTOCHEMICAL SCREENING OF

UNRIPE COCONUT FLUID

3.1.1. QUALITATIVE PHYTOCHEMICAL SCREENING TESTS

3.1.1.1 TEST FOR TERPENOIDS

Extract(5 mL) was mixed with chloroform(2 mL) followed by careful addition of conc. H2SO4(3

mL). A layer of reddish brown colouration was formed at the interface indicating a positive test

results for the presence of terpenoids103

3.1.1.2 TEST FOR FLAVONOIDS

Extract(5 mL) was heated with ethyl acetate(10 mL) in a test tube over a stream bath for 3 min.

The mixture was filtered and filtrate(4 mL) was shakened with NH4OH (1 mL). Yellow

colouration was formed indicating presence of flavonoid104

49

NaOH TEST FOR FLAVONOIDS

Extract (5 mL) was diluted with distilled water (10 mL). To this NaOH (2 mL, 10 %) was later

added to produce a yellow colouration. A change in the colour from yellow to colourless on

addition of dil HCl acid was an indication of flavonoids.104

3.1.1.3 TEST FOR ALKALOIDS

a) Extract (5 mL) was stirred with HCl (5 mL, 1 %) on a steam bath. Few drops of picric acid

solution were added to the extract (5 mL). The formation of a reddish brown precipitate was taken

as a preliminary evidence for the presence of alkaloids. 20

b) Extract (1 mL) was taken individually into two test tubes. To the first portion, few drops of

Dragendorff’s reagent were added; occurrence of orange-red precipitate was taken as positive test

for alkaloids. 20

c) To the second portion, Mayer’s reagent was added; an appearance of buff-coloured precipitate

was an indication for the presence of alkaloids.20

3.1.1.4 TEST FOR TANNINS

Extract(5 mL) was boiled in distilled water(20 mL) in a test tube and filtered. FeCl3(0.1%) was

added to the filtrate. The appearance of brownish green colouration indicated the presence of

tannins105

.

50

3.1.1.4 TEST FOR SAPONINS

Extract(5 mL) was boiled in distilled water(20 mL) in a test tube in a boiling water bath and

filtered. Filtrate(10 mL) was mixed with distilled water(5 mL) and vigorously shaken to form a

stable persistent froth. The frothing was mixed with 3 drops of olive oil and shakened vigorously.

The formation of emulsion indicates the presence of saponins106

.

3.1.2QUANTITATIVE PHYTOCHEMICAL SCREENING TEST

3.1.2.1 TERPENOIDS

Extract (5 mL) was macerated with ethanol (20 mL) and filtered. Filtrate (1 mL) was

pipetted and gradually added and the solution was allowed to stand for 30 min before adding

ethanol (2 mL). The absorbance of the solution was measured at the wavelength of 700 nm in the

spectrophotometer as.103

3.1.2.2 FLAVONOIDS

Extract (5 mL) was macerated with ethylacetate (20 mL). The solution was filtered

using whatman filter paper. Filtrate (5 mL) was pipetted and dilute NH3(aq)(5 mL) was added. The

upper layer was collected and the absorbance was read at the wavelength of 490 nm in the

spectrophotometer.104

3.1.2.3 ALKALOIDS

Extract (5 mL) was macerated with ethanol (20 mL), H2SO4(20 %, 1:1) and filtered.

Filtrate (1 mL) was pipetted and H2SO4(60 %, 5 mL) was added. After 5 min formaldehyde (0.5

51

%) in H2SO4(60 %) was added. The solution was mixed and allowed to stand for 3 h. The

absorbance was measured at the wavelength of 565 nm in the spectrophotometer.20

3.1.2.4 TANNINS

Extract (5 mL) was macerated with distilled water (20 mL) and filtered. Filtrate (5

mL) was pipetted and FeCl3 (0.3 mL, 0.1 M) in HCl (0.1 M) was added. Potassium ferrocyanide

(0.3 mL, 0.008 M) was added. The absorbance was read at the wavelength of 720 nm in the

spectrophotometer.105

3.1.2.5 SAPONINS

Extract (5 mL) was macerated with petroleum ether (20 mL). The solution was decanted

into a beaker and washed again with petroleum ether (10 mL). The filtrate was combined and

evaporated to dryness. Residue was dissolved in ethanol (6 mL). Solution (2 mL) was transferred

into a test tube and chromogen solution (2 mL) was added. The solution was allowed to stand for

30 min before measuring the absorbance at the wavelength of 55 nm in the spectrophotometer .106

3.2ANTIMICROBIAL SCREENING TEST

The liquid extract was screened for its antimicrobial activities. Following the method of

Bauer et al.107

using Ciprofloxacin, an antibacterial drug and Ketoconazole which is an antifungal

drug as reference drugs. The extract was screened against six micro-organisms namely: Bacillus

subtilis, Staphylococcus aureus, Pseudomonas aeruginosa,Escherichia coli, Candida albicans and

52

Aspergillus niger.This was carried out for sensitivity test and minimum inhibitory concentration

(MIC).

Required Materials:

Empty sterile petri dishes, sterile test tubes,test organisms suspension (5M Farland standard), cork

borer (8 mm diameter), weighing balance, molten nutritent agar/sterile Saboround Dextrose

Agar(SDA) media (prepared by suspending 65g of the medium in one litre of purified water. The

solution is boiled for one minute to dissolve the medium completely and autoclaved at 121oC for

15 mins), graduated pipette of 1 and 2 capacities and sterile droppers.

3.2.1 MINIMUM INHIBITORY CONCENTRATION (MIC)

Agar cup diffusion method was applied to determine the minimum inhibitory concentration (MIC)

of the extract and Ciprofloxacin and ketoconazole as standard drugs for bacteria and fungi

respectively. Fourdrops of the of each of the dilute solution were introduced into the corresponding

cup previously marked out in the agar seeded with microorganisms and the agar (MHA) plate. The

cork borer used to make the cup was 8 mm in diameter. The plates were incubated at 37 oC for 24

h for bacteria and 48 h for fungi tests.The diameter of zones of inhibition were measured and the

value subtracted from the diameter of the borer (8mm) to give the inhibition zone diameter (IZD).

The procedure was repeated for ciprofloxacin and ketoconazole reference drugs.

This is the minimum concentration of the liquid extract that inhibits the visible growth of

bacteria and fungi after incubation periods108

. Liquid extract (30 mL) was further diluted to 15, 7.5,

3.75, 1.875 and 0.937 per mL of distilled water. This serial dilutions were used following the

procedure outlined by Chemical Laboratory Standards Institute (CLSI)109

.

53

3.3. ACUTE TOXICITY SCREENING TEST

Wistar albino mice of either sex weighing 20–34 g were housed in separate cages, acclimatized for

one week and then divided into five groups of five mice each. The route of administration was via

oral route with the aid of an incubation tube.110

3.4INNOCULATION OF THE PARASITAEMIA

Parasitaemia was maintained in the laboratory by the method of David111

.Ten drops of the

parasitized bloodobtained with the aid of a capillary tube through the ocular region of the mice,

was diluted with normal saline (1 mL). Thereafter diluted parasitized blood (0.2 mL) was used to

infect the three mice that served as the host from where other experimental animals were infected.

Group I(positive control): was inoculated with malaria parasite (Mp+) and treated with 5 mL/kg

body weight of normal saline

Group II(normal control): was not inoculated with malaria parasite (Mp+) and treated with 5

mL/kg body weight of normal saline

Group III(standard control):was inoculated with malaria parasite (Mp+) and treated with 5

mg/kg body weight of Artesunate (standard drug)

Group IV: was inoculated with malaria parasite (Mp+) and treated with 200 mL/kg body weight

of theunripe coconut fluid

Group V: was inoculated with malaria parasite (Mp+) and treated with 300 mL/kg body weight of

theunripe coconut fluid.

54

3.4.1 DETERMINATION OF MALARIA PARASITE (MP+)

The determination of the malaria parasite (Mp+) was carried out according to the method of Dacie

and Lewis112

. A pair of scissors was used to cut the tail which was squeezed gently to obtain a

small drop of blood that was placed on the centre of a microscope slide. Immediately the thin film

was spread using a smooth edged slide spreader. The slide was labeled with marker. This method

was repeated for different infected mice. The slide with the blood was stained using Leishman’s

stain (in order to differentiate the parasites from the red blood cells when viewed under

microscope) , sprinkled with water after 2 min of the stain and air–dried in horizontal position.

3.5DETERMINATION OF ANTIOXIDANT ACTIVITIES

The effect of unripe coconut fluid on Superoxide Dismutase, Vitamin C and Vitamine E

in blood serum were also determined.

3.5.1 DETERMINATION OF SUPEROXIDE DISMUTASE (SOD)

Reagents: a) Phosphate buffer (0.05M) pH 7.8

This was prepared by dissolving K2HPO4(6.97 g) and KH2PO4(1.36 g) in distilled water and

making up to 1000 mL with distilled water. The pH was adjusted to 7.8 using pH meter.

b) Adrenaline solution(0.059%)

This was prepared by dissolving adrenaline (0.01 g) in distilled water (17 mL)

c) The blank was prepared with adrenaline (0.3 mL) in buffer (25 mL)

55

Procedure for the Determination of SOD

The post mitochondrial fractions were properly diluted. Each of the diluted sample (2 mL) was

added to phosphate buffer (2.5 mL, 0.05 M) pH 7.8. The mixture was equilibrated in the

spectrophotometry before adding adrenaline solution. The reaction started with the addition of

freshly prepared adrenaline solution (0.3 N) to the mixture followed by quick mixing by inversion

in the cuvette. The reference cuvette therefore contains buffer (2.5 mL), adrenaline (0.3 mL) and

extract (0.2 mL). Absorbance measurements were taken at 450 nm for 150 sec at 30 sec interval.

Increase in absorbance perimeter =∆a−∆o

�.�

∆a = Absorbance after 150 sec

∆o = Absorbance after 30 sec

2.5 = Slope of the plot of absorbance against concentration.

3.5.2 EFFECT OF THE FLUID ON VITAMIN C

Reagents: Indaphenol reagent, prepared by adding Indaphenol(10 mL) and making up to 90 mL

with distilled water, Oxalic acid(0.4%) and distilled water.

Procedure

Extract (5 mL) was macerated with oxalic acid(20 mL, 0.4%) and filtered. Indaphenol(9 mL) was

added to the filtrate and the absorbance was read at the wavelength of 520nm in the

spectrophotometer as reported in literature 113.

56

3.5.3 EFFECT OF THE FLUID ON VITAMIN E

Reagents: Ethanol, ferric Chloride(0.2 %) in ethanol and distilled water

Procedure

Extract(0.1 mL) was added to ethanol(2 mL). Solution (1 mL) was pipetted into an empty test tube

and ferric Chloride(1 mL, 0.2%) in ethanol was added. The solution was diluted to 5 mL with

distilled water and the absorbance was measured at the wavelength of 520 nm in the

spectrophotometer as reported in literature 114

.

3.6 THE HAEMATOLOGICAL TEST

Blood was collected from the animal into an EDTA tube to prevent coagulation of the

blood sample. The collected blood samples were put in a capillary tube which were centrifuged for

5 min at the speed of 4000 rpm (revolution per minute) to separate the blood from the serum as

reported in literature.115

3.6.1 DETERMINATION OF TOTAL WHITE BLOOD CELL COUNT

Reagents: Turks solution, Acetic acid, Gential Violet (G.V) and distilled water. Turks solution

was prepared by mixing distilled water (49 mL) with acetic acid (1 mL) and with a drop of G.V.

Turks solution destroys red blood cells in a blood sample and stains the nuclei of the white blood

cells, making it easier to see and count.

Procedure

Turks solution (380 µL) was pipetted into an empty EDTA tube and the blood sample (20µL) was

also pipetted into the same tube and shaken. The count was done with the aid of a microscope.

57

3.6.2 DETERMINATION OF TOTAL RED BLOOD CELL COUNT

Reagents: Normal saline, EDTA tube and distilled water. Normal saline was prepared by

dissolving NaCl(9g) in distilled water and making up to 1000cm3 with distilled water.

Procedure:

Normal saline(2 mL) was pipetted into an empty EDTA tube and blood sample(4 mL) was added

into the tube and shaken. The count was done with the aid of a microscope.

3.6.3 DETERMINATION OF HAEMOGLOBIN (HB) CONCENTRATION

Reagents: EDTA tube, Drabkin’s solution and distilled water.

Procedure:

Drabkin’s solution (4 mL) was added into an empty EDTA tube after which blood sample (20 mL)

was also added to the same tube and shaken. The mixture was allowed to stay for 10 min before

taking the absorbance at the wavelength of 540 nm in the spectrophotometer as reported in

literature.116

3.7 LIVER FUNCTION TEST ACTIVITY

Determination of the effects of unripe coconut water on AST, ALT and total protein

activities:

58

3.7.1 DETERMINATION OF THE ASPARTATE AMINOTRANSFERASE (AST)

ACTIVITY

Reagents: R1=[(Phosphate buffer(100mmol/L),L-aspartate(100 mmol/L) and α-oxoglutarate(2.0

mmol/L)] pH 7.4, R2 = 2,4-dinitrophenylhydrazine(2 mmol/L), NaOH(0.4 M) and distilled water.

Procedure:

Blood serum sample(100 µL) was pipetted into a sample tube and distilled water (100 µL) was

also pipetted into a blank tube. R1(500 µL) was added to the sample tube and the bank tube, the

solutions were allowed to stand for 30 min before adding R2 to both the sample tube and the bank

tube. The solutions were also allowed to stand for another 20 min before adding NaOH(0.4 M).The

absorbance was read at the wavelength of 546nm in the spectrophotometer as reported in

literatures.117.118

3.7.2 DETERMINATION OF THE ALANINE TRANSAMINASE (ALT) ACTIVITY

Reagents: R1=[(Phosphate buffer(100mmol/L),L-alanine(200 mmol/L) and α-oxoglutarate(2.0

mmol/L)] pH 7.4, R2 = 2,4-dinitrophenyl hydrazine(2 mmol/L), NaOH (0.4 M) and distilled

water.

Procedure:

Blood serum sample(100 µL) was pipetted into a sample tube and distilled water(100 µL) was also

pipetted into a blank tube. R1(500 µL) was added to the sample tube and the bank tube, the

solutions were allowed to stand for 30 min before adding R2 to the sample tube and the bank tube.

The solutions were also allowed to stand for another 20 min before adding NaOH(0.4 M).The

59

absorbance was read at the wavelength of 546nm in the spectrophotometer as reported in

literatures.132,134

3.7.3 DETERMINATION OF TOTAL BILIRUBIN

Reagents: R1 = Sulphanilic acid(29 mmol/L) and Hydrochloric acid(0.17N), R2 = Sodium

Nitrite(38.5 mmol/L), R3 = Caffeine(0.26 mol/L) and Sodium benzoate(0.52 mol/L), R4 =

Tartrate(0.93 mol/L) and Sodium hydroxide(1.9 M).

Procedure:

R1(200 µL) was pipetted into the sample blank and sample test tube, R2(50 µL) was added to the

test tube only and R3(1000 µL) was added to both the sample blank and the test tube. The solution

was thoroughly mixed, incubated for 10 min at 25oC. R4(1000 µL) was added to both the sample

blank and the sample test tube. The solution was thoroughly mixed, incubated for 5 min at 25oC

after which the absorbance was measured at the wavelength of 560nm in the spectrophotometer as

reported in literature.117

3.8 THE KIDNEY FUNCTION TEST ACTIVITY

To determine the effects of unripe coconut water on Acid Phosphatase(ACP), Urea,

Creatinine and Uric acid:

60

3.8.1 DETERMINATION OF THE UREA

Reagents: R1 = EDTA(116mmol/L), Sodium nitroprusside (6 mmol/L) and Urease(1 g/L), R2 =

Diluted phenol(120 mmol/L), R3 = Diluted Sodium hypochlorite(27 mmol/L) and Sodium

hydroxide(0.14 M)

Procedure:

Blood serum sample(10 µL) was pipetted into a test tube, standard reagent (10 µL) was pipetted

into a standard tube and distilled water (10 µL) was also pipetted into the blank tube. R1(100 µL)

was added to all the tubes, R2(2.5 mL) was added to all the test tube and R3 was also added to all

the test tubes. The mixtures were allowed to stay for 15 min before taking the absorbance at the

wavelength of 546nm in the spectrophotometer as reported in literatures.110,117

3.8.2 DETERMINATION OF URIC ACID

Reagents: Distilled water, standard reagent and R1 = Hepes buffer or 3,5-Dichloro-2-

hydroxybenzenesulfonic acid(50 mmol/L) pH= 7.0

Procedure:

Blood serum sample(20 µL) was pipetted into a test tube, standard reagent(20 µL) was pipetted

into a standard tube and distilled water (20 µL) was also pipetted into the blank tube. R1(1000 µL)

was added to all the tubes. The absorbance was taken at the wavelength of 546nm in the

spectrophotometer as reported in literature.110

61

3.8.3 DETERMINATION OF ACID PHOSPHATASE (ACP)

Reagents: R1 = Citrate buffer(7.5 mmol/L) pH=5.2, R2 1-naphthyl phosphate(10mmol/l) and R3=

Sodium Tartrate(135 mmol/L)

Procedure:

Blood sample(100 µL) was pipetted into a test tube and R2 (1000 µL) was added to the tube. The

mixture was incubated for 5 min at the temperature of 37oC. The absorbance was taken at the

wavelength of 546nmin the spectrophotometer as reported in literature.112

3.8.4 DETERMINATION OF CREATININE

Reagents: R1a=Picric acid(35 mmol/L), R1b=Sodium hydroxide(0.32 mmol/L)

Procedure:

Blood serum sample(100 µL) was pipetted into a test tube, standard reagent(100 µL) was pipetted

into a standard tube and equal volume of R1a and R1b(1000 µL) was added to all the tubes after

which the absorbance was read at the wavelength of 510nmin the spectrophotometeras reported in

literatures 110,113

3.9DETERMINATION OF TRACE ELEMENTS

3.9.1 SERUM CHLORIDE DETERMINATION

Reagents: Chloride reagent(1.5 mL), distilled water(10 µL) and standard reagent(10 µL)

62

Procedure:

Blood serum(10 µL) was pipetted into a test tube, distilled water(10 µL) was pipetted into a bank

tube and standard reagent(10 µL) was also pipette into a standard tube. Chloride reagent(1.5 mL)

was added to all the tubes. The mixture was left for 5 min before taking the measuring the

absorbance at the wavelength of 520nmin the spectrophotometer as reported in literature.114

3.9.2SERUM SODIUM DETERMINATION

Reagents: Sodium filtrate reagent (1000 µL), distilled water(50 µL) and standard reagent(50 µL),

Sodium acid reagent(50 µL) and colour reagent(50 µL)

Procedure:


tube and standard reagent(50 µL) was also pipette into a standard tube. Sodium filtrate reagent

(1000 µL) was added to all the tubes and the mixture was centrifuged for 5 min. Sodium acid

reagent(50 µL) and colour reagent(50 µL) were added after which the absorbance was measured at

the wavelength of 550nmin the spectrophotometer as reported in literatures.115,116

3.9.3SERUM POTASSIUM DETERMINATION

Reagents: Potassium filtrate reagent (1000 µL), distilled water(10 µL) and standard reagent(10

µL)

Procedure:

63


tube and standard reagent(10 µL) was also pipette into a standard tube. Potassium reagent (1000

µL), was added to all the tubes. The absorbance was measured at the wavelength of 520nmin the

spectrophotometer as reported in literature.117

3.9.4SERUM ZINC DETERMINATION

Reagents: Ammonia-ammonium chloride buffer(10 mL), distilled water(25 mL),

hydroxylamine(12 %, 2 mL), 2 drops of Erichrome-black-T indicator and EDTA(0.01 M)

Procedure:

Blood serum (10 mL) was pipetted into a test tube, distilled water (25 mL), Ammonia-ammonium

chloride buffer (10 mL), hydroxylamine(12 %, 2 mL) and 2drops of erichrome-black-T indicator

were added in the test tube.The mixture was titrated with EDTA(0.01 M) to blue colouras reported

in the literature as reported in literatures.118,119

64

CHAPTER FOUR

RESULTS AND DISCUSSION

4.1 RESULTS OF PHYTOCHEMICAL SCREENING OF UNRIPE COCONUT FLUID

Table 1: Results of Qualitative phytochemical screening of unripe coconut fluid

TEST OBSERVATION INFERENCE INTENSITY IN EXTRACT

1) Terpenoids H2SO4 test

Reddsish brown

colouration observed

Terpenoids present +

2) Flavonoids NaOH test

Yellow coloration

observed which

changed to clourless

on addition of dil HCl

Flavonoids present +

3) Alkaloids a) Picric Acid test

b) Dragendoff”s test

c) Mayer’s reagent

Reddish brown

precipitate was

observed

Orange-red precipitate

observed

Buff-coloured

precipitate observed

Alkaloids present

Alkaloids present

Alkaloids present

++

++

++

4) Tannins Ferric chloride test

Appearance of

brownish-green

colouration

Tannins presnt ++

5) Saponins (a) Emulsion test

(b) Frothing test

Emulsion formed

A stable froth was

observed

Saponin present

Saponin present

+

+

+ (Present in low concentration), ++ ( Present in moderate concentration)

65

Table 2: Results of Quantitative Phytochemical Screening of unripe coconut water

Terpenoids (%) 3.677 3.815 3.649

Flavonoids (%) 0.139 0.329 0.417

Alkaloid (%) 5.081 5.170 5.064

Tannins (%) 6.757 6.732 6.697

Saponins (%) 0.107 0.110 0.108

The presence of various phytochemicals in unripe coconut fluid revealed the potentials of unripe

coconut fluid as a therapeutic liquid. Alkaloids present in moderate concentrations is a bioactive

constituent of plants may be responsible for the medicinal value of the respective plant foods120

.

The presence of saponins in the unripe coconut fluid could imply that consumption of coconut

fluid has the potential to lower cholesterol levels in humans due to the hypocholesterolemic effect

of saponins121

. This study also observed the presence of terpenoids in low concentration. The

presence of terpenoids in the unripe coconut fluid could be responsible for their antioxidant and

antimalarial properties122

. These antioxidants are compounds that reduce the formation of free

radicals or they react with and neutralize them thus potentially protecting the cell from oxidative

damage123

. Flavonoids present in low concentration are also important antioxidants, and promote

several health benefits124

. This study also revealed the presence of tannins in moderate

concentration. Some alkaloids and terpenoids have antimalarial activities also10

.

66

4.2 RESULTS OF ANTIMICROBIAL ACTIVITY

Table 3:Results of Antimicrobial Sensitivity Testing of the unripe coconut fluid

Compound

Gram-positive bacteria Gram-negative

bacteria

Fungi Organisms

B.subtilis S. aureus P. aeruginosa E. coli C. albicans A. niger

Unripe

coconut

fluid

- - - - - -

RF1 ++ +++ ++ + - -

RF2 - - - ++ ++ +++

+ = sensitive

++ = moderately sensitive Rf 1 (Ciprofloxacin, antibacteria)

+++ = highly sensitive Rf 2 (Ketoconazole, antifungi)

-= resistance

Table 4: Results of the Inhibition Zones Diameter(mm)

Compound

Gram-positive bacteria Gram-negative bacteria Fungi Organism

B.subtilis S. aureus P. aeruginosa E. coli C. albicans A. niger

Unripe

coconut

fluid

- - - - - -

RF1 17 20 13 8 - -

RF2 - - - - 16 21

67

The compounds with the IZD > 17 and 8 were considered to be sensitive & active against the B.

subtilis and E. coli respectively, and upon serial dilution, gave the MIC. The higher the IZD

values, the higher the activity.

The choice of ciprofloxacin and ketoconazole as standards is due to the fact that they possess

broad spectra of antibacterial and antifungal activities respectively.125

The antimicrobial results

(Table 3) shows that unripe coconut fluid is inactive against Bacillus subtilis, Staphylococcus

aureus, Pseudomonas aeruginosa,Escherichia coli, Candida albicans and Aspergillus niger.

4.3 RESULT OF ACUTE TOXICITY TEST(LD50)

Table 5: Acute toxicity result table

GROUPS

DOSAGE MICE 1 MICE 2 MICE 3 MICE 4 MICE 5

PHASE 1

GROUP 1

10 mL/Kg ND & NST ND & NST ND & NST ND & NST ND & NST

GROUP 2


GROUP 3

1000 mL/kg ND & NST ND & NST ND & NST ND & NST ND & NST

PHASE 2

GROUP 1


GROUP 2


GROUP 3


ND: No Death

NST: No Sign of Toxicity

Unripe coconut fluid is not toxic to the body system

68

4.4RESULT OF PERCENTAGE PARASITAEMIA

Table 6: 4 days after inoculation of malaria parasite

SN

GRP 1 (UN) GRP 2 (NC) GRP 3 (SC) GRP4 (200

mL/kg)

Grp5 (300

mL/kg)

1

3

+ 0 5+ 4

+ 5

+

2

4

+ 0 6

+ 4

+ 7

+

3

3

+ 0 4

+ 5

+ 4

+

4

2

+ 0 7

+ 7

+ 6

+

5

2

+ 0 3

+ 3

+ 2

+

AP

2.8+ 0 5.6

+ 4.6

+ 4.8

+

Table 7: 7 days treatment of malaria parasite

SN

GRP 1 (UN) GRP 2 (NC) GRP 3 (SC) GRP4 (200

mL/kg)

Grp5 (300

mL/kg)

1

5

+ 0 2+ 2

+ 2

+

2

5

+ 0 2

+ 1

+ 4

+

3

6

+ 0 1

+ 3

+ 1

+

4

4

+ 0 3

+ 4

+ 3

+

5

5

+ 0 2

+ 1

+ 0

+

AP

5+

0 2+

2.2+ 2

+

UN: Untreated Control (Inoculated with malaria parasite but not treated)

NC: Normal Control (Was not inoculated with malaria parasite at all)

SC: Standard Control (Inoculated with malaria parasite and treated with Artesunate which is

the reference drug)

200 mL/kg and 300 mL/kg: Represent different doses of unripe coconut water.

69

AP: Average Parasitaemia

An understanding of the life cycle of malaria parasite is the fundamental method of treatment and

eradication of the disease. Malaria is mosquito-borne, caused by unicellular protozoan parasites of

the genus plasmodium and transmitted only by the female Anopheles mosquito.

After inoculation of parasitaemia, the parasite enters into the bloodstream of the host in the

form of haploid sporozoite127

. It immediately moves to the liver and invades the liver cell

(hepatocytocyte) where it reproduces by mitosis. The sporozoite transforms into schizont which

contains thousands of haploid cells called merozoits. The merozoites that are released from the

ruptured hepatocyte or liver cell into the blood stream quickly invade erythrocytes which is the

stage of malaria. The parasites matures into trophozoites that feed on the haemoglobin found in the

erythrocytes resulting in high increase of average parasitaemia as recorded in GRP1(UN) of Table

8.

Most anti-malaria drugs and natural products with antimalarial activities are stage-specific blood

schizonticides, since they act principally on the mature trophozite stage of parasite development.

Unripe coconut water is believed to be one of the natural products with antimalarial properties

since it produced similar effect with Artesunate which served as the standard reference drug. This

is illustrated in Table 8in Grps 3,4 and 5 above.

4.5 RESULTS OF ANTIOXIDANT ACTIVITIES

Presence of antioxidant in unripe coconut fluid helps in fighting free radicals and may also

play an important role in reducing the blood pressure in arteries

70

Table 8: Results of Determination of Superoxide Dismutase (µmol/L)

GRP1(UC) GRP2(NC) GRP3(SC) GRP4(200 mL/kg) GRP5(300 mL/kg)

26.375 89.370 89.376 66.926 72.427

30.337

76.397 79.387 74.344 70.046

34.391

50.677 82.397 70.404 78.593

32.814

56.805 65.452 67.655 69.667

28.976 48.967 70.667 65.789 63.578

Av=30.579 Av=64.443 Av=69.024 Av=69.023 Av=70.862

UC: Untreated Control

NC: Normal Control

SC: Standard Control

200 mL/kg and 300 mL/kg: Represent different doses of unripe coconut fluid.

Av= Average.

SODS are enzymes that alternatively catalyze the partitioning of superoxide (O2-) radical to

either O2 or H2O2128

. Groups 4 and 5 showed increase in SOD activities when compared to

Group 2.This shows that the unripe coconut fluid has good SOD activities in the blood serum.

However, Group 1 showed a decrease in SOD activities when compared to Groups 2, 3, 4 and

5. This agrees with the investigation of Mc cord129

where this decrease may be attributed to the

exhaustion of SOD in a bid to scavenge excess production of reactive oxygen caused by

oxidative stress of the malaria parasite.

71

Table 9: Results of Determination of Vitamin E (µmol/L)


0.409

0.665 0.554 0.476 0.532

0.476

0.632 0.620 0.576 0.587

0.404

0.654 0.609 0.643 0.576

0.413

0.644 0.509 0.587 0.622

0.402

0.661 0.600 0.698 0.501

Av=0.421 Av=0.651 Av=0.578 Av=0.596 Av=0.564

Table 10: Results of Determination of Vitamin C (µmol/L)

GRP1(UC) GRP2(NC) GRP3(SC) GRP4 (200 mL/kg) GRP5 (300 mL/kg)

0.094

0.220 0.319 0.735 0.466

0.076

0.206 0.245 0.166 0.384

0.083

0.154 0.198 0.062 0.214

0.066

0.192 0.156 0.468 0.339

0.089

0.063 0.247 0.357 0.145

Av=0.08 Av=0.167 Av=0.233 Av=0.358 Av=0.310

UC: Untreated Control

NC: Normal Control


200 mL/kg and 300 mL/kg: Represent different doses of unripe coconut fluid.

Av = Average.

72

Vitamin E is an important antioxidant that protects unsaturated oil from being destroyed in

the body by oxygen and also a potent water-soluble antioxidant in humans. Because of this

property, increase amount of it is required. Vitamin C is also a potent water-soluble antioxidant in

humans along with vitamin E. Group 1 showed a decrease in vitamins E and C concentrations

when compared to GRP 2. This agrees with the investigation of Das et al130

where these decrease

may be attributed to the exhaustion of these antioxidants in a bid to scavenge excess production of

reactive oxygen caused by oxidative stress of the malaria parasite. Groups 4 and 5 showed increase

in vitamins E and Cconcentration when compared to Group 2.These increase show that the unripe

coconut fluid has appreciable vitamins E and C activity in the blood serum.

4.6 RESLUTS OF HAEMATOLOGICAL TEST

Table 11: Results of determination of Total White Blood cell count (mL/mg)


10800

10300 10200 10200 10600

10600

10100 10400 10600 10100

10400

10200 10300 10400 10100

10600

10400 10100 10100 10000

10700

10200 10500 10000 10400

Av=10620 Av=10240 Av=10300 Av=10260 Av=10240

UC: Untreated ControlAv = Average.

NC: Normal Control


200 mL/kg and 300 mL/kg: Represent different doses of unripe coconut water.

73

White blood cells also known as leukocytes or immune cells which form a component of the

blood. They help to defend the body against infectious diseases and foreign bodies and

formpart of the immune system.GRP1 showed an increase in the number of white blood cells

when compared to GRP2, the normal control. This is because more white blood cells are

produced in a bid to defend the body system against diseases and infections that were caused

by the parasite in the blood. Alternatively, GRPS 3,4 and 5showed decreases in the number of

white blood cells when compared to GRP 1. The decrease may be due to the death of the

parasites that caused the malaria.

Table 12: Results of Determination of Total Red Blood Cell Count (mL/mg)


5.31

8.14 6.66 6.45 10.61

4.61

10.31 10.28 7.92 8.45

4.93

7.69 9.08 8.92 10.42

5.73

7.54 7.49 9.45 7.41

4.80

6.72 8.36 7.66 6.35

Av=5.08

Av=8.08 Av=8.37 Av=8.08 Av=8.65

74

Table 13: Results of Determination of Haemoglobin Concentration (mL/mg)

GRP1(UC) GRP2(NC) GRP3(SC) GRP4(200 m/Lkg) GRP5(300 mL/kg)

9.357

11.923 16.817 10.121 15.442

7.269

11.371 15.714 14.205 14.867

9.049

10.755 11.872 13.750 17.749

8.650

12.458 10.366 10.463 11.457

9.748

11.546 10.457 11.578 10.248

Av=8.815

Av=11.611 Av=13.045 Av=12.023 Av=13.953

Haemoglobin is a component of the red blood cells. It is the iron-containing substance in the

red blood cells that transports oxygen from the lungs to the rest of the body. It consists of a

protein (globin) and haem (a porphyrin ring with an atom of iron at its centre).131

When the

parasite that causes malaria infects a red blood cell, it consumes haemoglobin within its

digestive vacuole which results in decrease in the number of red blood cells and haemoglobin

concentration as seen in Tables 12 and 13 for GRP1(UC). As the parasites that consume

haemoglobin in the red blood cell die as a result of antimalarial effect exerted by these drugs,

the number of red blood cells and haemoglobin concentration increased above the normal

control(GRP2). This effect is most pronounced in GRP5 in Tables 12 and 13

75

4.7 RESULTS OF LIVER FUNCTION TEST ACTIVITIES

Table 14: Results of Aspartate Aminotransferase (AST) Activity(U/L)


21

11 15 18 17

25

15 14 19 11

20

14 15 17 19

21

12 13 12 11

36

11 12 11 15

Av=24.6 Av=12.6 Av=13.8 Av=15.4 Av=14.6

Table 15: Results of Alanine Aminotransferase (ALT) Activity(U/L)


49

20 26 28 25

50

21 22 24 30

55

25 21 26 23

46

29 30 20 21

47

30 25 21 22

Av=49.4 Av=25.0 Av=24.8 Av=23.8 Av=24.2

AST and ALT tests are used to evaluate the health of the liver. These tests are used to detect liver

damage and liver injury. The amount of AST and ALT in the blood is directly proportional to

extent of tissue damage132

. GRP1(UC) showed high level of AST and ALT indicating high liver

76

injury caused by the malaria parasite in the liver. GRPS 4 and 5 showed a decrease in AST and

ALT level similar to GRP2. These decrease show that unripe coconut fluid is not toxic to the liver.

Table 16: Results of Determination of total bilirubin (U/L)


0.0756

0.1972 0.159 0.197 0.119

0.0432

0.0648 0.168 0.176 0.238

0.0432

0.108 0.2052 0.276 0.183

0.0552

0.123 0.137 0.147 0.145

0.0379

0.097 0.046 0.165 0.169

Av=0.0510 Av=0.118 Av=0.143 Av=0.192 Av=0.171

Bilirubin is a yellow pigment that is found in the blood133

. Bilirubin is made in the body

when the old red blood cells are broken down. The breakdown of old cells is a normal healthy

process. After circulation in the blood, bilirubin is then transported to the liver. In the liver,

bilirubin is excreted into the bile duct and stored in the gall bladder. Eventually, the bilirubin is

released into the small intestine as bile to help emulsify fat. Bilirubin can effectively inhibit

parasite growth through development of oxidative stress can lead to its death or destruction.

GRPS 3,4 and 5showed little increase in the level of bilirubin compared to the normal control

GRP2. This increase in the level of bilirubin is necessary in order to inhibit the growth of malaria

parasites in the liver. GRP1 showed a decrease in the level of bilirubin compared to GRP2. This

decrease can be attributed to high growth of the malaria parasite in the liver with low inhibition.

77

4.8 RESULTS OF KIDNEY FUNCTION TEST ACTIVITIES

Table 17: Results of Determination of Creatinine (mmol/L)


3.047

1.00 1.O11 1.640 1.712

2.127

1.213 1.106 1.713 0.986

3.275

1.441 1.220 1.410 1.616

4.156

1.525 1.01 1.051 1.593

3.784

1.096 1.128 1.211 1.367

Av=3.278 Av=1.255 Av=1.095 Av=1.405 Av=1.455

Table 18: Results of Determination of Urea (mmol/L)


9.431

4.764 6.431 6.669 4.193

8.954

3.907 5.955 5.002 6.669

9.002

5.682 5.246 5.955 5.478

7.685

4.776 4.026 4.748 4.765

9.876

3.823 3.125 5.866 4.932

Av=8.990 Av=4.590 Av=4.957 Av=5.648 Av=5.207

78

Table 19: Results of Determination of Uric acid (mmol/L)


6.431

2.764 3.431 2.669 3.193

5.954

2.907 2.955 3.002 2.669

5.002

2.568 3.246 3.955 3.478

7.554

3.426 2.547 2.566 2.579

6.742

2.812 2.362 2.687 2.699

Av=6.337 Av=2.895 Av=2.908 Av=2.976 Av=2.924

Table 20: Results of Determination of Acid phosphate (mmol/L)

GRP1(UC) GRP2(NC) GRP3(SC) GRP4(200 mL/kg) GRP5(300kg/mL)

5.743

1.086 1.229 2.486 1.400

4.458

1.229 1.715 1.320 2.201

6.229

1.500 1.892 1.687 1.488

5.276

1.364 1.206 1.206 1.207

4.658

1.257 1.146 1.448 1.345

Av=5.273 Av=1.287 Av=1.438 Av=1.629 Av=1.528

Creatinine is a chemical waste that is generated from muscle metabolism. It is transported through

the blood stream to the kidney. Uric acid is a chemical waste produced when the body breaks

down food that contain organic compounds. They are dissolved in the blood, filtered through the

kidney and expelled in the urine. Urea is formed when ammonia produced from the liver reacts

79

with CO2. The kidney filters out most of the creatinine, urea and uric acid and disposes them in the

urine. Elevated levels of creatinine, urea, uric acid and acid phosphate signify impaired kidney

function or kidney disease. GRP1 (UC) showed elevated level of creatinine, urea uric acid and

acid phosphate compared to GRP2 (NC). This increase is due to the level of malaria parasites in

the blood.

GRPS 4 and 5 showed decreases in creatinine, urea, uric acid and acid phosphate level similar

to GRP2. These decrease suggest that unripe coconut fluid is not toxic to the kidney.

4.9RESULTS OF TRACE ELEMENT DETERMINATION

Table 21: Serum Potassium Determination (mmol/L)


37.333

62.047 71.33 64.067 54.167

38.667

61.333 59.642 68.00 66.667

48.667

60.145 65.333 66.668 69.333

47.238

62.065 67.861 75.659 72.854

39.632

61.081 70.661 72.887 75.645

Av=42.307 Av=61.334 Av=66.966 Av=69.456 Av=67.733

80

Table 22: Serum Sodium Determination (mmol/L)


137.50

151.250 181.250 168.750 231.250

142.50

150.000 187.750 218.750 256.250

125.60

156.087 206.250 168.750 181.250

138.40

161.281 190.881 186.450 166.130

140.52

152.781 189.766 195.320 152.630

Av=136.904 Av=154.780 Av=191.179 Av=187.604 Av=197.502

Table 23: Serum Chloride Determination (mmol/L)


60.364

76.818 71.818 98.182 81.364

51.818

85.909 91.364 92.273 91.364

58.182

84.567 88.636 105.455 86.913

55.162

79.223 83.763 87.321 96.722

57.761

83.097 79.677 77.541 89.987

Av=56.657 Av=81.923 Av=83.052 Av=92.154 Av=89.270

81

Table 24: Serum Zinc Determination (mmol/L)


0.095

0.159 0.115 0.164 0.299

0.017

0.104 0.202 0.231 0.278

0.014

0.124 0.142 0.254 0.172

0.020

0.108 0.152 0.198 0.144

0.016

0.119 0.164 0.175 0.161

Av=0.0324 Av=0.1228 Av=0.155 Av=0.2044 Av=0.2108

The result on Tables 21, 22, 23 and 24 showed that the levels of trace elements in serum were

significantly lower in GRP1 when compared to GRP2. This decrease may be attributed to the

damageable effect of malaria parasite on the quantity of trace elements. This is in agreement with

the works of authors Muller and kappes134

. In their opinion, levels of trace elements in serum is

inversely proportional to the level of malaria parasite in the blood. GRPS, 4 and 5 showed a slight

increase in the concentrations of K+, Cl

-, Zn

2+ and a significant increase in the concentration ofNa

+

when compared to GRP2. This increase may be due to the low level of malaria parasite in the

blood and the minerals present in the unripe coconut fluid.

82

CHAPTER FIVE

CONCLUSION

Unripe coconut fluid is believed to be one of the natural products with antimalarial properties

since it produced similar effect with Artesunate which served as the standard reference drug. This

unripe coconut water is seen to have good antioxidant capacity in the serum part of the blood. The

effect of the sample on body organs (kidney and liver) was also tested and was found to be non-

toxic. The unripe coconut fluid sample was screened against six (6) micro-organisms, viz: Bacillus

subtitis, Staphylococcus aureus, Pseudomonas aeruginosa,Escherichia coli, Candida albican and

Aspergillus niger; and was found to show no activity on these mico-organisms. The antimicrobial

test showed that unripe coconut fluid can be used as a control in antimicrobial test since it is

inactive to the bacteria and fungi above.

FURTHER STUDIES

Although coconut fluid is already well studied in terms of its chemical content, there is need to

extract, isolate and characterize solutes which contribute to its special biological effects especially

solutes responsible for its antimalarial activities. With the development of more advanced

detection techniques, screening can be intensified to detect novel compounds of medicinal

relevance present in unripe coconut fluid.

Furthermore, there is need to find out why unripe coconut fluid shows no activity against

bacteria and fungi whereas ripe coconut water does.

83

REFRENCES

1. Carlo, G.D; Mascolo, N; Izzo, A.A; Capasso, F.(1999). Flavonoids: Old and New aspects

of a class of Natural Therapeutic Drugs. Life Sciences 65: 337-353.

2. Bimlesh Kumar et al. ((2001).A Review of Phytochemistry and Pharmacology of natural

products

3. British Pharmacopoeia (2001). Introduction, general notices and medicinal and

pharmaceutical sciences. Her majesty stationary office, United Kingdom, 1: 720-736.

4. Campbell-Flack,D; Thomas, T; Falek, T.M; Tutuo, N; Clem K.(2000). Nutritional benefits

of coconut water. Am J Emerg Med 18(1):108-111.

5. Yong, J.W.H; GeL, N; Tan, S.N.(2009). The chemical composition and biological

properties of Coconut water. Molecules 14(12) 5144-5164.

6. Saat, M; Singh, R.G ;Sirisinghe and M Nawawi, (2002). Rehydration after exercise with

fresh young coconut water, carbohydrate electrolyte beverage and plain water. J Physiol

Anthropic App, Human Sci, 21: 93-104.

7. Campbell-Flack, D. J; Thomas, T.M; Falek, Tutuo N and K Clem.(2000). The intravenous

use of coconut water. Am J Emerg Med, 18: 108-111

8. Miller, AL. (1996). Antioxidant flavonoids: Structure, function and Clinical Usage.

Alternative Medicine Review 1: 103-111.

9. Bhagya, D; Prema, L; Rajamohan T. (2010). Beneficial effect of tender coconut water on

blood pressure and lipid levels in experimental hypertension. Journal of Cell and Tissue

Research 10(1): 2139-2144.

84

10. Edeoga, H.O; Okwu, D.E; Mbaoble, B.O.(1996). Phytochemical constituents of some

Nigerian Medicinal Plants. African J Biotechnol 2005; 4(7): 685-688.

11. Kindersley, D.(2006). Nutrition for life. Lark and Deen Publishers, UK, p.213.

12. Dichter, M.A, Delanty, N.(2000). Antioxidant therapy in neurological disease. Arch.

Neurol., 57: 1265- 126.

13. Osman, A.M; Younes, M.E; Sheta,A.E .(1974). Triterpenoid of the leaves of psidium

guajava .Phytochem 13:2015-2016

14. Long, B. H; Rose, W. C; Vyas, D. M; Matson, J. A; Forenza, S. (2002).Current Medicinal

Chemistry: Anti-Cancer Agents, 2, 255 266.

15. Prudhomme, M. (2003).European Journal of Medicinal Chemistry, 38, 123–140.

16. Hoehn, P; Ghisalba, O; Moerker, T; Peter, H. H. (1995). Journal of Antibiotics (Tokyo),

48, 300–305.

17. Yang, S.W; Cordell, G. A. (1997).Journal of Natural Products, 60, –790.

18. Sańchez, C; Meńdez, C; Salas, J. A. (2006). Natural Product Reports, 23 1007–1045.

19. Akinaga, S; Sugiyama, K; Akiyama, T. (2000). Anticancer Drug Design, 15, 43– 52.

20. Oguntoye, S.O; Duwade K. A.(2010). Phytochemical screening and antibacterial activities

of coconut fruits, MSc. Thesis, University of Uyo, pp 65-71

21. Cook, N.C; Samman, S. (1996).Phytochemicals: Chemistry, metabolism, cardioprotective

effects and dietary sources. Nutritional Biochemistry 7: 66-76.

22. Hemingway, R.W; Karchesy, J.J. (1989). Chemistry and significance of condensed tannins.

Plenum Press, New York, 47‐60.

23. Trease. K.E; Evans, W.E. (1985). Textbook of Pharmacognosy. 12th edition. Balliere

Tindall Publication. London pp.53 7-541.

85

24. Obidoa, O; Joshua, P.E; Eze, N.J.(2010). Phytochemical Analysis of Cocos Nucifera L. J.

Pharm. Res., 3(2): 280-286.

25. Haslam, E.(1989).Plant Polyphenols – Vegetable Tannins Revisited –Chemistry and

Pharmacology of Natural Products, CambridgeUniversity Press, Cambridge, p. 9.

26. Porter, L. J. (1989). in Methods in Plant Biochemistry-Plant Phenolics, Series Academic

Press, London vol. 1, p. 389

27. Antherden, L.M. (1999). Textbooks of Pharmaceutical Chemistry. 6th edition. Ox ford

University Press London. Pp. g J 0-814

28. Osagie, A.U.(1998). Nutritional Quality of plant foods in Benin city, Nigeria: Post Harvest

Research Unit, University of Benin, pp. 228-229.

29. Akinleye .O, Fabunmi. A.O. (1997). Screening of coconut water for antimicrobial

activities. Biotechnology for Development in Africa. 1st edition. FADI B. Ochumba Press

Ltd. Enugu pp.300-304.

30. Sofowora, A. (1984). Medicinal Plants and Traditional Medicine in Africa. John Wiley and

Sons. Ltd, London. pp.100–102

31. Brian, S.F; Antony, J.I ; Peter, G.S; Austin, R.T.(1989). Vogel's Tcstbook of Practical

Organic Chemistry 5th edition. Longman Group UK Ltd. Pp.1 225-1247.

32. Mitscher, L.A;Drake, S;Golloapudhi, S.R;Okwute,S.K. (1987). A modern look at folkloric

use of anti infective agents. Journal of Natural Product, 50: 1025- 1040

33. Narayana, K.R; Reddy, S.R; Chaluvadi, M.R; Krishna, D.R. (2001). terpenoids

classification, pharmacological, biochemical effects and therapeutic potential. Indian

Journal of Pharmacology 33: 2-16.

86

34. Okwu, D.E. (2005). Phytochemicals, Vitamins and Mineral contents of two Nigerian

Medicinal Plants. Int J Mol Med Adv Sci; 1(4): 375-381.

35. Stray, F.(1998). The Natural Guide to Medicinal Herbs and Plants. Tiger Books

International. London, pp. 12-16.

36. Pascaline,J;Charles,M; Lukhoba, C. (2008). Phytochemical constituents of some medicinal

plants used by the Nandis of South Nandi district Kenya. Journal of Animal & Plant

Sciences, Vol. 9(3): 1201- 1210.

37. Koche, D; Shirsat, R; Imran; S. (2010). Phytochemical screening of eight traditionally

used ethnomedicinal plants from akola district (ms) India. International Journal of Pharma

and Bio Sciences, Vol. 1(4),

38. Pascaline,J; Charles,M; Lukhoba, C.(2011). Phytochemical constituents of some medicinal

plants used by the Nandis of South Nandi district Kenya. Journal of Animal & Plant

Sciences, Vol. 12(3): 1117- 1210.

39. Singh, S; Gautam, A; Sharma, A.(2010). Centella asiatica L.: a plant with immense

medicinal potential but threatened. International Journal of Pharmaceutical Sciences

Review and Research, Volume 4(2): Article 003

40. Kartnig, T. (1998). Herbs, Spices and Medicinal Plants, L.E. Cracker, J.E. Simon (Eds.),

Oryx Press, Arizona, USA, vol. 3: 145-173 1998.

41. Ghasemzadeh, A; Jaafar, H.Z.E; Rahmat, A.( 2010). Antioxidant activities, total Phenolics

and flavonoids content in two varieties of Malaysia Young Ginger ( Zingiber officinale

Roscoe). Molecules 15: 4324-4333.

42. Edward, C. (2006). Species Profiles for Pacific Island Agroforestry.; 1-27.

87

43. Martin, Diane, M.; Gershenzon, Jonathan; Bohlmann; Jörg.(2003)."Induction of Volatile

Terpene Biosynthesis and Diurnal Emission by Methyl Jasmonate in Foliage of Norway

Spruce". Plant Physiology 132 (3): 1586–159

44. Pichersky, E. (2006). "Biosynthesis of Plant Volatiles: Nature's Diversity and Ingenuity".

Science 311 (5762): 808–811.

45. Günata, Ziya; Wirth; Jérémie, L.; Guo; Wenfei; Baumes; Raymond, L. (2001).

"Carotenoid-Derived Aroma Compounds". ACS Symposium Series. ACS Symposium

Series 802: 255–261.

46. Sandhya, V.G;Rajamohan,T. (2008). Comparative evaluation of hypolipedimia effects of

coconut water and lovastin in rats fed fat-cholesterol enriched diet. Food chemtoxicol, 25:

3585-3592

47. Thimmappa, R.; Geisler, K; Louveau, T; O’Maille, P; Osbourn, A. (2014). "Tetraterpene

biosynthesis in plants". Annu Rev Plant Biol. 65: 225–57.

48. Zelena, K; Hardebusch, B; HüLsdau, B; Berger, R.; Zorn, H.(2009). "Generation of

Norisoprenoid Flavors from Carotenoids by Fungal Peroxidases". Journal of Agricultural

and Food Chemistry 57 (21): 9951–5.

49. Tripoli, E; Guardia, M.L; Giammanco, S; Majo, D.D; Giammanco, M.(2007). Citrus

flavonoids: Molecular structure, biological activity and nutritional properties: A review.

Food Chemistry 104: 466-479

50. Dewick, P. M.(2009). in Medicinal Natural Products, 3rd ed., John Wiley & Sons Ltd,

Chichester, p. 187.

88

51. Boutanaev, A.M; Moses, T; Zi, J; Nelson, D.R.; Mugford, S.T; Peters, R..J; Osbourn, A.

(2015).Investigation of terpene diversification across multiple sequenced plant

genome"Natural product academy Science USA. 112 (1): E81–8.

52. Murlidhar, A; Babu, K.S; Sankar, T.R; Redenna, P; Reddy, G.V; Latha, J.(2002).

Antiinflammatory activity of flavonoids from stem bark of Butea monosperma : A

mechanism based study. International Journal of Phytopharmacology ; 1: 124-132.

53. Babu, S; Nair, G.M. (2008). Short communication : Enhanced accumulation of

triterpenoids and flavonoids in cell suspension cultures of Azadiracta indica with an

extended stationary phase. Indian Journal of Biotechnology 7: 270-272.

54. Miller, AL. (1996). Antioxidant flavonoids: Structure, function and Clinical Usage.

Alternative Medicine Review; 1: 103-111.

55. Saslowsky. D; Winkel-Shirley, B.(2001). "Localization of flavonoid enzymes in

Arabidopsis roots". The Plant Journal: for cell and molecular biology 27 (1): 37–48.

56. Fernandez, S.P; Wasowski, C; Loscalzo, L.M; Granger, R.E; Johnston, G.A.R; Paladini,

A.C; Marder, M. (2006). Central nervous system depressant action of flavonoid glycosides.

European Journal of Pharmacology; 539: 168-176.

57. Rijke, E.D; Niessen, P.(2006). Analytical separation and detection methods for flavonoids.

Journal of Chromatography; 1112: 31-63.

58. Jiang, C; Schommer, C; Kim, S.Y; Suh D.Y.(2006). "Cloning and Characterization of

Chalcone Synthase from the moss Physcomitrella patens". Phytochemistry67 (23): 2531–

2540.

59. David, S.(2007).Studies force new view on biology of flavonoids"EurekAlert!; Adapted

from a news release issued by Oregon State University.

89

60. Si, D; Wang, Y; Zhou. Y.H.(2009). Mechanism of CYP2C9 inhibition by flavones and

flavonols. Drug Metab. Dispos. 37 (3): 629–34.

61. Larget, R; Lockhart, B; Renard, P; Largeron, M.(2000). "A convenient extension of the

Wessely-Moser rearrangement for the synthesis of substituted alkylaminoflavones as

neuroprotective agents in vitro".Bioorg. Med. Chem. Lett. 10(8): 835–8.

62. Tohge, T; Yonekura-Sakakibara, K; Niida, R; Wantanabe-Takahasi, A; Saito, K.(2007).

"Phytochemical genomics in Arabidopsis thaliana: A case study for functional

identification of flavonoid biosynthesis genes". Pure and Applied Chemistry 79 (4): 811–

23.

63. Ruidavets, J; Teissedre, P; Ferrières, J; Carando, S; Bougard, G; Cabanis,

J.(2000)."Catechin in the Mediterranean diet: vegetable, fruit or wine?".Atherosclerosis

153 (1): 107–17

64. Nibbs, A.E; Scheidt, K.A.(2012)."Asymmetric Methods for the Synthesis of Flavanones,

Chromanones, and Azaflavanones". European journal of organic chemistry 2012 (3): 449–

462.

65. Winkel-Shirley, B. (2001). Flavonoid biosynthesis: A colourful model for genetics,

biochemistry, cell biology and biotechnology. Plant physiol. 126, 485-493

66. Mazza, G; Francis, F.J. (1995). Anthocyanins in graps and grape products. Crit. Rev. Food

Sci. Nutr, 35, 341-371

67. Wroslstad, R.E; Durst, R.W.; Lee,J. (2005). Tracking colour and pigment changes in

anthocyanin products. Trends food Sci. Tech, 16, 423-428.

68. Bitis, L; Kultar, S; Melikoglu, G; Ozsoy, N; Can, A. (2003). Isoflavonoids and antioxidant

activity of Rosa agrestis leaves. Natural Product Radiance 24: 580-589.

90

69. Salucci, M; Stivala, L.A; Maiani, G; Vannini, V. (2012). Flavonoids uptake and their effect

on cell cycle of human colon adenocarcinoma cells. British Journal of Cancer 86: 1645-

1651.

70. Williams, R.J; Spencer, J.P.E; Rice-Evans, C. (2004). Serial review: Flavonoids and

isoflavonones (Phytoestrogens): Absorption, Metabolism and Bioactivity. Free Radical

Biology and Medicine 36: 838-849.

71. Skerget, M; Kotnik, P; hadolin, M; Hras, A.R; Simonic, M; Knez, Z.(2005). Phenols,

proanthocyanidins, flavones and flavonols in some plant materials and their antioxidant

activities. Food Chemistry;89: 191-198.

72. Francis, G; Zohar, K; Harinder, P;Makkar,S; Klaus, B.(2002). "The biological action of

saponins in animal systems: a review". British Journal of Nutrition 88 (6): 587–605.

73. Sun, H; Xie, Y; Ye, Y.(2009)."Advances in saponin-based adjuvants".Vaccine27 (12):

1787–1796.

74. Skene, C.D; Philip, S.(2006). "Saponin-adjuvanted particulate vaccines for clinical use".

Methods40 (1): 53–9

75. Zentner, E.(2011)."Effects of phytogenic feed additives containing quillaja saponaria on ammonia

in fattening pigs". Retrieved 27 November 2012.

76. Asl, M; Nassiri; Hossein; Hosseinzadeh.(2008). "Review of pharmacological effects of

Glycyrrhiza sp. and its bioactive compounds". Phytotherapy Research22 (6): 709–24.

77. Jonathan, G; Cannon, R. A; Burton, S.G; Wood; Noel, L; Owen.(2004)."Naturally

Occurring Fish Poisons from Plants",J. Chem. Educ.81 (10): 1457.

78. Saponin from quillaja bark". Sigma-Aldrich. Retrieved 23 February 2009

91

79. Li, X; Shi, C; Deng, Z; Fu, H; Proksch, P; Lin, W.;Pongamone, A.E. (2006). saponins from

the stems of mangrove plant, Trigonella foenum-graecumPhytochemistry 67: 1347-1352.

80. Kemde, H;Zeevaart, J.(1997). The five “classical” plant hormones. Plant cell; 9: 1197-

1210.

81. Tucker, G.A; Roberts, J.A. (2000).Plant Hormone Protocols; Humana Press Inc. Totowa,

NJ, USA,

82. Haberer, G; Kieber, J.J.(2002). New insights into a classic phytohormone. Plant

Physiol.128, 354–362.

83. Letham, D.S.(1963). Zeatin, a factor inducing cell division isolated from Zea mays. Life

Sci, 2: 569-573.

84. Sheu, J.R; Hsigo, G; Shen, M.Y; Chou, C.Y; Lin, C.H; Chen, T.F; Chou, D.S.(2004).

Inhibitory mechanism of kinetics, a growth promoting plant Hormone, 14: 189 – 196.

85. Ofem, O.E, Antai, A.B; Essien, N.M; Oka, V.O.(1994). Enhancement of some sex

hormones concentrations by consumption of leaves extract of Viscum album (mistletoe) in

rats. Asian Journal of Medical Sciences 2014; 5 (3): 87-90

86. Miller, C.O;Skoog, F;Von Saltza,M.H;Strong, F.M.(1955). Kinetin, a cell division faction

from deoxyribonucleic acid. J Am Chem Soc, 77: 1392-1393.

87. Chen., J; Sun, Z; Zhang, Y; Zeng, X; Qing, C; Liu, J; Li, L.;Zhang, H.(2009). Synthesis of

gibberellin derivatives with anti-tumor bioactivities. Bioorg. Med. Chem.Lett., 19, 5496–

5499.

88. Yong, J.W.H; Tan, S.N; Hua, L; Ong, E.S.(2008). Analyses of gibberellins in coconut

(Cocosnucifera L.) water by partial filling - micellar electrokinetic chromatography - mass

spectrometry with reversal of electroosmotic flow. Electrophoresis 29, 2126–2134.

92

89. Heo, H.J. (2002). Inhibitory effects of zeatin isolated from Fiatoua Villosa, on acetyl

cholinesterase activity from PC12 cells. Mol Cell, 13: 113-117.

90. Zhang, S.M; Willett, W.C; Selhub, J; Hunter, D.J; Giovannucci, E.L; Holmes, M.D;

Colditz, G.A; Hankinson, S.E .(2003). Plasma folate, vitamin B6, vitamin B12,

homocysteine, and risk of breast cancer. J. Natl. Cancer Inst., 95, 373–380.

91. Depeint, F; Bruce, W.R; Shangari, N; Mehta, R.; O'Brien, P.J. (2006). Mitochondrial

function and toxicity: Role of B vitamins on the one-carbon transfer pathways. Chem. Biol.

Interact., 163, 113–132

92. Thakur, M.K; Paramanik, V.(1986).The role of steroid hormone coregulators in health and

disease. Hormone Research 2009; 71(4)194-200.

93. Jackson, J.C;Gordon, A;Wizard, G;Mc Cook,K; Rolle, R.(2004). Changes in chemical

composition of coconut (Cocos nucifera L) water during maturation of the fruit. J Sci food

Agric; 54: 1049-1052.

94. Msengi, A.E;Mbise, R.L;Msuya, P.M;Doamsi, D.M.(1985). Biochemistry of water from

unripe coconut obtained from two locations in Tanzania. East Afr J, 62: 725.

95. Kuberski, T; Robert, A;Linehan, B;Brayden, R.N;Teburae,M.(1979). Coconut water as

rehydration fluid. New Zealand Med J, 90: 98-100.

96. Farnsworth, N.R. (1984). The role of medicinal plants in drug developmemt p. 17-30

97. Gen,X.P; Lin F.S.(1986). Traditional antiparasitic drugs in china, parasitol today, 2:353-

355

98. Gutteridge, W.E.(1989). parasite vaccines,versu anti-parasite drugs rivals, parasitol 98 :

587-597

93

99. Klayman, D.L.(1985). Ginghaosu(Artemisinin): an antimalarial drug from china. Science,

228: 1049-1055

100. Marques, A.C.(1987). Human migration and spread of malaria in Brazil. Parasitol

today, 3:166-170.

101. Ferreira, J.F.S; Luthria, D.L; Sasaki, T; Heyerick, A.(1999). Flavonoids from

Artemisia annua as antioxidants and their potential synergism with Artemisinin against

malaria and cancer. Molecules 15: 3135-3170.

102. Li, Y; Yu, P.L; Chen, Y.X; Li, L.Q; Gai, Y.Z; Wang, D.S; Zheng, Y.P.(1979).

Synthesis of some derivatives of artemisinine. Chin Sci Bull, 24: 667-669

103. Pascaline,J; Charles,M; Lukhoba, C.(2012). Phytochemical constituents of some

medicinal plants used by the Nandis of South Nandi district Kenya. Journal of Animal &

Plant Sciences, Vol. 13 (3): 1201- 1210,

104. Harborne, J.B.(1973). Phytochemical Methods – A Guide to Modern Technique of

Plant Analysis. Chapman and Hall. pp. 49-188.

105. Dey,P. M; Harborne, J. B. (1989).Plant Polyphenols – Vegetable Tannins Revisited

–Chemistry and Pharmacology of Natural Products, CambridgeUniversity Press,

Cambridge, p. 165.

106. Johnston, C.S; Gaas, C.A. (2006).Test for saponins. Medscape General Medicine,

8(2), 61.

107. Bazerque, P; Perez, C; Pauli, M. (1990).Antibiotic assay by the Agar-well Diffusion

Method, Acta Biologiae et Medicine Experimentalis, 15: 113-115.

108. Vardanyan, R.S; Hurby, V.J. (2006). Synthesis of Essential Drugs. Elsevier B.V.,

pp. 426

94

109. Chemical Laboratory Standards Institute (CLSI) (2002). Performance for

Antimicrobial Disc and Dilution Susceptibility Test for Bacteria Isolated from Animals. 22:

13-14.

110. Ochei, J; Kolhatkar A. (2007). Care and use of experimental animals. In: Medical

Laboratory Science Theory and practice, Tata McGraw-Hill India 1213-1232

111. David, K. (2006). Inoculation and Maintenance of Parasitaemia. Bioorg Med Chem

14:875-874

112. Dacie and Lewis.(2006). Evaluation of antimalarial effects of medicinal plants

extracts in male and female albino rats. Asian J Pharm Clin Res; 6: 217-220.

113. UNESCO (2003). Trace elements, minerals and vitamins : news functional and

chemical in human. News of trace elements ; 10 : 30.

114. Sergeant, G.R; Grandison, Y; Mason, K; Sergeant, B; Sewell, A; Vaidya, S.(1980).

Heamatological indices in normal negro children : a Jamaican cohort from birth to five

years.Clin. Lab. Haematol. 2: 169-178.

115. Nussemblatt, V; Semba, K.(2002). Micronutrient malnutrition and pathogenesis of

malarial anemia. Acta.Tropica. 82: 321-337.

116. Wander, K; Shell-Duncan, B; McDade, T. W.(2009). Evaluation of health of the

liver and kidney deficiency as a nutritional adaptation to infectious disease: An

evolutionary medicine perspective, Am. J.Hum. Biol., 21: 2, 172.

117. Bremen, J. (2001). The ears of the hippopotamus : manifestation, determinants, and

estimates of the malaria burden. Am. J. Trop. Hyg. 64 (1, 2 S): 1-11.

118. Oppenheimer, S.J.(2001). Iron and its relation to immunity and infectious disease.

J. Nutr.131: 616S-635S.

95

119. Trape, J.F.(1985). Rapid evaluation of malaria parasite density and standardization

of thick smear examination for epidemiological investigations. Trans. R. Soc. Trop. Med.

Hyg. 79(2):181-184.

120. Anurag, P; Rajamohan,T.(2003). Cardioprotective effects of tender coconut water

in experimental myocardial infarction. Plants food Hum Nutr, 58: 1-12.

121. Alleyne, T.(2005). The control of hypertension by use of coconut water and mauby:

two tropical food drinks. West Indian Med J;54:3-8.

122. United Nations Food and Agriculture Organization. (2012). Top production

coconut- 2011. Retrieved September 7, 2012, from www.faostat.fao.org.

123. Santoso, U; Kubo, K; Ota, T; Tadokoro, T; Maekawa, A. (1996). Nutrient

composition of kopyor coconuts (Cocos nucifera L.). FoodChemistry, 57(2), 299-304.

124. Akorum, S; bendjeddou, D; Satta, D; Lalaoui, K. (2009). Antibacterial activity and

acute toxicity effect of flavonoids extracted from Mentha longifolia. American-Eurasian

Journal of Scientific research; 4: 93-99.

125. Ajani, O.O; Nwiniyi, O.C. (2009). Synthesis and Evaluation of Antimicrobial

activity of phenyl and furan-2-yl[1,2,4]triazol[4.3-9] quinoxaline-4-(5H)-one and their

Hydrazone precusors. Canadian J. of pure and App. Science 3(3): 983-992

126. White, N.J.(2004). Antimalarial drug resistance J. Clin. Invest. 113(8). 1084-92

127. Lim, P; Alker A.P, Whim, N.(2009). Arteminisin derivatives combination therapy

failure in falciparum malaria in Cambodia Malaria J. 8:11

128. Mc cord, J; Fridovich;M.(1969). Superoxide dismutase An enzyme function for

erythrocuprein. The journal of Biological Chemistry 224(22): 6049-55

96

129. Mc cord, J; Fridovich;M.(1998). Superoxide dismutase : The first twenty years

(1968-1988). Free Radical Biology and Medicine 5(5-6): 363-9

130. Das, B.S; Thurnham, D.I; Das, D.B.(1996). Plasma alpha-tacopherol, retinol and

carotenoids in children with Falciparum malaria. Am. J. Clin Nutr. 64:94-100.

131. Epstein, F.H; Hsia, C. C.W. (1998). Respiratory Function of Haemoglobin. New

England Journal of Medicine 338(4): 239-247

132. Johnston, D.E.(1999). Special consideration in interpreting liver function test. Am

fam physician 59 (8) : 2223-90

133. Pirone,C; Quike, J; Martin, E; Priestap, H;lee ,A ; David, W.(2009). Animal

Bilirubin Discovered in plants. Journal of the American Chemical Society 131 (8) : 2830.

134. Muller, S; kappes, B.(2007). Vitamin and co-factor biosynthesis pathway in

plasmodium and other apicomplexan parasites Trends in parasitology 2(4) : 1-177

Date post:	24-Mar-2020
Category:	Documents
Upload:	others
View:	0 times
Download:	0 times

FACULTY OF PHYSICAL SCIENCE - University of Nigeria, Nsukka · 2017-02-22 · 2 tittle page...

Documents