1

CHAPTER ONE

BACKGROUND OF THE STUDY

Human' poses great ability and capacity to adapt physiologically to different types of

foods. On the basis of these adaptability, nutrition science has demonstrated that there are

certain foods that cannot be compromised such as fruits and vegetables (pamplona-

roger ,2008). Most fruits and vegetables have low energy density and are recommended for

weight management (Rools and Ello-martins etal.,2004).

A variety of fruits and vegetables are consumed in Nigeria daily, forming an integral

part of our diet. Fruits contain a high proportion of water averaging 85%, fats and proteins in

very small amount along with a fair proportion of carbohydrates present in the forms

cellulose, starch and some sugars. The optimally recommended by both world health

organization and food and agricultural organization is a low fat and fibre diet rich in

complex carbohydrates characterized by a frequent consumption of fruits and vegetables at

least 400g daily along with whole grain, cereals and legumes at least 30g daily. (WHO/FAO,

2003).

One of such fruits is water melon (citrillus lanatus or c. lanatus). Water melon is an

important curcubit crop accounting for 7% of the world wide areas devoted to vegetable

production. (Shaogui Guo et al.,2013). Water melon belongs to the family Cucurbitaceae.

About three (3) genera of Cucurbitaceae bear the common name 'Melons'. They are Cucumis,

Citrillus, and Cucumeropsis. The genus Cucumis includes: C.melon (true melon), while the

genus Citrillus includes: C. lanatus. C. lanatus is one of the major underutilized fruits grown

in the warmer part off the world. The juice or pulp from water melon is used for human

consumption while the seed may be consumed roasted.

C. lanatus plays a very vital role in Africa as it is used traditionally to quench thirst

when there is shortage of water owing to its high water percentage of 85%. Furthermore,
 2

watermelon seed are useful for oil production at the subsistence level in several African

countries and in the middle east (Mandel etal., 2005). The seeds have been documented to

contain to contain high percentage of polyunsaturated fatty acids such as Oleic, linolenic,

and Linoleic acid amongst other fatty acids with varying percentage composition. It will be

therefore interesting to determine and evaluate how the ingested seed oil affects the

metabolism of lipid and other biochemical parameters in the human body.

C.lanatus is both edible fruit and vine-like plant. It is an annual climbing herb up to

3metres long (Evans, 2009). C.lanatus originally originated from the southern part of Africa

from where it spread all through the tropics and sub-tropical regions. It is mainly a dry season

crop and requires only limited rainfall. The fruits varies in size from about 7cm in diameter to

over 200cm. The fruit shape varies in size from round to long marrow. The outside

colouration is usually a variegation of green stripes , while the inside flesh may be red or

pink. The seeds are black or pale coloured.(Maynard, 2001). The fresh water melon fruit

owes about 65% of the whole fruit to its flesh and about 95% to water. Water melon plant

flowers from July to August, and the flowers are monoecious ( individual flowers either male

or female although both sexes can be found on the same plant) and pollinated by insects. The

wild unselected fruits tend to be slightly bitter, due to the presence of Cucurbitacin.

In African countries such as Nigeria, Senegal, Mali etc, the fruits and seeds are fed to

Cattles, Birds, as well as Pigs. (Rakhi, et al.,2000). Each territory seems to have its own

special cultivars and dry conditions are essential to raising the sweetest fruits as high

atmospheric humidity apparently seems to suppress the formation of sugars.

STATEMENT OF RESEARCH PROBLEM

Fruits have become an integral part of man's diet worldwide. These fruits are

consumed fresh, canned or processed for the varying nutritional and medical roles they play

in the human body. Such roles range from beefing the immune system and combating ailment
 3

to the provision of high fibre, vitamin and mineral content in the diet (Rolls and Ello-Martin

etal.,2004). One of such fruits commonly eaten in Nigeria is water melon (citrillus lanatus or

C.lanatus) which is readily available during the dry season in the market. The fruit of water

melon (C.lanatus) are mostly consumed in the dry season and its gives chilling effect and

reduces thirst. The parts of water melon usually consumed are the succulent pulp (with

varying colour due to species and climatic factor), the rind, the leaves (serve as green

vegetable) and the seed which may be eaten roasted.

In several parts of Africa, seed extracts (seed oil) of water melon is widely gaining

recognition and use. The seed oil has been documented to contain a high percentage of

polyunsaturated fatty acids such as Oleic, Linoleic, and Linolenic acid, amongst other fatty

acids, with varying percentage composition.

Despite the vital role the seed oil of water melon plays nutritionally, there is dearth of

knowledge about the metabolic consequences of the ingested oil in lipid profile in the body.

It will therefore be interesting to determine and evaluate how the ingested seed oil

affects the lipid profile in the body. Additionally, it will be useful to find out the effect they

ingested seed oil has on other biochemical parameters in the body.

Hence, the present study is aimed at determining the effect of water melon seed oil on

serum lipid profile and other biochemical parameters in serum such as haematological

indices, liver enzymes, creatinine and electrolytes in the human body using female albino

wistar rats as model.

RESEARCH QUESTION

1. Does the chloroform extract of citrillus lanatus seed contain the same photochemical

as earlier reported in literature?

2. Does the chloroform extract(seed oil) of citrillus lanatus affect lipid profile in human

body using wistar rats as model?

 4

3. Does the chloroform extract (seed oil) of citrillus lanatus affect the haematological

indices?

4. Does the chloroform extract (seed oil) of citrillus lanatus affect biochemical

parameters in this case liver enzymes such as Alanine amino transferase(ALT),

Aspartate amino transferases(AST) and Alkaline Phosphatase (ALP)?

5. Does the chloroform extract (seed oil) of citrillus lanatus affect the concentrations of

creatinine in the body?

6. Does the chloroform extract (seed oil) of citrillus lanatus affect electrolytes?

7. If items 2,3,4,5 and 6 above are positive, what is the nature of the effect?

8. How do these data correlate with the physiological and pathological state of the liver,

kidney and general biochemistry of the experimental animals?

9. Can these findings be extrapolated to humans?

OBJECTIVES OF THE STUDY

The objectives of the study are:

- Measurement of serum lipid profile including:

Very low Density Lipoprotein cholesterol (VLDLc)

High Density Lipoprotein cholesterol (HDLc)

Low Density Lipoprotein cholesterol (LDLc)

Total Cholesterol (TC)

Triglycerides (TG)

- Measurement of hematological indices including:

White Blood Cell Count (WBC)

Red Blood Cell Count (RBC)

Hemoglobin count (HGB)

Hematocrit count(HCT)
 5

Mean Corpuscular Volume (MCV)

Mean Cell Hemoglobin (MCH)

Mean Corpuscular Hemoglobin Concentration (MCHC)

Platelet Count (PLT)

Lymphocyte Count in percentage (LYM%)

Mixed Cell percentage (MXD)

Neutrophil Count in percentage (NEUT%)

Lymphocyte Count in percentage (LYM#)

Mixed Cell (MXD#)

Neutrophil Count in (NEU#)

Red Blood Cell Distribution Width (RDW-SD)

Red Blood Cell Distribution Width (RDW-CV)

Platelet Distribution Width (PDW)

Mean Platelet Volume (MPV)

Platelet Larger Cell Ratio (P-LCR)

- Measurement of serum liver enzymes including:

Alanine amino transferases(ALT)

Aspartate amino transferases(AST), and

Alkaline Phosphatase (ALP)

- Measurement of Creatinine as a renal function index

- Measurement of electrolytes including:

Bicarbonate (HCO3)

Sodium (NA+)

Potassium (K+)
 6

Chloride (CL-)

- Histopathological study of liver.

- Histopathological study of kidney.

RESEARCH HYPOTHESIS

NULL HYPOTHESIS: There is no significant effect between the chloroform extract (seed

oil) of C.lanatus on the lipid profile, hematological indices, liver enzymes, creatinine and

electrolyte.

ALTERNATE HYPOTHESIS: There is a significant effect between chloroform extract

(seed oil) of C.lanatus on the lipid profile, hematological indices, liver enzymes, creatinine

and electrolyte.

SIGNIFICANCE OF THE STUDY

SCOPE AND LIMITATIONS OF THE STUDY

This research was limited to the following

1. Plant material collection/seed oil extract process

2. Administration of extract to the experimental animals for 21 days

3. Evaluation of serum lipid profile

4. Assessment of hematological parameters

5. Assessment of serum liver enzymes

6. Assessment of creatinine concentrations

7. Assessment of electrolyte concentrations

8. Histopathological studies of liver

 7

9. Histopathological studies of kidney

DEFINITION OF KEY CONCEPTS

PULP: The pulp of a citrus fruit are the stringy content of the fruits endocarp. The pulp

contains juice of the fruit. The pulp is usually removed from the fruit juice by filtering it out.

The colour of the pulp can change depending on the species and the ripening stage. The

juiciness of the pulp depends on species, variety, season, and even the tree on which it grew.

SEED: The seed can be defined as a mature plant ovule containing an embryo.

CUCURBITACIN: Cucurbitacin refers to any class of biochemical compound that some

plants notably members of the family Cucurbitaceae that includes the common pumpkin,

gourds, water melon develop in order to defend themselves from herbivores.

 8

CHAPTER TWO

2.1 Citrillus lanatus historical background and description

2.2 Citrillus lanatus botanical classification

2.3 Ecology/geographical distribution of Citrillus lanatus

2.4 Citrillus lanatus seed

2.5 Photochemical properties/constituents of Citrillus lanatus seed and nutrient

composition

2.6 Lipid composition

2.6.1 Amino acid composition

2.6.2 Vitamins, carbohydrate and mineral composition

2.6.3 Uses of Citrillus lanatus

2.7 Uses of Citrillus lanatus

2.7.1 Ethno-medicinal uses/relevance

2.7.2 Medicinal uses

2.1 CITRILLUS LANATUS HISTORICAL BACKGROUND AND DESCRIPTION

Watermelon (C. Lanatus) is an important Cucurbit crop, accounting for 7% of the

worldwide area devoted to vegetable production (Shaogui Guo.et al 2013). Watermelon is a

fruit crops of the family Cucurbitaceae along with other cucurbit crops including the melon,

cucumber and Zucchini

Historical, watermelon in thought to have originated in southern Africa, where it is

found growing wild because it reaches maximum genetic diversity there resulting in sweet,

bland and bitter forms. Citrillus Lanatus (Egusi melon) in the ancestor of the watermelon now

found all over the world, but originated from West Africa. Though Citrillus Colocynthis often

also considered to be a wild ancestor of watermelon is now found native in North and West

Africa. Furthermore, Fenny Dane and Jiarong Liu on the basis of the chloroplast DNA
 9

investigation suggested that the cultivated and wild watermelon appeared to have diverged

independently from a common ancestor possibly C. Ecirrhouse from Zambia (Dame. F and

Jiarong. Liu, 2007).

DESCRIPTION

Watermelon is an annual herbaceous vime with long stems (up to 10m) Lying or

creeping on the ground, with curly tendrils Citrillus Lanatus (watermelon) produce a fruit that

is about 93% water, hence the name “water” melon. The “melon” part come from the fact that

the fruit in large and round and has a sweet pulpy flesh.

The scientific name of watermelon (Citrillus Lanatus) is derived from both Greek and

Latin roots. The Citrillus part comes from a Greek word “Citrus” which is a reference to the

fruit. The Lanatus part is Latin and has the meaning of being wholly, referring to the small

hairs on the stems and leaves of the plant (Baker, et al, 2012). The leaves of Citrillus Lanatus

are 5 – 20 by 3 – 19 cm, and hairy, usually deeply palmately lobed with 3 – 5 Lobes, leaf

stalks are 2 – 19cm long male flower on 1.2 – 4.5cm long pedicels.

The flower are monoecious usually 1 – 2.5cm long, and pale green on pedicels up to

4.5cm long; with 5 shortly united petals which are pale green. Fruits of the wild plants are

1.5-20cm in diameter, nearly spherical, greenish, mottled with darker green of cultivated

plants up to 30 x60cm spherical or ellipsoid, yellowish, evenly coloured or stripped. Fruits

vary considerably in morphology like colour, odour and taste. (Dane. F, and Jiarong. Liu,

2007).

They have common features, large leave, creeping or climbing stems usually with

simple or branched tendrils, flesh fruits called pepo, with health exocarp, containing

numerous seed, and a woody roof stock. About three genera of Cucurbitaceae bear the

common name, melons. They are; Cucumis citrillus, cucum and Cucumeropsis. The genus

cucum includes: C.melon L (7m Melon) whereas the genus Citrillus include C.lanatus Thrub.
 10

Table.1: Characteristics of Various Species of Watermelon from Different Region

Species Origin Fruit Fruit Shape Fruit Rind Seed Size Seed

Weight Colour Colour Colour

(kg)

C.Var Korea 7.0 Round Red Black Normal Black

Lanatus size

C.Var Korea 7.0 Elongated Red Jubilee Tomato Black

Lanatus stripe seed size

C.Var Korea 7.0 Oblong Deep Dark green Small size Black

Lanatus red

C.Var China 6.0 Round Red Jubilee Normal Black

Lanatus stripe size

C.Var India 6.0 Round Red Grismon Normal Dotted

Lanatus stripe size brown

C.Var USA 12.0 Elongated Red Crimson Normal Dotted

Lanatus Stripe size brown

C.Var Zambia 3.0 Round White Green Giant size Black

Lanatus

c. Iran 0.1 Round White Dotted Small size Grey

colocynthi Greed

Source: (Ji Hyun Hwanga, etal, 2011)

 11

2.2 CITRILLUS LANATUS BOTANICAL CLASSIFICATION

The botanical and scientific nomenclature of water melon is Citrillus Lanatus.

Citrillus part comes from a Greek word “citrus” which is a reference to the fruit while the

Lanatus part is a Latin word meaning wooly referring to the small hairs on the stems and

leaves of the plant (Baker et al, 2012). Furthermore, Citrillus Lanatus has vernacular names

which include the following; Egusi melon and Egusi water melon, others include; West

African watermelon (En). Watermelon, dessert watermelon, cooking melon, (Rhodes and

Zhang, 1999).

However, botanically, watermelon is classified thus;

Kingdom - Plantae

Sub-kingdom - Tracheobionta - Vascular Plants

Super division - Spermatophyte - Seed Plants

Division - Magnoliophyta - Flowering Plants

Class - Magnoliophyta - Dicotyledonous

Sub class - Dellenidae

Order - Violales - Cucurbitales

Family - Cucurbitaceae - Cucumber Family

Genus - Citrillus Schrad - Watermelon

Species - Citrillus Lanatus

Variety - Citrillus Lanatus

 12

2.2 PHYLOGENY TREE OF CITRILLUS LANATUS BROAD

CLASSIFICATION

1. ORIGINAL ANCESTOR

ARCHAEA BACTERIA

EUKARYA

EXCARATA UNIKONTA
RHIZARIA

CHROMALVEOLATA ARCHAEPLASTIDA

RED ALGAE

LAND PLANT GREEN ALGAE

ANGIOSPERM SEEDLESS
GYMNOSPERM

MONOCOTYLEDONEAES MAGNOLLIDS
VASCULAR NON-VASCULAR
DICOTYLEDONEAES
 13
2. CUCURBITACEAES

ZANONIODEAE CUCURBITOIDEAES

MELOTHRIEAE JOLLIFIEAE CUCURBITEAE SICYEAE

SCHIZOPEPONEAE TRICHOSANTHEAE
BENINCASEAE

COGNIAUXIA LAGENANIA BENICASA ACANTHOSIYOS BAN BEKEA

RUTHALICIA CITRILLUS PRAECITRILLY EUREIANDRA

COLOCYNTHIS LANATUS REHMIL ECIRRHOUS

Source: Cambell and Reece, 2006. Phylogeny Tree of Citrillus Lanatus, Biology. 8th e
 14

CLASSIFICATION OF WATER MELON AS A FRUIT

Water melon is the fruit of a plant originally from vine of southern Africa. The

watermelon fruits loosely considered a type of melon has a smooth exterior rind and a juicy,

sweet, usually red interior flesh.

Interestingly with reference from Webster’s dictionary, Citrillus Lanatus is termed a

fruit because it development meets the definition of a fruit which is the ripened ovary of a

seed plant and its content. Nutritionally, consumers prefer water melon primarily as a fruit

because of its sweet flavor and refreshing qualities.

WATER MELON AS A VEGETABLE

A vegetable by definition is anything made or obtained from plant. It is planted from

seeds or seedlings, harvested and then cleared from the field like other vegetables. Since

watermelon is grown as a vegetable crop using vegetative production systems, watermelon is

thus considered a vegetable (Woldford, 2004).

2.3 ECOLOGY/GEOGRAPHICAL DISTRIBUTION OF CITRILLUS LANATUS

Citrillus Lanatus widely grows across many tropical, subtropical and warm

temperature (hot summers) region of the world, including African countries where it is one of

the major commercial crop.

Citrillus Lanatus from the agriculturally perspective with respect to soil prefers a rich

sandy-loamy soil and very sunny position (humidity) (Zhang, 2006). It is a drought resistant

plant and sensitive, intolerant to wet soils. (Sanders, 2009). Tolerates a PH in the range of

5.3-8. Critillus Lanatus requires a long hot summer for it to fruit well with sweet fruits.

Excessive rainfall and high humidity give excessive growth and promote disease

infection mainly leaf and fruit rot; with consequent low yields. Although irrigated dry season

cropping gives higher yields. Local famers plant seed melons during the rainy season, due to
 15

lack of irrigation facilities. Seed yield in the dry savanna zone is 2-3 times higher than in the

rain forest zone (Ladipo, 2000).

Although there is some watermelon production in virtually all states, about 3-4 of all

U.S watermelon is grown in Florida, California, Texas, Georgia and Indiana (Levi, 2002). On

global basis, China is by far the largest watermelon producing country and account for over

half of all world watermelon production (Schippers, 2002).

2.4 CITRILLUS LANATUS SEEDS

Citrillus Lanatus plant produces a special type of fruit known by botanists as a Pepo,

which has a thick rind (Exocarp) and fleshy centre (Mesocarp and Endocarp): Pepos are

derived from an inferior ovary and are characteristics of the cucurbitaceae. The water melon

seeds which are numerous ranging in size from small size, tomato seed size, normal size to

giant size are mostly coloured black, dotted brown and grey. The seeds are very nutritional

medicinal as the can be eaten roasted or raw from the pulp. The following phytochemicals

have been isolated from the seed of citrillus lanatus, which are of health relevance to humans.

Flavoniods:

Flavoniods are products of plants metabolism and have different phenolic structures

(Ohtsuki, 2003). They are class of plant secondary metabolites. These secondary metabolites

according to the IUPAC nomenclature (MC Naught, 1997), can be classified as follows;

a. Flavoniods or Biflavoniods

b. Iso flavoniods, derived from 3 phenyl chromen-4-one structure

c. Neoflavoniods, derived from 4phenyl cou-marine (4 phenyl-2-benzopyrone structure).

Consequently, these three (3) flavoniods classes are all ketone containing compounds

and as such are anthononthins, (Flavones and flavonols)

 16

STRUCTURE OF THE THREE (3) FLAVONIOD CLASSES AS CONTAINED IN

CITRILLUS LANATUS SEED

Molecular structure of flavon-backbone

(2-phenyl-1-4 Benzopyrone)

Flavonoids Structure Isoflavon Structure

FLAVONIODS ARE WIDELY DISTRIBUTED IN PLANTS FULFILLING MANY

FUNCTIONS

In higher plants, flavoniods are involved in UV-filtration, symbiotic nitrogen fixation

and floral pigmentation. They may also act as chemical messengers, physiological regulators,

and cell cycle inhibitors (Galeolti et al, 2008).

Flavoniods have been shown to have a wide range of biological and pharmacological

activities in in-vitro studies, some of which includes; Anti-allergic (Yamamoto et al, 2001),

anti-inflammatory effect (Cazarolli et al, 2008), antioxidant effect (Yamamoto et al, 2001),

anti cancer (de souse et al, 2007) etc. Flavoniods have also been shown to inhibit

topoisomerase enzyme (Essien et al, 2009) and to induce DNA mutations in the mixed

lineage leukemia (MLL) gene in in-vitro studies (Barjesteh et al, 2007).

 17

TANNINS

Tannins are large polyphenolic compounds containing sufficient hydroxyl and other

group such as carboxyl group to form strong complex with various macromolecules.

STRUCTURE OF TANNIC ACID, A TYPE OF TANNIN

0H
0H
0H 0H
0H 0H
0H

0H
0H
0H
0H 0H

Tannins are water-soluble polyphenols benzoic acid derivates that are present in many

plant foods. Tannin compounds are widely distributed in many species of plants, where they

play a role in protecting the plant from predators, and perhaps as pesticides as well as its use

in plant growth regulation (Katic et al, 2006)

Tannins have molecular weights ranging from over 500 to 3000 (gallic acid esters)

and up to 20,000 (pro-anthocyanidins) (Bate-Smith, 2002). They are classified into two

structural groups viz: hydrolysable and condensed tannins.

Hydrolysable tannins are susceptible to being cleaved into sugars and carboxylic acid

by hydrolysis where as condensed tannin are not susceptible to being cleaved by hydrolysis.

Condensed Tannins are polymers formed by the condensation reaction of flavans.

They do not contain sugar residues (Teresa et al, 2006). Tannin bind to both protein and

carbohydrate hence, have serious implication on commodities containing tannins.

 18

The also act as anti-nutritional factors in two ways:

1. They can provoke as astringent reaction in the mouth and make the food unpalatable

2. They can form complex with proteins, thus recipretate proteins in the GUTs, reducing

the digestibility or inhibiting digestive enzyme function as well as microorganism.

(Mucller Harrey, 2006).

ALKALOIDS

Alkaloids are a group of naturally occurring chemicals compounds that contain

mostly basic nitrogen atoms. Some synthetic compounds of similar structure are also

attributed to alkaloids (Robert, 1998)

Furthermore, In addition to carbon hydrogen, and Nitrogen, alkaloids may also

contain Oxygen, Sulphur and more rarely other elements such as Chloride, bromine and

phosphorus (Leland, 2006). Alkaloids are produced by a large variety of organisms including

bacteria, fungi, plants and are need as medications, as recreational drugs, or in etheogenic

ritual. Most Alkaloids are toxic to their organisms, and in human system almost uniformly

invoke a bitter taste. Alkaloid containing plants have been used by human since antiquity for

therapeutic and recreational purposes. (Essien et al, 2009).

SAPONINS

Saponins are glucosides which consist of polycyclic aglycones attached to one or

more sugar side chains with foaming characteristics.

STRUCTURE OF SAPONINS

CH3 CH3 Sugar

H3C
CH3

CH3
CH2OH
CH3
Sugar-0
 19

The foaming ability of saponins is caused by the combination of a hydrophobic (Fat-

soluble) sapogenin and a hydrophillic (water-soluble) sugar part. Saponins are phytochemical

which can be found in most vegetable, beans and herbs

Saponins have many health benefits studies have illustration the beneficial effects on

blood cholesterol levels, cancer, bone health and stimulation of immune system (Igile, 2006).

Saponins bind with bile salt and cholesterol in the intestinal tract. It causes a reduction of

wood cholesterol by preventing it re-absorption.

CYANOGENIC GLYCOSIDE

Hydrogen cyanide is released from the cyanogenic glycoside when fresh plant

material in macerated as in chewing which allows enzymes and cyanogenic glycoside to

come together releasing hydrogen cyanide as product (Ajayi et al, 2011).

Cyanogenic glycoside is present in a number of food plants and seeds. Cyanide is one

of the most potent, rapidly acting poisons known. It inhibit the oxidative processes of cells

causing them to die very quickly.

Studies have shown that cyanide inhibit the cytochrome oxidase and hydrophenol

oxidase enzyme through combination with copper (Cu) and Iron ions respectively as co-

factors. (Seawright, 2005).

OXALATES

Oxalates (IUPAC: ethandioate) in the dianion with the formula (204 2-, also written

(C00) 2
2-
. Many metal ions form insoluble precipitation with oxalates, a prominent example

being calcium oxalate the primary constituent of the most common kind of kidney stones

(Ajayi et al, 2011). Oxalate occurs in many plants, where it in synthesized via the incomplete

oxidation of carbohydrate.
 20

In the body, oxalic acid combines with divalent metallic cations such as calcium

(Ca2+) and Iron II (Fe2+) to form crystals of the corresponding oxalates which are then

excreted in urine as minute crystals. This oxalate can form larger kidney stones that can

obstruct the kidney tubes. Those with kidney disorders, gout, rheumatoid, arthritis are

typically adused to avoid foods in high oxalic acid.

2.6 NUTRIENT COMPOSITION

Every aspect of the fruit of watermelon has nutritional value, including the seeds and

the rind. Citrillus Lanatus contain about 6% sugar and 92% water by weight. As with many

other fruits, it is a source of vitamin C. the composition of dried egusi seed (watermelon)

without shell per 100g includes; Water – 5.lg, Energy 2340j (557 kcal), Protein 28.3g, Fat

47.4g, Carbohydrate 15.3g, Calcium 54mg, Phosphorus 775mg, Iron 7.3mg, Thiamine

0.19mg, riboflavin 0.15mg, Niacin 3.55mg and Folate 58µg. The seed being an excellent

source of energy and contains no hydrocyanic acid, making it suitable an life stock feed. The

seed oil contains glycosides of linoleic, oleic, palmitic and stearic acids. The fruit flesh

contains bitter cucurbitacins (Schippers, 2002).

The composition of watermelon per 100g edible portion (50-70% of the mature fruits

include: Water 91.5g, Energy 134kj (32kcal) Protein 0.69, fat 0.4g, Carbohydrate 7.2g,

Calcium 8mg, Phosphorus 9mg. iron 0.17g, Thiamine 0.08mg 0.08mg Riboflavin 0.02mg,

Niacin 0.2mg, Folate 2mg, and acorbic acid 9.6mg (USDA, 2002). Watermelon is a rich

natural of lycopene, a carotenoid of great interest because of its antioxidant capacity and

potential health benefit (Rhodes and Zhang, 1999). Cucuribitaceae plants are known to have

bioactive compound such as cucuribitacin, triterpenes sterols and alkaloids. (Yuan, et al,

2006).

Furthermore, amino-acid Citruline has been extracted from watermelon and analyzed

(Wada, 1930) watermelon with red flesh is a significant source of Lycopene (Mandel, et al
 21

2005). Citrillus Lanatus contain polycopene while some varieties (Salmon yellow tangerine

type) contain small amount of polycopene and multiple carotenoids, Lutein and B-carotene.

(Davis, A.R. et al., 2007).

Table 2: Distribution of Nutrients of Citrillus Lanatus (Fresh Fruits)

Nutrient Content Per 100g

Energy 149kj (135kcal Vitamins

Water 93.2g Carotine 440 μg

Protein 0.6g Vitamin E 50 μg

Lipids 0.2g Vitamin B1 45 μg

Carbohydrates 7.7g Vitamin B2 50 μg

Fibre 0.2g Nicotinamide 150 μg

Minerals 0.4Ng Pantothenic acid 1600 μg

Vitamin B6 70 μg

Minerals Folic acid 5 μg

Sodium 1mg Vitamin C 6mg

Potassium 160mg

Magnesium 3mg Lipids

Calcium 10mg Palmitic acid 45mg

Manganese 20-200 μg Stearic acid 9mg

Iron 400 μg Oleic acid 22mg

Copper 70 μg Linolenic acid 25mg

Zinc 100 μg Linoleic acid 40mg

Phosphorus 11mg Salicylic acid – 480 μg

Chloride 8mg
 22

Amino Acid

Fluoride 11 μg Phenylalamine – 10mg

Iodine 1 μg Tryptophan – 7mg

Tyrosine – 10mg

Carbohydrates

Glucose – 1800mg

Fractose – 3500mg

Sucrose – 2400mg

Source: Deutsche Forschungsanstalt fur Stuttgard, 1991).

Table 3: Comparism between percentage content of

watermelon seed and the recommended daily allowance as reported by

Okunrobo, O. Lucky, et al., 2012 on metal analysis quantitative

determination.

CONTENT RESULT RECOMMEDED FEMALES

DIETARY ALLOWANCE IN

MALES (40-50 YRS)

40-50Yrs

Moisture content 4.80 + 0.05

Ash 3.40 + 0.80

Crude protein 21.50 + 0.03 56g/day 46g/day

Crude fibre 2.50 + 0.10 38g/day 25g/day

Crude fat 35.40 + 1.00 20-35% of calories 20-35% of calonies

Carbohydrate 32.40 + 0.70 130g/day 130g/day

Source: Dietary Reference intake: http:llen:Wikipedia.org/wiki/dietary reference intake

(accessed 05/10/11)
 23

PHYSICO-CHEMICAL PROPERTIES OF CITRILLUS LANATUS SEED

Watermelon is a popular fruit in the world and substantial amount of these fruits are

being produced by local fruit farmers in order to fulfill the demand existing in the market.

Watermelon fruits are delicious, with the seeds possibly a good source of either edible or

industrial grade oil. Below are tables showing the physic-chemical properties of water melon.

Table 4: Result of physico-chemical properties of water

melon according to the methods described in ISO and AOAC official

methods and outcome as reported by S.B. Navartne etal, 2014.

The physic-Chemical Properties Watermelon Seed Oil

Melting point 26.1 +0.10C

Specific gravity g 1-cm3 0.91 + 0.01 at 400C

Refractive index 1.68 at 400C

Viscosity 60 at 240C

Smoke point 1720C

Odor Fruity

Acid value 1.2 +0.1

Free fatty acid % 2.38 +0.18

Iodine value 39.5.5+0.5

Peroxide value 2.8 + 0.1

Specification value 148.5 + 0.5

 24

Table 5: Physico-chemical effect of citrillus Lanatus as reported by Arinola,

Stephen Olanrewju and Ogunbusola Enmic, 2013

Physico-Chemical Characteristics (Seed) Citrillus Lanatus

Percentage oil recovered (%) 36.50 + 2.50a

Specific gravity 0.87 +0.01a

Refractive index 1.5/8+ 0.100a

Iodine value (mgf2lg 98.26 + 1.00b

Saponification value 183.73 + 3.10b

Thiobabituric Acid no(Mg MALKG) 2.19+ 3.10b

Free fatty acid no value (%) 5.31+o.50b

Peroxide value (Meq/kg) 0.09+0.01b

From the table, the 36.50% oil recovered from citrillus Lanatus indicates that

watermelon can be a good source of oil.

 25

Table 6: Physico-chemical properties of Citrillus Lanatus

(Egun Melon) seed oil as reported by 0. M Oluba et al 2008 who carried

the study between October and November, 2007. The oil from the seeds

were extracted by Continuous extraction is soxhlet apparatus for 8hr

using petroleum either (60-80co boiling rang) as solvent according to the

method described by AOAC, 1980

Parameters Values

Acid value (Mg kohg-1) 3.5+ 0.3

Iodine value (Mg iodine g-1) 110.0+8.2

Peroxide value 8.3+4.6

Saponification value (Mgkohg-1) 192.0+ 43.7

Colour Yellow

Specific gravity 0.93

Refractive index 1.45

Valued are means + standard deviation of triplicate determination

2.6.1 Lipid Composition of Citrillus Lanatus Seed

The Fatty Acid composition of citrillus Lanatus seed oil is represented below from

the following reports.

S.B. Negative an D. J. S. Subasinghe, (2014) using GCMS

Method report the following as composition of fatty acid in citrillus Lanatus seed oil
 26

Table 7: Composition of Fatty Acid of Watermelon Seed Oil

IUPAA Name General Name % of Fatty Acid

Hexadecanoic acid Palmitic acid 37.3206 453

9,12-octadecandienon acid Linoleic acid 0.42875902

Hepatedecanoic acid Margarine acid 0.681266923

9,12-Octadecanoic acid Lainoleric/Omega6 21.68968234

9,octadecanoic acid Oleic acid/omega 9 3.597835603

Octadecanoic acid Stearic acid 30.38960251

6,9,12 Octadecatrionu Omega 6 0.117120248

Acid

Ricinoleate Omega 9 1.964244198

11-Econcenoic Acid Omeg 9/Gondoic acid 0.80621311

Econcenoic Acid Arachidic acid 1.665230151

18-Methylicosanoic Acid 0.015532032

Bicyclo(2,21) heptanes 0.011194075

2-Acetu Acid

Pentacosanoic 0.12318298

2,6,10.14,18,22-tetra co- o.770608201

Sahexane

Cholesterol 0.06937472

Gama-tocopherol Vitamin E 0.21523869

Hexacosanoic acid 0.134324244

Furthermore, the Citritus Lanatus seed oil lipid contents as reported by Garg V. K and

Mes, W. R. (1986) is said to poses high proportion of A 5- sterols with Codisterol present has
 27

a trace component having 25 (27) – dehydroporiferasterol, 28.7% Clerosterol, Campesterol,

2.9% - 3.12% Isofucosteril 15.5% Sitosterol and 13.9% Stigmaterol.

The total lipid content of Citritus Lanatus seed was found to be 50.23% ie 50.23 +

1.35a under statistical analysis mainly Oleic and Linoleic with fatty acid values to be 2.56%1.

As reported by Anhwange, Benjamin et al, 2010.

2.6.2 Amino Acid Composition Citrillus Lanatus Seed:

The amino acid composition of Citrillus lanatus seed is shown below:

Amino Acid (Essential) as reported by Godwin C. Ojieh et al; 2007

Amino acid Cone (g/100g Protein)

Alanine 2.0

Arginine 5.6

Lysine 9.0

Glycine 0.4

Serine 2.2

Threonine 2.4

Methionine 3.1

Aspartic Acid 0.3

Iso Leucine 16.3

Leucine 4.8

Glutamic Acid 4.2

Proline 16.9

Phenylalanine 3.2

Tyrosine 3.2

Valine 1.3
 28

Cystine 1.1

Amino acid Cone (g/100g Protein)

Alanine 1.31

Histidine 0.78

Leucine 2.12

Lysine 0.84

Isoleucine 0.99

Methionine 0.7

Phenylanine 2.28

Cystine 0.37

Threonine 1.11

Tryptophan 0.32

Valine 1.48

Aspartic Acid 2.88

Glutamic Acid 6.01

Serine 1.43

Tyrosine 10.64

Arginine 4.29

Water melon also has a high content of citrulline

 29

2.6.3 Vitamins, Carboyhdrate and Mineral Composition of Citrullus Lanatus

Seed

Citrillus Lanatus seeds and fruits are highly rich in various vitamins,

carbohydrate and mineral which necessitate their forming part of humans’ diet. There are

highly rich in vitamins such as Vitamins A, B, C etc. their vitamins composition is shown

below:

Vitamins Percentage (100%)

Vitamin A 3%

Vitamin B ranging from 1 – 3%

B1 Thiamine

B2 Riboflavin

B3 Niacin

Pantothenic acid (B5)

Pyridoxine (B6)

Folate (B9)

Vitamin C 14%

Source: Mathias et al., 2001

Furthermore, from Habibur, Rahman et al., 2001 water melon is said to contain

Vitamin C, Caroterwids in the forms Lycopene, Phytofluene, Phtoene, Beta Carotene, Lutein

& Neunesporene.
 30

From the USDA Nutrient Database watermelon seed has the following vitamins;

Carotin 440 μg

Vitamin E 50 μg

Vitamin B1 45 μg

Vitamin B2 50 μg

Nicotinamicle 150 μg

Pantotheric Acid 1600 μg

Vitamin B6 70 μg

Folic Acid 5 μg

Vitamin C 6mg

Source: Deutsche Forsehungsanstalt Fur Lebansmittel chemic

Stuttgant 1991

Mineral Composition

The mineral composition of Citrillus lanatus are thus as reported by Habibur.

Rahman, et al., 2013.

Magnesium - 11.4mg

Calcuium - 16.8mg

Potassium - 7.8mg

Sodium - 5.7mg

Zinc - 1.2mg

Furthermore, Mathias et al., 2001 reported the following as mineral content of

Citrillus lanatus seed with respect to %.

Calcium - 1%, Potassium - 2%, Iron – 25%

Magnesium – 3%, Phosphorous – 2%, Zinc – 1%

 31

From the reports of Erhirhic, E. O and Ekene, N. E. 2013, watermelon seed was found

to posses the following mineral I milligram concentration.

Calcium – 8mg, Phosphorous – 9mg, Iron – 0.17mg

From the report of Godwin et al., 2008. Compositional studies, watermelon has the

following minerals:

(Na) Sodium (13.0 + 0.2)

(Ca) Calcium (28.2+0.2)

(K) Potassium (96.1+0.2)

(Mg) Magnesium (31.4+0.2)]

(Mn) Maganese (1.7+0.1)

(Cu) Copper (0.4+0.1)

(Fe) Zinc (1.2+0.1)

(P) Phoshorous (125.3+3.1)

From the available literature regarding mineral content of Citrillus lanatus seed; it can

be deduced that the seeds are rich in the following minerals potassium, calcium, phosphorus,

magnesium which are of relative health benefits.

Carbohydrate Composition

The carbohydrate composition of Citrillus lanatus seed is said to contain fructose,

glucose and carbohydrate composition of Citrillus lanatus based on Habibur, Rahman, etal.,

2013 publication shows that:

Fructose - 3500mg

Glucose - 1800mg

Sugar cane - 24000mg. in the same vein, Citrillus lanatus was said to contain

7.2g of carbohydrate by the publications of Erhirhic, O. E. and Ekene N. E. (2013).

2.7 Uses of Citrillus Lanatus

 32

2.7.1.1 Ethnomedicinal Uses/Relevance

With reference to the publication of Erhirhic E. O. and Ekene, N. E. on medicinal

values of Citrillus lanatus seed, 2013, Citrillus lanatus fruits is eaten as a febrifuge when fully

ripe or even when putrid (Grieve and Leyel, 1984). The root of Citrillus lanatus is a purgative

and is high dose can also serve as an Emetic (Grieve and Liyel, 1984). The seed is demulcent,

pectoral and tonic (Duke and Ayensu, 1985). Most often at times, Citrillus lanatus is

employed in the treatment of urinary tract infection as well as bed wetting (Moerman, 1998).

The Citrillus lanatus seed is a good and effective vermifuge having a hypotensive

action. Fatty oil in the seed from aqueous or alcoholic extracts, had been reported to paralyze

tapeworms and roundworms (Chopra, 1958). The fruit rind is usually prescribed in cases of

alcoholic poisoning and diabetes (Duke and Ayensu, 1985).

In Northern part of Sudan, Citrillus lanatus is used in treatment of burns, swellings,

rheumatism, gout and as laxative (Schippers and Budd, 1997), whereas in Sengal, the fruits

are drastically used as purgative. In Nigeria, Citrillus lanatus is used in treatment of diarrhea

and gonorrhea. Tar is extracted from the seed and used for the treatment of scabies and skin

tanning (Schippers and Budd, 1997). The seed oil has an antihelmintic action which is better

than that of pumpkin seed oil (Jackson, 1990).

2.7.2 Medicinal Uses Cum Bioactivities of Citrillus Lanatus

The use of herbal products is of global importance because of their low side effects,

accessibility and affordability when compared with conventional medicine Citrillus Lanatus

is popular in indigenous system of folk medicine and it is known to contain bioactive

compounds such as cucurbitaim, triterpenes, sterols and alkaloids, vitamin, minerals.

Traditionally (Trado-medically), Citrillus Lanatus has been reportedly used as purgative and

emetic in high dose, vermifuge, demulcent, diuretic and tonic. The seeds have been
 33

implicated in the treatment of urinary track infection, bed welling, renal stones, alcohol

poisoning, hypertension, diabetic, diarrhea and gonorrhea.

Furthermore, current medicinal uses of Citrillus Lanatus are illustrated below with

respect to their Bioactivities of the plant secondary metabolites

Bioactivity of Citrillus Lanatus:

 Anti-inflammatory activity

 Anti-Gardial

Anti-Inflammatory Activity

The anti-inflammatory activity of Citrillus Lanatus seed as reportedly published by

Erhuhie, E.O. and Ekene, N.E with reference from (Madlavi, etal, 2012) show that inutro and

in-vivo inflammatory activity of citrillus Lanatus seed oil in carrageenan induced paw edema

in rat model. The potency of the oil compared with standard diclofenac (10mg/kg) showed

significant reduction of edema in paw induce rat model maximum at 3hr (percentage reducing

in paw volume 44.44%, 55.56% and 63.11% for Citrillus Lanatus seed oil (50mg/kg), citrillus

Lanatus seed oil (100mg/kg) and diclofence (10mg/kg) respectably and citrillus Lanatus seed

oil at concentration 42.35%, 68.48% and 78.50% protection of HRBC in hypotonic solution

respectably.

Anti-Giardial Activity

An in-vitro antigiardial activity of Citrillus Lanatus fruits, as well as cucurbitacin E

and L 2-0- - glucoside pure isolated compounds from C. Lanatus was carried out cucurbitain

E and L 2-0- glucoside were revealed to have strong potent antigiardial activity against

Giardia Lamblia in vitro with Ic50 = 2 and 5ng/ml after 5 days respectively. The finding

suggested that with the crude extract is petroleum either, ethyl acetate etc along with other
 34

isolated compounds were active against Giardia Lamblia hence the citroides of Citrillus

Lanatus being recommended as new source for treatment of Giardiasis. (Loiy, etal, 2011).

Other medicinal use of Citrillus Lanatus include the following:

Anti-oxidant usage

Anti-prostatic power usage

Anti-ulcerogenic usage

Hepato-protective usage

Cardiac protective effect due to its rich source in lycopene

Laxative usage

It use in diabetes treatment amongst other uses.

 35

CHAPTER THREE

METHODOLOGY

RESEARCH DESIGN

A total of 24 female wistar rats were used for the study, and the rats were randomly

selected into four (4) groups of six (6) animals each. The average weight of rats in each group

was determined.

The grouping is as follows:

GROUP 1 - CONTROL GROUP

GROUP 2 - EXPERIMENTAL TEST GROUP

GROUP 3 - EXPERIMENTAL TEST GROUP

GROUP 4 - EXPERIMENTAL TEST GROUP

The administration lasted for a period of 21 days, after which the animals were

sacrificed. The experimental procedures used in this study are approved by the animal care

committee of College of Health Science, University of Uyo, Uyo, Akwa Ibom State, Nigeria.

In the course of the administration, the animals in various groups received extracts

(seed oil of C. Lanatus) based on their body weight, in kilograms, as shown below:

Group Body Weight/ml/kg Feeding type

GROUP 1 CONTROL No extract will be given Normal feed + water

Group 2 0.2ml (extract) =932.0mg/kg body Feed + water+ extract

weight (0.2ml)

Group 3 0.4ml(extract) = 1864.0mg/kg body Feed + water+

weight extract(0.4ml)

Group 4 0.6ml(extracts) =2796.0mg/kg body Feed + water +

weight extract(0.6ml)
 36

The administration lasted for 21 days, body weight of rats were recorded on alternate

days after which, the animal were sacrificed and appropriate procedures for evaluation of

lipid profile, hematological indices, liver enzyme, creatinine, and electrolyte along with

Histopathological studies of liver and kidney employed.

APPARATUS AND EQUIPEMENT

MANUAL GRINDER

WEIGHING BALANCE

CENTRIFUGE

SPECTROPHOTOMETER

RAT CAGES

SAMPLE BOTTLES

PIPPETE

CONICAL FLASK

DISSERTING SET

BEAKER

CUVETTE

SYRINGE

NEEDLE

TEST TUBE

TEST TUBE RACK

WATER BATH

SPATULA

EMBEDDING OVEN

EMBEDDING CASETTE

EMBEDDING MOULD
 37

MICROTOME

MICROSCOPE

PLANT MATEERIAL COLLECTION/SEED OIL EXTRACT

The plant material used for this study is watermelon (C.Lanatus) seeds. The water

melon seeds were taken from matured, fresh water melon fruits purchased from Mbak Itam

market in Uyo L.G.A of Akwa Ibom State, Nigeria.

The seeds were washed and sun dried for a period of about 1 week. After drying, the

seeds were stored in a dry place to avoid fungal growth which may contaminate the seed,

hence yielding poor result. The seed thereafter were grinded with a manual laboratory grinder

in the Department of Biochemistry Laboratory, University of Uyo, Uyo.

Chloroform extraction was performed (in a fume cupboard) by adding 200ml

chloroform to the sample (grinded C.lanatus seeds). The resulting mixture was mashed very

well before filtration with cheesecloth and later with white filter paper to obtain a clear

filtrate. The filtrate was then placed in a water bath at temperature 60˚c to evaporate. The

evaporated extract (oil) was then decanted into a clean container closed tightly and

refrigerated till it is read for use.

ANIMAL TREATMENT

Twenty Four (24) female albino rats of the Wistar strain with weight range between

179g to 257g were used in this study. The animals were obtained from the Animal House,

Faculty of Basic Medical Science, University of Uyo, Uyo, Akwa Ibom State. The animals

were randomly selected into Four (4) groups of Six (6) animals each.

The animals were acclimatized for a period of One (1) week; and were given rat mash

and water ad libitum. After acclimatization period, the rats were subjected to different
 38

dosages of the seed extract (seed oil) based on their body weight with Group 1 (control)

animals receiving an equivalent volume of distilled water as placebo.

The experimental test groups 2, 3 and 4 received 932.0mg/kg (0.2ml seed oil extract),

1864.0mg/kg (0.4ml seed oil extract) and 2796.0mg/kg (0.6ml seed oil extract) respectively

for a period of twenty one (21) days via oral gavaging. All animal experiments were carried

out in line with the guidelines of Institutional Animal Ethical Committee as approved by the

Post Graduate School, University of Uyo, Uyo, Akwa Ibom State.

ANIMAL SACRIFICE AND PREPARATION OF SERA FOR ANALYSIS

At the end of the extract administration (21days), all the experimental animals were

fasted overnight and anaesthetized by dropping each animal in a transparent glass chamber

saturated with chloroform fumes. Incision were made on the thorax and blood samples were

collected by cardiac puncture into sterile plain tubes for sera preparation and anticoagulant

(EDTA 0.77M) bottles were used for whole blood for analysis.

Serum samples were obtained from the clotted blood into sterile plain tubes after

centrifugation at 2000 rpm for 10 min using a bench top centrifuge (MSE Minor, England).

The sera were stored in the refrigerator for analysis. All biochemical analysis were carried

out within 72hours of sample collection

TISSUE PROCESSING FOR HISTOLOGICAL STUDIES: HAEMATOXYLIN AND

EOSIN (CLOE, 1943)

 The liver and kidney were carefully dissected out, trimmed of all fat and blotted dry to

remove any blood. They were weighed and volume determined by water displacement

and the fixed in neutral buffer formalin (fixation). The fixed tissues were transferred

to a graded series of ethanol (dehydration). On day one (1), they were placed in 70%

alcohol for 7 hours, then transferred to 90% alcohol and left in the latter overnight. On
 39

day two (2), the tissue were passed through three changes of absolute alcohol for an

hour each then cleared in xylene (clearing). Once cleared, the tissues were infiltrated

in molten paraffin wax in the oven at 58˚c. Three intervals were made, after which the

tissue were embedded (embedding) in wax and blocked out. Prior to embedding, it

was ensured that the mounted section to be cut by the rotary microtome were oriented

perpendicular to the long axis of the tissue. The sections were designated "vertical

sections". Serial section of 5µm thick were obtained from a solid block of tissue

(microtomy) fixed on clean albuminized slides to prevent sections from pulling off the

slides and later stained with Haematoxylin and Eosin staining techniques, after which

they were passed through graded doses of alcohol, cleared in xylene, and mounted in

DPX moutant and allowed to dry at room temperature and observed Histopathological

under digital light microscope.

BIOCHEMICAL ANALYSIS

ESTIMATION OF TRIGLYCERIDE ASSAY PRINCIPLE

This involves hydrolysis of triglycerides by Lipase. The glycerol concentration is then

determined by enzymatic assay coupled with Trinder reaction that terminates in the formation

of a Quinoneimine dye. The amount of the dye formed determined by its absorbance at

520nm, is directly proportional to the concentration of triglycerides in the sample.

Triglycerides Lipase Glycerol + fatty acds

Glycerol + Atp glycerol Kinase Glycerol-1_phosphate+ Adp

Glycerol-1-phosphate + 02 G-1-Phosphate Oxidase DAP +H202

H2O2 + 4-aMINOPHENAZONE + 4CHLOROPHENOL PEROXIDASE Quinonemine

 40

ESTIMATION OF TOTOAL CHOLESTEROL

ASSAY PRICNCPLE

The cholesterol is determined after enzymatic hydrolysis and oxidation. Hydrogen

peroxide is then produced from the oxidation of cholesterol by cholesterol oxidase.

Quinoneimine dye is formed from hydrogen peroxide and 4-aminoantipyrine in the presence

of phenol and peroxidase. The absorbance at 556nm of the solution of this dye is proportional

to the concentration of the cholesterol in the sample.

The enzymatic reaction sequence employed in the assay of cholesterol is as follows;

Cholesterol esters Cholesterol esterase Cholesterol + Fatty acids

Cholesterol + Cholesterol Oxidase Cholestene-3-one + H202

2H2O + 4-Aminoantipyrine + Phenol Peroxidase Quinoneimine dye+4H20

ESTIMATION OF HDL-CHOLESTEROL

ASSAY PRINCIPLE

After centrifugation, the cholesterol level in the High Density Lipoprotein fraction

(HDL), which remains in the supernatant, is determined by the same method as in total

cholesterol above

ESTIMATION OF LOW DENSITY LIPOPROTEIN (LDL)

Low Density Lipoprotein-CHOLESTEROL (LDL-c) and Chylomicron fractions are

precipitated quantitatively by the addition of Phosphotungstic acid in the presence of

magnessium ion. Low density lipoprotein cholesterol was obtained by calculation using the

formula provided in Randox HDL-cholesterol Kit booklet:

LDL-CHOLESTERIL (mg/dl) = TC -TG⁄5 - HDL-cholesterol.

 41

ESTIMATION OF ALANINE AMINO TRANAMINASES (ALT)

The enzyme alanine amino transferase is widely reported in a variety of tissue

sources. The major source of ALT is of hepatic origin and has led to the application of ALT

determination in the study of hepatic disease.

Levels of ALT are only slightly elevated in patients following a myocardiac infarction

(Henry, J. B., 1974). Ultraviolet (uv) method for ALT determination were first developed by

Wroblewski and Ladue in 1956. The method was based on the oxidation of NADPH by

lactate dehydrogenase (LDH).

PRINCIPLE

The enzymatic reaction sequence employed in the assay of ALT is as follows:

L-Alanine + α-ketoglutarate ALT Pyruvate + L- Glutamate

Pyruvate + NADH + H+ LDH LACTATE +NAD+ H2O

The pyruvate formed in the first reaction is reduced to lactate in the presence of

lactate dehydrogenase and NADH. The activity of ALT is determined by measuring the rate

of oxidation of NADH at 340nm. Endogenous sample pyruvate s converted to lactate by

LDH during the lag phase prior to measurement.

ESTIMATION OF ASPARTATE AMINO TRAANSFERASES (AST) PRINCIPLE

Aspartate amino transferase (AST) catalyzes the transfer of the amino group from

Aspartic acid to α-ketoglutaric acid. This enzyme is found in practically every tissue of the

body, including red blood cells. It is in particular high concentration in the liver, heart, and

skelectal mucsls.

Acute destruction of tissue results in the release of AST into the blood stream.

Following myocardiac infarction there is a significant increase in serum AST activity in

about 6 to 8 hours with peak values reached after 48 to 60 hours. However, serum ALT
 42

activity remains within normal limits or only marginally increased. In hepatitis and other

forms of liver disease associated with hepatic necrosis, both ASTY and ALT are elevated.

Elevated levels of serum AST activity are also observed in infectious mononucleosis,

muscular dystrophy, dermatomyositis, and in other forms of muscle and liver injury.

The method presented here is an UV-kinetic method based on the rate of NADH

oxidation in a coupled Malic dehydrogenase reaction. The TC matrix system analyzer system

automatically proportions the appropriate sample and reagent volumes into the cuvette. The

system monitors the change in absorbance at 340nm. This change in absorbance is directly

proportional to the activity of aspartate amino transferase in the sample and is used by the TC

matrix system to calculate and express aspartate aminotransferas activity.

L-ASPARTATE + α-Ketoglutarate AST Oxaloacetate + L- Glutamate

Oxaloacetate + NADH + H+ MDH L-Malate + NAD+

ESTIMATION OF ALKALINE PHOSPHATASE

ASSAY PRINCIPLE

Alkaline phosphatase is determined by measuring the rate of hydrolysis of various

phosphate esters. P-Nitrophenyl phosphate is one such ester that was used as a substrate by

FUJITA in 1939. (Fujita, H., 1939). The present method is a modification of the reference

method of the above committee and the American association for clinical chemistry. (Tietz,

N.W 1983)

PRINCIPLE

The enzymatic sequence employed in the assay of Alkaline phosphatase is as follow:

P-Nitrophenyl Phosphate (P-NPP) + H20 Alk. phosphatase

P-Nitrophenol + H3P04
 43

P-NPP is colourless but P-Nitrophenol has strong absorbance at 405nm. The rate of

increased absorbance at 405nm is proportional to the enzyme activity.

ESTIMATION OF SERUM CREATININE

Creatinine, an anhydride of creatinine is a waste product formed by the spontaneous

dehydration of kidneys. (Henry, J.B.1974). Most of the creatinine is found in muscle tissue

where it is present as creatine phosphate and serve as a high-energy storage reservoir for

conversion of ATP.

Independent of diet, serum creatinine concentrations depends almost entirely upon its

excretion rate by the kidneys. The assay of creatinine has been based on the reaction of

creatinine with alkaline picrate as described by Jaffc. In the endpoint method, acetic acid is

used to destroy the creatinine picrate complex, resulting in a loss of colour, the non creatinine

serum constituents retain their picrate derived colours and thus, the differences in

absorbencies gives the creatinine concentration. (Heinegard, D and Tiderstrom, G., 1973)

PRINCIPLE

Creatinine + sodium picrate alkali creatinine picrate complex (yellow orange)

Creatinine reacts with picric acid in alkaline conditions to form a colour complex,

which absorbs at 510nm. The rate of formation of colour is proportional to the creatinine

concentration in the sample. In the end point method the difference in absorbance

measurement after colour formation yields a creatinine value corrected for interfering

substances.

DETERMINATION OF ELECTROLYTE

The electrolytes bicarbonates ion (HC03-), sodium ion (Na+), potassium ion (K+) and

chloride ion (Cl-) was done suing an electrolyte analyzer called Landwind LW E60B.
 44

STATISTICS

Statistical analysis was carried out using window SPSS. One way analysis of variance

was adopted for comparison and the results were subjected to a student T test. The data were

expressed as mean± standard deviation and values of p<0.05 were considered significant.