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1.0 CHAPTER ONE

1.1 Introduction and literature review

Phoenix dactlifera L., date palm, is among the most important species in the palm family

(Arecaceae), which encompasses about 200 genera and more than 2,500 species (El-Hadrami and

El-Hadrami, 2009; Jain and Johnson, 2011). The specie name was inspired by the finger like

shape of the fruit and the genus from the legendary bird of ancient Greece (El-Hadrami and

Jameel Al-Kayri 2012). It is a long lived monocotyledonous specie and one of the tallest

domesticated tree. This perennial and dioecious species represents a cornerstone of the economy

in many producing countries, especially in North Africa and Middle East (El-Hadrami and

Jameel Al-Kayri 2012) Over 100 million trees are currently grown worldwide on an estimated

area of 1 million ha. Date palm provides fruit, fuel, fibre and shade for other essential cover

crops. Date fruit is a highly nutritious food product, rich in simple sugars such as glucose and

fructose (65% - 80%), a good source of fibres and some essential minerals and many vitamins

but low in fat and protein with no starch. In date fruits, proteins are in the range of 1-3% and

even though their amino acid pattern is favorable and per human needs. Date seed (pits)

constitute approximately 10% of the fruit on the average date seeds contained 5-7% protein, 7-

10% fat, 10-20% Fibre, 1-2% ash, and 55-65% Carbohydrate on a dry weight basis. Mineral

analysis showed higher concentration of K followed by P, Mg, Ca, and Na. Commonly Date

palm pits are used as animal feed as they are rich in tannins and resistant starch in all parts of the

Date palm, apart from the roots which are used for a purpose best suited to them.

Diabetes is a group of metabolic disease in which a person has high blood sugar, either because

the pancrease does not produce enough insulin\, or because cell do not response to the insulin

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that is produce. There are three types of diabetes; Type one which results from the body’s failure

to produce insulin. Type two which result from the insulin resistance. Type three (gestational

diabetes) occurs when pregnant women without a previous diagnosis of diabetes develop a high

blood glucose level.

1.1 Overview of Date medjool

The characteristic of Date medjool palm can be truly called ‘tree of life’ and is distributed

throughout the Middle East, North Africa, South Sahel, areas of East and South Africa and even

certain parts Europe and USA. The fruit plays important roles in nutrition especially of human

population. Several products are made from the fruit which can generate employment and thus

influence socio- economic aspect of the life of people. This crop has a great potential as a source

of renewable energy, an alternative source of fossil energy, by producing bio-fuel since the fruits

are high in carbohydrate containing about 44-88% total sugars. The most basic way to grow a

date medjool palm is to plant a pit and wait for the pit to sprout, it can take up to 20 years for a

sprouted pit to yield a tree. Date palms that give rise to medjool fruit are believed to be

indigenous to the North African coast and Arabic Peninsula, and the land between this countries

remains the primary growing area (Mohan Jain 2012).

1.1.1 Geographical distribution of Date palm

Phoenix dactylifera is a widely distributed species occurring in diverse geographic, soil and

climate areas (El Hadrami et al., 2011a). the vast majority of the tree are located in the Middle

Eeast and North African although the crop has been established in California, Arizona and

Mexico in the Americas (Emirate Journal. 2012). The common requirement among the date palm

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growing area is the high temperature (35oC). Date palm grows in nearly rainless regions at 9-39o

North latitude, which are represented by Sahara and Southern fringe of Near East (Emir. J 2012)

1.1.2 Botanical calssification

Date medjool is classified according to Dransfield and Uhl, (1986) as follows

Kingdom: plantae

Group: Spadiciflora

Order: Palmea

Family: Palmaceae

Sub family: Coryphyoideae

Tribe: Phoeniceae

Genus: Phoenix

Species: Dactylifera L.

1.2 Chemical composition of Date palm.

Dates has been shown to contain calcium, magnesium, phosphorus, potassium, iron, zinc,

copper, manganese, selenium, vitamins A, A1, B, B1, B2, B3, B5, B6, and C as well as a variety

of amino acids (El-Hadrami, Al-Kayri and Jameel 2012). Dates also contain thiamine, riboflavin,

niacin, and pantothenic acid. These vitamins and minerals help the body produce haemoglobin,

which is a protein in red blood cells that binds to oxygen and carries oxygen from the lungs to

tissues (Emirate Journal. 2012). Potassium is an essential mineral that the body needs to maintain

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proper muscle contractions including contractions of the heart muscle. Potassium also promotes

a healthy nervous system and efficient metabolism in the body. One serving of Dates contains

240 milligrams of potassium, which is more than the amount of potassium found in bananas.

1.2.1 Carbohydrate

Dates also contain carbohydrates that include 3 grams of dietary fibre and 29 of naturally

occurring sugars such as fructose, glucose, and sucrose. In other words, one serving of Date

contains 31 of carbohydrates, which supplies the body with large amounts of energy. Dates in

addition to being a good source of dietary fibre are sodium free, fat free, and cholesterol free.

Each of these factors are important for reducing the risk of developing heart disease and cancer.

The fibre found in Dates comes in two forms, soluble and insoluble. Soluble fibre have been

shown to help control diabetes by decreasing high blood sugar as well as lowering high

cholesterol, specifically low density lipoprotein (LDL) cholesterol. Insoluble fibre increases the

body’s ability and rate at which food is processed through the digestive system.

Moreso, Dates have phenolic acids like gallic acid, protocatechuic acid, caffeic acid, p-coumaric

acid, o-coumaric acid, vanillic acid, syringic acid, and ferulic acid. However, a sufficient amount

of carotenoids and antioxidants is lost when Dates are sun-dried.

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1.3 Phenolic content and other secondary metabolite

In Dates, three main families of phenolics (hydroxycinnamates;flavonols, flavan-3-ols, flavan-

3,4-diols, proanthocyanidines) can be detected (El-Hadrami et al.,1998; Daayf et al., 2003; El

Hassni et al., 2004; J’Aiti et al., 2009).

Mansouri et al. (2005) and Biglari et al. (2008a) reported that the total phenolic content ranged

from 2.49 to 8.36 mg gallic acid equivalent per 100g of fresh weight of Algerian and Iranian

dates, respectively. A number of factors affect phenolic content in Date and may justify the

variations observed in the different studies. These include the cultivar, geographic origin,

growing conditions, maturity of the tested Dates, season fertilizers, soil types, amount of sunlight

received and condition of storage, sampling and extraction among others. Dates are rich in

carotenoid and provitamin A. Boudries et al. (2007) analyzed the content of these two

components in cvs. The major carotenoid pigments detected were lutein and beta carotene.

Variation in total carotenoid content were detected among cultivars in different ripening stages.

Provitamin A. value also varied with cultivar and ripening stage (El-Hadrami, and Jameel, 2012).

1.4 Nutritional value of Dates

Dates represent an important nutritional element in the diet of local populations where the trees

are grown. Dates contain a high percentage of carbohydrate (total sugar, 44-88%), protein (2.3-

5.6%), fats (0.2-9-3%), essential salts and minerals, vitamins and an elevated proportion of

dietary fibre(6.4-11.5%) (El Hadrami and El Hadrami, 2009). They also contain oil in the flesh

(0.2-0.5%) and the seed (7.7-9.7%). The seed represent the entire fruit weight.

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1.4.1 Carbohydrates, proteins and fats

Dates are particularly rich in sugars, especially glucose, fructose, mannose, maltose and other

non-reducing sugars such as sucrose. Glucose and fructose ratio varies between 1 and 2

depending on the cultivar and the ripening stage. A small amount of carbohydrate found in Dates

is represented by polysaccharides such cellulose and starch (Shinwari, 1993).

Deglet Noor and Allig showed the presence of 17 different amino acids, including glutamic acid

that is the foremost in the seeds,representing 17-18% of total amino acids. Other essential amino

acid detected in the seed include lycine, isoleucine, leucine, methionine, threonine, valine and

phenylalanine.

Fatty acids occur in both the flesh and seed of Dates as a range of saturated and unsaturated

acids. In seeds, at least 14 different fatty acids were detected, while in the flesh only occurred at

very low concentrated.

1.5 Health benefits of dates

Natural dietary antioxidants from fruit such as Dates are believed to activate the enzymatic and

non-enzymatic antioxidant system (El Hadrami et al., 2005). Epidermiological evidence suggests

that a diet rich in fruits and vegetables promotes a lower incidence of chronic disease such as

cancer, cardiovascular disorder and diabetes (Block et al., 1996; Tapiero et al.,2002; Duthie et

al., 2003). Phenolics account for most of antioxidant properties. The direct of phenolics as free

radical, prevents nuclei acids, proteins and lipids oxidative damage (Gotz et al., 1996; Rice-

Evans et al., 1997; Offen et al., 1998; Morel and Barouki, 1999; Jakus, 2000; Droge, 2002).

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1.5.1 Anti-microbial properties of Date medjool

Date being rich in phenolics, are known to exhibit anti-viral, anti-bacteria and anti-fungal

properties, making them a remedy for certain diseases and prevention of chronic inflammations

(El-Hadrami and Jameel M. 2012) The fruit and its by-products are rich in dietary fibers,

selenium, carotenoids, ascobate, and other essential antioxidant which may prevent the oxidative

damages caused as a result of lymphocytes phagocytosis activity of invasion pathogens and pest

(El-Hadrami and Jameel, 2012)

1.5.2 Anti-tumoral and anti-ulcer properties Date medjool

Phenolics were shown to be responsible for the decrease in carcinogenic potential resulting from

mutagen exposure (Bravo, 1998). Phenolics such as caffeic and ferulic acids, highly present in

dates, are known to react with nitrite and inhibit the in vivo formation of nitrosamine, hence

inhibiting skin tumours (kaul and khanduja, 1998). Yoshida et al (1990) examined the effect of

quercetin which is one of the flavonoid commonly found in Dates and in cell growth of human

malignant cell derived from gastro-intestinal tract and on cell cycle progression result that

quercetin inhibit the growth of human gastric cancer cells with an IC50 value of 32-

55micrometer.

1.5.3 Immune-modulatory properties of Date medjool

Dates being rich in both phenolics and dietary fibres can play an important role in the modulation

of the immune system and prevention of cardiovascular diseases (El-Hadrami and Jameel, 2012).

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Lower incidences of cardiovascular disorders are expected in populations relying on a regular

intake of Dates. This is believed to occur through the inhibition of the oxidation of low-density

lipoprotein (Frankel et al., 1993) and through the prevention of platelet aggregation (El-Hadrami

and Jameel, 2012). Phenolics contained in Dates may also be able to reduce blood pressure and

have antithrombotic and anti-inflammatory effects as shown for other fruits (Gerritsen et al.

1995; Muldoon and Kritchvesky, 1996). In addition, phenolics are known to inhibit α-amylase

and α-glucosidase activities behind the postprandial increase in blood glucose level, often

manifesting in type-II diabetes (Andlauer and Furst, 2003; McCue and Shetty, 2004).

The immune modulatory activities of phenolics derived from Dates include anti-allergic

(Noguchi et al., 1999) properties able to suppress the hypersensitive immune response. It also

includes anti-inflammatory responses triggered by the suppression of the tumor necrosis factor-

α- mediated pro-inflammatory pathways (Ma and Kinneer, 2002).

1.6 BIOCHEMICAL PARAMETERS

. Biochemical parameters are measurable biological factors required in understanding the

pathological implications of each abnormal result in a biological experiment. Together with the

normal results these form a pattern which reflects one or more underlying disease process( ).

Investigative biochemical profiles are designed to provide all the data necessary for a broad

investigation of internal abnormality. Parameters with limited data are best used for monitoring

an established diagnosis for which the results of a more wide ranging profile have already been

obtained. Individual biochemical evaluations may be used, for example, for therapeutic drug

testing (phenobarbitol, bromide, digoxin)( ), Here we look at some enzymes that are subjected to

oxidative stress when attacted by some reactive oxygen specie.

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1.7 The Liver

Liver is a self regenerating organ that plays important roles in the body. It functions not only in

metabolism and removal of exogenous toxins and therapeutic agents responsible for metabolic

derangement but also in the biochemical regulation of fats, carbohydrates, amino acids, protein,

blood coagulation and immunomodulation function (Ram and Goel, 1999). Due to its ability to

regenerate, even a moderate cell injury is not reflected by measurable change in its metabolic

function. However, damage caused by lipid peroxidation on the membrane of the hepatocytes

allows the leakage of some cytosolic enzymes of the liver into the blood stream (Plaa and Hewitt,

1982). An elevation in the liver enzymes such as alanine transaminase, aspartate transaminase

and alkaline phosphatase, into the blood stream is an indication of damage on the liver . These

enzymes are elevated to distinguish and assess the extent and type of hepatocellular injury (Ram

and Goel, 1999

1.7.1 Assessing Liver Function

There are several enzyme markers that come with the routine blood chemistry test. One of them

is called Alkaline Phosphatase (ALP). It is very specific for liver disease and if it is elevated at

all, it indicates a problem that needs further evaluation. Another enzyme is called Alanine

Transferase (ALT) ( ). It can be elevated because of a variety of reasons and we want you to

understand its significance. ALT is released from the liver cell when the cell swells from being

tweaked by anything that causes it to be inflamed. Any time the body is dealing with

inflammation anywhere, the ALT can go up ( ).

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1.7.2 Aspartate aminotransferase (AST) is present in many tissues and is useful in evaluating

muscle and liver damage in small and large animals. AST is not liver specific in any domestic

animal species and the reference range in horses is rather broad. Skeletal muscle is the second

largest source of AST in animals. It is an absolute prerequisite to eliminate extrahepatic tissue

damage as a possible source of serum AST when evaluating the enzyme in relation to the liver.

In combinations with the physical examination and history, the evaluation of other serum

enzymes should aid in differentiating the source of increased AST levels. AST is present in both

the cytoplasm and mitochondria of hepatocytes (and many other cells) and will elevate in states

of altered membrane permeability. In such cases, levels are expected to be less than in states of

frank necrosis, when both cytoplasmic and mitochondrial enzymes are released.

1.7.3 Alanine aminotransferase (ALT) is considered to be liver specific in small animals. This

enzyme is present in high concentrations in the cytoplasm of hepatocytes. Plasma concentrations

increase with hepatocellular, damage/necrosis, hepatocyte proliferation, or hepatocellular

degeneration. ALT is a cytoplasmic enzyme, and is considered to be liver specific in primates

and some other small animal species. There is little hepatic ALT activity in large domestic

animals. Thus, further comments regarding ALT will relate only to dogs and cats.

Elevation of serum levels of both AST and ALT can occur with states of altered hepatocellular

membrane permeability. Because ALT is located only in the cytoplasm, serum levels tend to be

relatively higher than AST, as a result of membrane leakage from the hepatocyte( ).

Mitochondrial enzymes are less likely to be released with most of the conditions which result in

increased membrane permeability. Many causes of altered membrane permeability are

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potentially reversible but some may progress to hepatocellular necrosis which is essentially an

irreversible change. Causes of increased cell membrane permeability include:

Anoxia/circulatory hypoxia

The magnitude of both AST and ALT elevations in serum is generally related to the number of

hepatocytes affected.

1.7.4 Alkaline phosphatase (ALP)

The alkaline phosphatases are a group of enzymes which catalyze the hydrolysis of a phosphate

group from an organic molecule at an alkaline pH. They are called isoenzymes because they

catalyse the same reaction in the same species but have different biochemical properties ( ).

ALP is primarily bound to cell membranes. The physiological functions of these isoenzymes are

not fully understood although recent information suggests that one of the biological roles of ALP

is detoxification of endotoxin. ALP is found, to some extent, in all tissues and is relatively stable

in serum. However, only a few organs actually contribute to the circulating enzyme level.

An elevated alkaline phosphatase concentration is generally due to cholestasis in most adult

domestic animals ( ). A mild elevation in immature animals is likely to be the result of normal

bone growth. In dogs, when an elevated ALP value is seen, liver disease, Cushings disease, and

recent steroid therapy should all be considered. Prolonged steroid therapy resulting in iatrogenic

Cushings disease can be diagnosed on the basis of low pre and post ACTH cortisol levels.

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The liver isoenzyme will be elevated in any active liver disease. In acute hepatocellular necrosis,

ALT, AST and GLDH are markedly elevated while ALP is only minimally elevated. Intrahepatic

and extrahepatic biliary obstruction causes more dramatic elevations of ALP, which in some

cases can be 10-20 times the normal level ( ). This is due to recycling as well as increased

synthesis of the liver isoenzymes. Extrahepatic biliary obstruction can be caused if the hepatic or

common bile duct is obstructed either partially or completely. Possible causes include tumour,

granulomatus inflammation, abscesses, pancreatitis and duodenitis.

The anticonvulsant drugs phenobarbitol, diphenyl hydantoin (phenytoin) and primidone can

cause minimal to marked elevations of the liver isoenzymes in dogs. The activity of ALT is also

usually increased in such situations. In cats, the liver contains much less ALP per gram of tissue

than dogs, and it is cleared from serum much more rapidly ( ). This causes the normal value to be

lower than in dogs, and mild elevations can be significant.

Elevations of the bone isoenzyme can be seen in young animals as a result of normal bone

growth, and occasionally with bone tumours. These elevations are usually minimal and are

seldom more than 2-3 times the normal value ( ).

1.8 Kidney

Kidney are a pair of organs located in the back of abdomen and serve several essential regulatory

role in most animals including vertebrate and some invertebrate. They are essential in the urinary

system and also serve homeostasis function such as regulation of electrolyte, maintenance of

acid-base balance and regulation of blood pressure (Wikipedia atom feed). They serve the body

as a natural filter of the blood, and remove waste which are diverted to the urinary bladder. In

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producing urine, the kidneys excrete waste such as urea and ammonium, and they are also

responsible for the absorption also produce hormone including calcitroil, erythropoietin and the

enzyme renin.

1.8.1 Function

The kidney participates in whole body homeostasis regulating acid-base balance, electrolyte

concentration extracellular fluid volume and regulation of blood pressure, is also used to filter

the blood, as the kidney filter blood, they create urine, which collects in the kidney funnel-shape

structure that drain down tubes called ureters to the bladder.

1.9 Diabetes

Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from

defects in insulin secretion, insulin action, or both (American Diabetes Association, 2000; Wilds

et al., 2004). Insulin is a hormone synthesized by the beta cells of the pancreas, which is

required to utilize glucose from digested food as an energy source (Alberti, 1996). The chronic

hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of

various organs, especially the eyes, kidneys, nerves, heart, and blood vessels (American Diabetes

Association, 2007).

1.9.1 Alloxan

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Alloxan (2,4,5,6-pyrimidinetetrone) is an oxygenated pyrimidine derivative. It is present as

alloxan hydrate in aqueous solution (Frode and Medeiros, 2008). Alloxan is a toxic glucose

analogue, which selectively destroys insulin-producing cells in the pancreas (that is beta cells)

when administered to rodents and many other animal species (Szkudelski, 2001). This causes an

insulin-dependent diabetes mellitus (called "Alloxan Diabetes") in these animals, with

characteristics similar to type 1 diabetes in humans.

Alloxan is a strong oxidizing agent and it forms a hemiacetal with its reduced reaction product

dialuric acid (in which a carbonyl group is reduced to a hydroxyl group) which is called

alloxantin (Szkudelski, 2001) as seen in fig 1.2..

Alloxane (left) with dialuric acid (right) and alloxantin (center)

1.9.2 Signs and symptoms of diabetes

The sign and symptoms of diabetes include Excessive thirst (polydipsia), Excessive urination

(polyuria) and dehydration, Excessive hunger or appetite (polyphagia), Unexplained weight loss,

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Blurred vision, near sightedness or other vision problems, Slow healing of sores, Skin problems,

such as itchiness, Fatigue, lethargy or drowsiness, Shakiness or trembling, Mood swings or

irritability, Dizziness or fainting.

1.9.3 Management of diabetes

In treatment of diabetes, correct diagnosis is essential. Thus emphasis should be placed on using

appropriate diagnostic criteria (Alberti, 1996). Once the diagnosis is confirmed, an attempt

should be made to classify the type of diabetes.

The major components of the treatment of diabetes include:

1. Diet (combined with exercise)

2. Oral hypoglycaemic therapy

3. Insulin treatment

Education of the person with diabetes is an essential component of management in every case.

Aim/objective of the studies

The aim is to evaluate the effect of ethanol pulp extact of date medjool on kidney and liver

parameter on alloxan induce diabetic rats.

Specific objectives of the research

To determine the acute toxicity (LD50) of the ethanol pulp extract of Date medjool.

To determine the effects of ethanol pulp extract of Date medjool on blood glucose levels

of the rats.

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To assay the effect of ethanol pulp extract of Date medjoo on serum liver marker

enzymes.

To determine qualitatively and quantitatively the phytochemical constituent of ethanol

pulp extract of Date medjool .

CHAPTER TWO

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2.1: Materials

2.1.1: Chemicals/ reagents

Some of the reagent and their manufacturer are:

Chemicals Manufacturer

Ethanol BDH England

Creatinine reagent Randox

Alloxan Sigma Aldrich germany

Gibenclamide May and baker England

Sodium chloride BDH England

Urea reagent Randox

Ethyl actate BDH England

Distilled water Lion table water, UNN

Chloroform Sigma

Picric acid Lab.tech. chemical

Tween 80

2.1.2: Instrucment/equipment

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Refrigerator Harrier thermocool

One touch glucometer Lifescan USA

PCV tubes Pyres, England

Haematocrit centrifuge Vickers Ltd England

PCV reader Pyres, England

Pasteur pipette Pyrex, England

Test tubes Pyrex, England

Test tube rack Pyrex England

Colorimeter El scientific colridia

Syringes Mono-ject china

Measuring cylinder Pyrex England

Beaker Pyrex England

Centrifuge Vickas Ltd England

Micropipette Perfect USA

Weighing balance Metler HAS

Volumetric flasks Pyrex England

Filter paper Watchman no 1

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Evapouring dish Pyrex England

Lumba counting chamber Quijing, china

ALP Kit Randox, UK

ALT Kit Randox, UK

AST Kit Randox, UK

Chol cal (chol kit) Randox, UK

Sugar level kit Randox, UK

Masking tap ABRO, USA

Plain and EDTA bottle

Mortal and pestle

Cheese cloth

Blender

Funnel

2.1.3: Plant materials

Date medjool fruits (phoenix dactylifera) were bought from Minna in Niger State and identified

by the Department of Botany, University of Nigeria Nsukka. A voucher specimen was deposited

in the Department’s Herbarium.

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2.1.4: Animals

Twenty-one (21) adult rats of both sexes weighing 100 – 200g were purchased and kept under

room temperature in department of animal house and were acclimatized in the new environment

for a period of two weeks with adequate feed and clean water.

2.1.5: Parameters

Glucose level

Aspartate aminotransferase (AST)

Alanine aminotransferase (ALT)

Alkanine phosphatase (ALP)

Urea

Creatinine

2.2: Methods

2.3: Preparation of plant extracts

The dried date medjool pulp was grounded into fine powder with a grinding machine. 250g of

pulverized pulp was subjected into three different chesta bottle and 500ml of enthanol, methanol

and acetone were poured into diiferent bottle and keep for seven two hours (three days), the filter

was concentrated using rotary evaporator to obtain slurry of the extact; the semi-pastry extract

was stored in a refrigerator until when needed.

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2.4 Preparation of Liquid Extract

2.0g of the slurry ethanol pulp extract of date medjool was dissolved in 96ml of distilled water

and 4ml of tween 80.

2.4.1: Estimated of percentage yield of extract

After evaporating the solvent (ethanol), the extract was obtained in the slurry form. The extract

was weighed with a weighing balance and the weight recorded.

The percentage of the extract yield was calculated as:

Percentage yield = weight (g) of extract

______________________________ x 100

Weight (g) of puverised pulp

2.4.2: Experiment design

The twenty-one wistar albino rats were randomly divided into seven (7) different groups and

given feed and water for two weeks before the experiment started and the reason is for the

acclimatization, the group were as follows:

Group 1: Normal feed and clean water

Group 2: Alloxan induced and treatment + Glibenclamide

Group 3: Alloxan induced without treatment

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Group 4: Alloxan induced and treatment + 100mg/kg of extract

Group 5: Alloxan induced and treatment + 300mg/ kg of extract

Group 6: Alloxan induced and treatment + 500mg/kg of extract

Group 7: No alloxan and treatment + 500mg/kg of extract

Blood sample of the rats were collected through ocular puncture for biochemical analysis. After

the experiment, the animals were sacrificed by suffocating in a container containing chloroform

after which they were mass buried.

2.4.3: Acute Toxicity Test

Determination of LD50 of the Extract was done using Lorke’s method (1983).

Median Lethal Dose (LD50) is the log dose of a drug that kills 50% of the population to which the

drug is administered. Investigation on the acute toxicity study LD50 of the extract was determined

using the method described by Lorke (1983). Twenty-one experimental rats were distributed into

seven (7) groups and different doses of 100mg/kg, 300mg/kg and 500mg/kg of ethanol extract of

Date medjool were administered orally to the rats in the cage. The rats were observed for 72

hours for adverse reactions, abnormal behavior and general body conditions. The resulting

observation indicated that Date medjool ethanol extract was not toxic since no death of the rats

was recorded.

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2.5 preparation of reagents

2.5.1: Preparation of Normal saline

0.9g of sodium chloride was dissolved in distilled water and made up to 100ml with distilled

water in a measuring cylinder.

2.5.2 Preparation of Alloxan

0.7g of alloxan monohydrate was dissolved in 10ml of ice cold normal saline.

2.5.3 preparation of Glibenclamide

5mg of glibenclamide was dissolved in 20ml of distilled water

2.5.4: Determination of serum glucose

The one-touch blood glucose monitoring meter and test strips were used for the assay.

This method is based on the reaction of gluconic acid and hydrogen peroxide. Hydrogen

peroxidase subsequently oxidizes the dyes in a reaction mediated by peroxidase producing a blue

coloured product. The intensity of the colour, which is proportional to the glucose concentration

of the sample, was read from the one – touch meter.

2.6: Biochemical parameter

The parameters were measured spectrophotometrically, using enzymatic colometric assay kits

(Random, UK) as follows:

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2.6.1 Assay for Serum alanine aminotransferase enzyme:

Principle: ALT is measured by monitoring the concentration of pyruvate hydrazone formed with

2, 4-dinitrophenylhydrazine. The colour intensity is measured against the blank at 540nm.

Method: The blank and sample test tubes were set up in duplicates. 0.1ml of serum was pipetted

into the sample tubes. To these were added 0.5ml buffer solution containing phosphate buffer, L-

alanine and α-oxoglutarate. The mixtures were thoroughly mixed and incubated for exactly 30

minutes at 37 0C ml and pH 7.4. 0.5ml of reagent containing 2, 4-dinitrophenylhydrazine was

later added to both tubes while 0.1ml of sample was added to sample blank tube. The tubes were

mixed thoroughly and incubated for exactly 20 minutes at 25 0 C.5.0ml of sodium hydroxide

solution was then added to each tube and mixed. The absorbance was read against the blank after

5 minutes at 540nm.

2.6.2 Assay for Serum aspartate aminotransferase enzyme:

Principle: AST is measured by monitoring the concentration of oxaloacetate hydrazone formed

with 2, 4-dinitrophenylhydrazine. The colour intensity is measured against the blank at 546nm.

Method: The blank and sample test tubes were set up in duplicates. 0.1ml of serum was pipetted

into the sample tubes. 0.5ml of Reagent 1 was pipette into both sample and blank tubes. The

mixtures were thoroughly mixed and incubated for exactly 30 minutes at 37 0C ml and pH 7.4.

0.5ml of Reagent 2 containing 2, 4-dinitrophenylhydrazine was added into all the test tubes

followed by 0.1ml of sample into the blank tubes. The tubes were mixed thoroughly and

incubated for exactly 20 minutes at 25 0 C.5.0ml of sodium hydroxide solution was then added to

each tube and mixed. The absorbance was read against the blank after 5 minutes at 546nm.

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2.6.3: Determination of Serum alkaline phosphatase enzyme:

Principle: The principle of this method is based on the reaction involving serum alkaline

phosphatase and a colourless substrate of phenolphthalein monophosphate, giving rise to

phosphoric acid and phenolphthalein which at alkaline pH values, turn pink that can be

determined spectrophotometrically.

P-nitrophenylphosphate + H20 -------- ALP ------ PO42- + P-nitrophenol (pink at pH=9.8)

Method: The blank and sample test tubes were set up in duplicates. 0.05ml of sample was pipette

into the sample test tubes. 0.05ml of distilled water was pipetted into the blank tube. 3.0ml of

substrate was pipette into each tube respectively, which was then mixed and the initial

absorbance taken at 405nm. The stop watch was started and the absorbance of the sample and the

blank read again three more times at one minute intervals.

Calculation: alkaline phosphatase activity was calculated as follows:

Activity of ALP (in U/L) = Absorbance of sample x 3300

Absorbance of standard

2.6.4: Determination of serum urea concentration

Principle: urea in serum is hydrolysed to ammonia in the present of urease, the ammonia is then

measured photometrically by Berthelot’s reaction.

Method: three tubes were set up (blank, standard and sample), 0.1ml of sample and standard was

added to sample and standard test tube, 0.1ml of distilled water was also added to blank tube,

01ml of reagent was added to each tube and mixed and incubate for 20 minutes at 37OC. 2.50ml

26

of reagent 2 and reagent 3 was added and mixed and incubate for 15minutes at 37OC. The

absorbance of the sample and standard was taken against the blank.

2.6.5 Determination of creatinine

Principle: Creatinine in alkaline solution react with picric acid to form a coloured complex, the

amount of the complex formed is directly proportional to the creatinine concentration.

Method: Blank, sample and standard test tube were set in test tube rack, 0.5ml of water was

added to the blank test tube, 0.5ml of sample and standard were added to sample and standard

test tubes, also 0.5ml of working reagent were added to each tube and mixed and inserted into

the Rx Monza Flowcell holder and the reading was taken.

2.7: Statistical analysis

The data obtained were analyzed using statistical package for social sciences (SPSS) version

16.0 and the result expressed as mean + standard error of mean. Significant differences of the

result were established by one-way and two-way ANOVA and the acceptance level of the

significant was p<0.05 for all the result.

27

3.0 CHAPTER THREE

3.1: Acute toxicity of Date medjool

Result of the acult toxicity of (LD50) of the ethanol extract of date medjool pulp

Dose mg/kg body weight Number of animal before oral Number of death after

Administration Administration

100 3 _______

300 3 _______

500 3 _______

The acute toxicity studies (LD50) of the extract of date medjool pulp showed that no animal died

in any group after receiving increase dose (between 100-500mg/kg body weight)of the aqueous

ethanol extract of date pulp.

28

3.2 Effect of the ethanol pulp extract of date medjool on glucose concentration in normal,

standard, diabetic, induction and no induction rats.

Normal

Contro

l

Std Con

trol

Diabeti

c Con

trol

Indu

ction

+ 10

0mg/k

g

Indu

ction

+ 30

0mg/k

g

Indu

ction

+50

0mg/k

g

No Ind

uctio

n + 50

0mg/k

g0

50

100

150

200

250

300

350

400

450

500

GluB4InductionGluAfterInductionGluAfterTreatment

Treatment s

Glu

cose

Con

cen

trat

ion

(m

g/d

l)

From the chart above it was found out that there is an increase in group 4 and group 5 and a

decrease in group 6 and group 7 but the increase and decrease was not significant (p>0.05), it

was revealed also that there is a significant decrease in group 6 (p<0.05) compared to group 2

and a decrease in group 4, group 5 and group 7 compared to group 2 but decrease was not

significant (p>0.05). There is also a significant decrease in group 6 and group 7 compared to

group 3 (p<0.05) and a decrease in group 4 and group 5 but the decrease was not significant.

29

3.3 Effect of ethanol pulp extract of date medjool on ALP concentraction in normal,

standard, diabetic control and administration group.

Normal

Contro

l

Std Con

trol

Diabeti

c Con

trol

Indu

ction

+ 10

0mg/k

g

Indu

ction

+ 30

0mg/k

g

Indu

ction

+50

0mg/k

g

No Ind

uctio

n + 50

0mg/k

g0

10

20

30

40

50

60

70

80

90

Treatment Groups

AL

P (

IU/L

)

From the chart above it was observed that there is a significant decrease in group 4 and group 6

(p<0.05) compared to group 1 and a decrease in group 5 and group 7 but the decrease was not

significant (p>0.05). The result also revealed that there is a significant decrease in group 4 and

group 6 (p<0.05) and a decrease in group 5 and group 7 when compared to group 2 (p<0.05) but

the decrease was not significant. There is also a significant increase (p<0.05) in group 4, group 5,

group 6 and group 7 compared to group 3.

30

3.4: Effect of ethanol pulp extract of Date medjool on AST concentration in normal,

standard, diabetic control and administration group.

Normal

Contro

l

Std Con

trol

Diabeti

c Con

trol

Indu

ction

+ 10

0mg/k

g

Indu

ction

+ 30

0mg/k

g

Indu

ction

+50

0mg/k

g

No Ind

uctio

n + 50

0mg/k

g0

20

40

60

80

100

120

140

160

Treatment Groups

AS

T (

IU/L

)

From the chart above it was observed that there is a significant increase in group 6 and 7 when

compared to group 1(p<0.05), and there is a significant increase in group 6 and 7 when compared

with group 2 (p<0.05). there is also a significant increase in group 6 and 7 when compared with

group 3 (p<0.05).

31

3.5: Effect of ethanol pulp extract of Date medjool on ALT concentration in normal,

standard, diabetic control and administration group.

Normal

Contro

l

Std Con

trol

Diabeti

c Con

trol

Indu

ction

+ 10

0mg/k

g

Indu

ction

+ 30

0mg/k

g

Indu

ction

+50

0mg/k

g

No Ind

uctio

n + 50

0mg/k

g0

20

40

60

80

100

120

Treatment Groups

AL

T (

IU/L

)

There is a decrease in group 4, 5, 7 when compared with group 1 and increase in group 6 when

compared with group 1. But the decrease and increase was not significant (p>0.05). There is a

significant increase in group 6 when compared with group 2 (p<0.05) and increase in group 4, 5

and 7 but the increase was not significant (p>0.05). There is a significant increase in group 6

compared to group 3 and increase in group 4, 5 and 7, but the increase was not significant

(p>0.05).

32

3.6: Effect of ethanol pulp extract of Date medjool on urea concentration of normal,

standard, diabetic control and administration group.

Normal

Contro

l

Std Con

trol

Diabeti

c Con

trol

Indu

ction

+ 10

0mg/k

g

Indu

ction

+ 30

0mg/k

g

Indu

ction

+50

0mg/k

g

No Ind

uctio

n + 50

0mg/k

g0

50

100

150

200

250

Treatment Groups

Ure

a ()

There is a decrease in urea level of group 4, 5, 6 and 7 when compared to group 1, but the

decrease was not significant (p>0.05). a significant decrease in all administration groups when

compared to group 2 (p<0.05). There is a significant decrease in group 4, 5, 6 and 7 when

compared with group 3 (p<0.05).

33

3.7 Effect of ethanol pulp extract of Date medjool on creatinine contraction of normal,

standard, diabetic control and administration group.

Normal

Contro

l

Std Con

trol

Diabeti

c Con

trol

Indu

ction

+ 10

0mg/k

g

Indu

ction

+ 30

0mg/k

g

Indu

ction

+50

0mg/k

g

No Ind

uctio

n + 50

0mg/k

g0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Treatment Groups

Cre

atin

ine

()

There is a significant decrease in group 5 when compared to group 1 (p<0.05). There is a

significant decrease in group 4, 5, 6 and 7 (p<0.05) when compared with group 2, and a

significant decrease in group 4, 5, 6 and 7 (p<0.05) when compared to group 3

34

CHAPTER FOUR

4.1: DISCUSSION

The present research was conducted to determine the effect of the ethanol pulp extract of Date

medjool on liver and kidney parameters of alloxan-induced diabetic rats, this was done by

inducing diabetes in the rats using alloxan and treating the rats with the ethanol pulp extract of

Date medjool. Consequently, the glucose level, ALP, ALT, AST, urea and creatinine were

analyzed in alloxan induced diabetic rats using acute toxicity test which shows that the plant

extract was not harmful to the animal after receiving an increase dose of 100mg/kg to 500mg/kg.

From the study, it also shows that the percentage yield of the plant contain oil, carbohydrate

sugar such as glucose, fructose, sucrose, mannose, protein, low fats, soluble fibre, it also contain

phenolic compounds and phytochemical such as flavonoid, saponin, tannin etc. Our data also

showed that treatment of diabetic rat with ethanol pulp extract of Date medjool result in marked

increase in animal body weight as well as in life span of treated rats when compared with the

untreated diabetic rats. The basis for the application of Date medjool was found in glucose level

due to high concentration of sugar compounds present.

Alloxan has been shown to produce hyperglycemia which may be due to partial or complete

destruction of the beta cells and Insulin deficiency which lead to various metabolic alterations in

the animal via increase blood glucose level (Murugan et al., 2009). Ethanol pulp extract of Date

medjool was found to reduced blood glucose level in experimental rats compared to diabetic and

standard rats due to the soluble fibre present in Date medjool. The extract significantly (p<0.05)

35

reduced the glucose concentration at both test doses compared to the untreated diabetic group,

this was compared with the experiment done by Amreen F. et al., 2012 using ethanol extract of

Andrographis paniculata which shows that blood glucose level decrease within the 2 weeks of

the administration of the extract compared to untreated diabetic rats, showing that the ethanol

pulp extract of Date medjool act as a potential anti-diabetic drug, and can be developed further in

synergy with other agent to help in the fight against diabetes (ADA 2007).

AST plays a role in the liver and other tissue by evaluating muscle and liver damage in small

and large animal. From the studies carried out it was found out that the mean value in AST after

treatment is 1.2000E2 for normal control, 1.1667E2 for standard control, 1.1300E2 for diabetic

control, 1.0867E2, 1.1667E2, 1.4667E2, and 1.4400E2 for experimental rats, this shows that

Date madjool extract was used to increase the serum AST level of experimental rats In compared

to untreated diabetic rats but has a slight decrease compared to standard and normal control, this

is similar with the experiment done by Ameen F. et al., 2012 using ethanol extract of

Andrographis paniculata which restored aspartate (AST) in diabetic rats. This also indicate that

Date medjool have an anti-diabtic activity. this is as a result of the protein content present in

these plant.

ALT is a cytoplasmic enzyme and is considered to be liver specific in small animals, its plasma

concentration increase with hepatocellular, damage/necrosis, hepatocyte proliferation or

hepatocellular degeneration. From the studies it was found out that the mean value of ALT after

treatment is 82.6667mg/dl for normal control, 74.6667mg/dl for standard control, 71.3333mg/dl

for diabetic control and 76.6667mg/dl, 71.3333mg/dl, 97.3333mg/dl and 75.0000mg/dl for

administration group, these show that there is an increase in ALT level in rats administered with

extract compared to standard and diabetic group, as seen in experiment done by Amreen F. et

36

al., 2012 using ethanol extract of Andrographis paniculata which have an anti-diabetic

properties and it restore ALT level in diabetic rats, this show that date medjool ethanol extract

have an anti-diabetics activity.

ALP is an enzyme which catalyzes the hydrolysis of a phosphate group from an organic

molecule at an alkaline PH, It also plays a role in detoxification of endotoxin. From the studies

carried out the mean value of ALP after treatment was found to be 71.000mg/dl for normal

control, 77.0000mg/dl for standard control, 29.000omg/dl for diabetic control, 52.6667mg/dl,

63.3333mg/dl, 69.3333mg/dl and 60.0476mg/dl for the administration group (100mg/kg,

300mg/kg and 500mg/kg) from the data it was revealed that there is an increase in ALP level in

experimental group compared to diabetic control and this is compared to experiment done using

ethanol extract of Andrographis paniculata in diabetic rats by Amreen F. et al., 2012 which

indicate the restoration ALP level in diabetes, of which shows that Date medjool ethanol pulp

extract have anti-diabetic properties.

The study of the effect of ethanol extract pulp extract of Date medjool under low and high doses

were important in order to have more data for human risk assessment. The kidney and body

weight had a significant decrease with treated rat in high and low doses compared to untreated

rat. With respect to biochemical parameter on treated rats there is significant decrease in urea

level and creatinine level of experimental rats compared to untreated diabetic control group and

this decrease in urea and creatinine level in diabetic control group might be due to low protein

content in date which may lead to low urea and creatinine level in blood, this is compared with

the experiment done by Amreen F.et al., 2012 in diabetic rats using aqueous extract of Aegle

marmelos indicating the reduction of blood sugar, urea and creatinine. This suggest that ethanol

37

pulp extract is a good source of anti-diabetic drug and also reduce the liver disease by increasing

the liver enzyme which are need for the breakdown of toxic substances in the liver.

4.2: Conclusion

The ethanol pulp extract of Date medjool has been revealed from this study that they have an

increasing and decreasing effect in biochemical parameter of liver and kidney and also reduce

blood glucose level in diabetic rats, this suggests that this plant can be of great help in

biochemical system and can therefore be developed possibly in synergy with other agent to help

in the fight against diabetes.

4.3: Suggestion for further studies

Ethanol pulp extract of Date medjool has been found out in this study to have an increase effect

liver parameter and decrease effect in kidney and blood glucose, so I suggest that further studies

should be carried out on the plant extract to know the effect in lipid peroxidation and oxidative

stress in rats.