sativa - CORE

بسم االله الرحمن الرحيم

A Study on the Potential Hypoglycemic Effect of Feeding Nigella

sativa

(Black cumin) Seeds in Rats

By

Mawda Elmahi Ali Elmhadi

B. Sc. of Veterinary Medicine (2006)

University of Khartoum

Supervisor

Dr. Nabiela Musa Elbagir

Faculty of Veterinary Medicine

University of Khartoum

A Thesis submitted in partial fulfillment of the requirements

for the degree of Master of Science in Biochemistry

December 2010

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i

DEDICATION

To the candles which had brightened my days،

The soul of my mother …..

My father …..

My sisters and brothers…..

My friends…..

My nephews…..

With all love…..

Mawda

ii

ACKNOWLEGMENTS

Thanks first and eternal to God Allah، the gracious and the

compassionate who made this work possible and present.

My grateful thanks to my supervisor Dr. Nabiela Musa Elbagir for

the advice and guidance during the period of the work. Thanks are

extended to the technical staff of the department of Biochemistry

especially Ustaz Eltyeib Abass for the laboratory aid.

I am also indebted to Dr. Shawgi Mohamed Hassen for his helping

in data analysis and Dr. Ahmed Kamal for his helping in conducting

histopathological findings.

Finally، greet thanks to Ustaza Aisha Elmahi and to my friend

Maali Osman for their help and encouraging.

iii

LIST OF CONTENTS

DEDICATION i

ACKNOWLEGMENTS ii

CONTENTS iii

LIST OF TABLES vii

LIST OF FIGURES viii

ENGLISH ABSTRACT ix

ARABIC ABSTRAC xii

INTRODUCTION 1

CHAPTER ONE

Literature review 3

1.1 Nigella sativa 3

1.1.1 Scientific classification 4

1.1.2 Botanical description 4

1.1.3 Chemical composition 5

1.1.4 Distribution 6

1.1.5 Pharmacological properties 6

1.1.6 Toxicological properties 8

1.2 Effects of Nigella sativa on glucose metabolism 8

1.2.1 Hypoglycemic effect 8

1.2.2 Effects of Nigella sativa seeds on oral glucose tolerance

Test 10

1.2.3 Effects of Nigella sativa seeds on glucose-6-phosphate

dehydrogenase activity 11

1.3 Effects of N. sativa seeds on Hemoglobin level 12

iv

1.4 The effect of N. Sativa seeds on serum Alanine transaminase

(ALT) 12

1.5 The effects of N. sativa seeds on body weight 13

1.6 Glibenclamide 13

1.6.1 Uses of Glibenclamide 13

1.6.2 Mechanism of action 13

CHAPTER TWO

MATERIALS AND METHODS 15

2.1 Experimental details 15

2.1.1 Experimental animals 15

2.1.2 The basal rat diet 15

2.1.3 Equipment 16

2.2 Methods 16

2.2.1 Germination test 16

2.2.2 Experimental procedure 17

2.2.3 Blood samplings 18

2.2.4 The body weight 18

2.2.5 Histopathological examinations 18

2.3 Analytical methods 18

2.3.1 Determination of plasma glucose 18

2.3.2 Determination of glucose-6-phosphate dehydrogenase

activity (G6PD) 20

2.3.3 Determination of insulin 21

2.3.4 Determination of Alanine aminotransferase (ALT) 22

2.3.5 Hemoglobin estimation 23

2.4 Oral glucose tolerance test (OGGT) 24

v

2.5 Histopathological methods 25

2.6 Statistical analysis 25

CHAPTER THREE

RESULT 26

3.1 The effects of feeding N. sativa seeds on blood glucose

concentration 26

3.2 Blood insulin level 28

3.3 Oral glucose tolerance test (OGTT) 30

3.4 Glucose-6-phosphate dehydrogenase activity (G6PD) 31

3.5 Alanine aminotransferase activity (ALT) 31

3.6 The effects of N. sativa seeds on Hemoglobin concentration 32

3.7 The effects of N. sativa seeds on body weight 33

3.8 Histopathological findings 35

CHAPTER FOUR

DESCUSSION 41

4.1 The effects of feeding N. sativa seeds on serum blood

glucose concentration 41

4.2 The effects of feeding N. sativa seeds on insulin level 43

4.3 The effects of feeding N. sativa seeds on oral glucose tolerance

test 44

4.4 The effects of feeding N. sativa seeds on glucose-6-phosphate

dehydrogenase Activity 45

4.5 The effects of feeding N. sativa seeds on Alanine transaminase

(ALT) activity 46

vi

4.6 The effects of feeding N. sativa seeds on blood hemoglobin

level 46

4.7 The effects of feeding N. sativa seeds on body weight 47

4.8 Histopathological findings 47

CONCLUSIONS 49

RECOMMENDATIONS 49

LIST OF REFERENCES 51

vii

LIST OF TABLES

Table (1) The changes in the serum glucose concentration (mg/dl)

Among the different groups of rats at different time intervals. 27

Table (2) The changes in the serum insulin concentration (µIU/ml)

in the treated and control groups of rats. 28

Table (3) The effects of seeds on body weight (g). 33

Table (4) Biochemical and Hemoglobin concentration changes in

rats. 34

viii

LIST OF FIGURES

Fig. (1) The effect of N. sativa seeds on serum insulin level (µIU/ml)

day 14th and 28th. 29

Fig. (2) The effect of N. sativa seeds on oral glucose tolerance test. 30

Fig. (3) The effects of N. sativa seeds on Alanine aminotransferase

activity (U/L)in the different treated groups of rats. 32

Fig. (4) Liver from rats received normal basal rat’s diet. Notice no

changes (H and E X 400). 36

Fig. (5) Liver from rats received Glibenclamide (10mg/kg body weight)

on feed. Notice a focal infiltration of mononuclear cells especially

around vessels (H and E X 400). 37

Fig. (6) Liver from rats received N. sativa (50mg/day) on feed.

Notice no changes (H and E X 400). 38

Fig. (7) Liver from rats received N. sativa (50mg/day) + glucose

(2g/kg body weight) on feed. Notice no changes (H and E X 400). 39

Fig. (8) Liver from rats received 2g/kg body weight glucose on feed.

Notice that some hepatocytes exhibit fatty degeneration and focal

areas of necrosis (H and E X 400) 40

ix

ABSTRACT

The present study was designed to evaluate the hypoglycemic effect of

Nigella sativa (Black cumin) seeds and to explore how the induction of

hypoglycemia، by feeding seeds، can modulate glucose metabolism in

animals. This effect was compared with a reference of a known

hypoglycemic drug Glibenclamide.

Thirty female Wister albino rats were used as experimental animals and

divided into five groups (A، B، C، D and E) of six rats each. Each rat was

fed 8 g for 30 days as follows: In group A، the rats were fed basal diet

and kept as control ، group B was fed Glibenclamide (hypoglycemic

drug) at the rate of 10 mg/kg body weight، calculated as part from the rat

basal diet، group C was fed Nigella sativa (50 mg/day as part from the

basal diet)، group D was fed Nigella sativa (50 mg/day + glucose 2g/kg

body weight as part from the basal diet) and group E was fed glucose

(2g/kg body weight calculated as part from the rat basal diet). Blood

glucose levels were measured weekly. Insulin concentration was

recorded at days 14 and 28. At the end of the experimental period، blood

hemoglobin concentration was measured and oral glucose tolerance test

was conducted for all rats. Glucose-6-phosphate dehydrogenase and

Alanine aminotrasferase activities were estimated، and a

histophathological examination of the liver and the pancreas was done

for all groups. Animal's body weight was reported at the beginning and at

the end of the experiment.

x

All the treated groups showed significant reduction in blood glucose

concentration after one week of treatment، and it was more pronounced

in group B. At the second week and until the end of the experiment،

significant reduction compared to the control group، were maintained

only in groups B and C which received Glibenclamide and N. sativa،

respectively. By the end of the experiment، groups D and E showed

similar levels of glucose to the control group. In group D، feeding high

glucose dose was not associated with the application of N. sativa to the

diet. When insulin concentration was measured after two weeks، no

significant increase was observed in all groups except groups D and E.

By the end of the experimental period، all treated groups showed

significantly higher insulin levels and it was significantly higher in group

E.

After four weeks of treatment، no significant changes were noticed in

Glucose-6-phosphate dehydrogenase activity in all groups، only higher

values were found in groups D and E، being two folds higher than group

B. The activity of Alanine aminotrasferase was significantly increased in

the group receiving N. sativa but was significantly decreased in the

groups treated with Glibenclamide and glucose، respectively. The

performance of the groups receiving Glibenclamide or only the N. sativa

was similar، when Glucose Tolerance Test was carried out، but addition

of extra glucose to the rat basal diet plus the N. sativa abolished this

effect. Hemoglobin concentration and animal’s body weight were not

influenced by all treatments applied. No histopathological changes were

xi

noticed in the liver and pancreas of Nigella sativa treated groups

compared with the other groups and the control.

The results showed that Nigella sativa at 50 mg/day can exerts potential

hypoglycemic effects in rats. The hypoglycemic effect of the seeds may

be mediated، at least in part، by decreasing glucose concentration and

increasing insulin level. Glucose uptake and absorption were

significantly influenced by feeding N. sativa to the rats. The increase of

Alanine aminotrasferase activity in N. sativa treated groups، with no

histopathological changes، may indicate slight effects on the integrity of

liver cells.

xii

المستخلص

فى خفض سكر الدم، ولتوضيح آيفية ) الحبة السوداء(صممت الدراسة الحالية لتقييم تأثيرالكمون

البذور، ومقارنة هذا إحداث خفض سكر الدم بتغيير أيض الجلكوز فى الحيوانات التى أعطيت

.التأثير بدواء الجلبنكلاميد المعروف بخفضه لسكر الدم

أ، ب، (أنثى جرذ وستر ألبينو آحيوانات تجارب وقسمت إلى خمس مجموعات 30إستخدمت

أطعمت الجرذان فى : يوما آالأتى 30جرام لمدة 8أطعم آل جرذ . جرذان لكلٍ 6) ج، د، هـ

ستخدمت آمجموعة شاهد، وأعطيت المجموعة ب الجلبنكلاميد المجموعة أ الأآل الأساسى وإ

حسبت آجزء من الأآل (آجم من وزن الجسم /ملجرام 10بجرعه ) دواء خافض لسكر الدم(

اليوم آجزء من الأآل الأساسى، /ملجرام 50، وأعطيت المجموعة ج الكمون بمعدل )الأساسى

آجم من /جرام2(افة إلى الجلكوز اليوم إض/ملجرام 50وأعطيت المجموعة د الكمون بمعدل

آجم من وزن /جرام2(، وأعطيت المجموعة هـ الجلكوز )وزن الجسم آجزء من الأآل الأساسى

سجل مستوى جلكوز الدم إسبوعياً و ترآيز ). الجسم وتم حسابها آجزء من الأآل الأساسى

موغلوبين الدم و فى نهاية مدة التجربة سجل ترآيز هي. 28و 14هرمون الإنسولين فى اليوم

فوسفيت -6-تم تقدير نشاط إنزيم الجلكوز. إجرى إختبار تحمل الجلكوز لكل الجرذان

لكل ديهيدروجينيز والألنين أمينوترانسفيريز، آما فحصت الأنسجة المرضية للكبد والبنكرياس

.المجموعات وسجلت أوزان الحيوانات فى بداية ونهاية التجربة

الجة نقصاً معنوياً فى ترآيز جلكوز الدم بعد إسبوع من التجربة أظهرت آل المجموعات المع

المجموعة (وآان هنالك إنخفاضاً فى المجموعتين ب و ج . وآان أآثر وضوحاً فى المجموعة ب

فى الأسبوع الثانى وحتى ) المعالجة بالجلبنكلاميد والمجموعة المعالجة بالكمون على التوالى

بنهاية التجربة أظهرت مجموعتى الإختبار د و هـ . عة الشاهدنهاية التجربة مقارنة مع مجمو

إعطاء جرعة عالية من الجلكوز للمجموعة د غير . مستويات جلكوز مشابهة لمجموعة الشاهد

بنهاية مدة . مصاحبة بإعطاء الكمون معنويه فى آل المجموعات عدا المجموعتين د و هـ

هرمون الإنسولين وبزيادةً أآبر فى المعالجة أظهرت آل المجموعات زيادة فى مستوى

.المجموعة هـ

xiii

فوسفيت ديهيدروجينيز فى آل -6-لم تلاحظ أى تغيرات معنويه فى نشاط إنزيم الجلكوز

المجموعات بعد أربعة أسابيع من المعالجة، وقد لوحظت الزيادات فقط فى المجموعتين د و هـ

نشاط إنزيم الألنين أمينوترانسفيريز حدثت زيادة معنويه فى . التى بلغت ضعف المجموعة ب

فى المجموعة التى أعطيت الكمون بينما لوحظ نقص معنوى فى المجموعات المعالجة

عند إجراء إختبار تحمل الجلكوز آان الأداء متساوى فى . بالجلبنكلاميد والجلكوز على التوالى

لكوز لأآل الجرذان المجموعات التى أعطيت الجلبنكلاميد والكمون فقط ، لكن إضافة الج

لم يتأثر ترآيز الهيموغلوبين وأوزان الحيوانات . الأساسى بالإضافة للكمون الغى هذا التأثير

لم تلاحظ أى تغيرات أنسجة مرضية فى الكبد والبنكرياس للمجموعات المعالجة . بالمعاملات

.بالكمون مقارنة بالمجموعات الأخرى ومجموعة الشاهد

. اليوم قد يظهر تأثيراً بخفض سكر الدم فى الجرذان/ملجرام50كمون بمعدل أظهرت النتائج أن ال

وقد يكون تأثير البذور الخافض لسكر الدم ، على الأقل جزئياً، بخفض ترآيز الجلكوز وزيادة

وهنالك تأثير معنوى لإعطاء الكمون على إستهلاك وإمتصاص . مستوى هرمون الإنسولين

نشاط إنزيم الألنين امينوترانسفيريز فى المجموعات المعالجة زيادة . الجلكوز فى الجرذان

خلايا بالكمون، مع عدم وجود تغيرات أنسجة مرضية، قد يدل على التأثير الطفيف على سلامة

.الكبد

1

INTRODUCTION

Diabetes Mellitus is a serious, complex metabolic disorder of multiple etiologies. It

has a significant impact on the health, quality of life and life expectancy of patients,

as well as on the health care system (Kwon et al., 2008). Although different types

of hypoglycaemic agents such as thiazolidinediones, insulin, biguanides and

sulphunylurea are available, but there is growing interest in herbal remedies due to

the side effects associated with these therapeutic agents, beside their limitations in

managing the disease effectively. Diabetes Mellitus characterized by chronic

hyperglycemia with disturbances of carbohydrate, fat and protein metabolism

resulting from defects in insulin secretion (B-cell dysfunction), insulin action

(insulin resistant) or both (Kardesler et al., 2008).

Type 2 insulin-resistant diabetes mellitus accounts for 90-95% of all diabetes. Both

genetic and environmental factors play an important role in the onset of type 2

insulin-resistant diabetes (Lima et al., 2008). The microvascular and macrovascular

complications of diabetes including cardiovascular disease, blind-ness, renal failure

and peripheral nerve damage (Taskinen, 2002). Recent estimates indicate there

were 171 million people in the world with diabetes in the year 2000 and this is

projected to increase to 366 million by 2030 (Wild et al., 2004). Around 3.2 million

deaths every year are related to a complication of diabetes, which equals six deaths

each minute (WHO, 2007).

Renewed attention to alternative medicine and natural therapies has stimulated new

researches to look for more efficacious agents with lesser side effects (Kim et al.,

2006). According to the world ethno botanical information reports, almost 800

plants may possess antidiabetic potential. N. sativa is one such herbal product

which has been in use as a spice from ancient times. Its medicinal value to treat

various ailments is also well-known (Khanam and Dewan, 2008). N. sativa and

several species of plants have been described as having antidiabetic property

2

(Uddin et al., 2002; Uddin et al., 2005). Naturally occurring antidiabetic plant are

relatively non toxic, inexpensive and available in an ingestive form. It used where

low and middle-income populations are important (Gazioano et al., 2007).

The objectives of the present study are to investigate about the hypoglycemic effect

of N. sativa seeds on glucose absorption uptake and metabolism in Wister albino

female rats. The effects of feeding the seeds will be compared to the effect of the

Glibenclamide (hypoglycemic drug). The parameters to be measured include:

Plasma glucose concentration.

Serum insulin concentration.

Glucose-6-phosphate dehydrogenase activity.

Blood Hemoglobin (Hb).

To perform oral glucose tolerance test (OGTT).

To measure Alanine aminotransferase (ALT) activity.

To measure changes in animal’s body weight.

To examine histopathological changes related to treatments

3

CHAPTER ONE

LITERATURE REVIEW

1.1 Nigella sativa

Nigella sativa (N. sativa) commonly known as black seed or black cumin is a small

plant originating in the Middle East and is found widely in Egypt, Asiatic Turkey

and the Balkan states. It has been traditionally used in the Indian subcontinent

(Nadkarni, 1976), Arabian countries (Sayed, 1980) and Europe (Lautenbacher,

1997) for culinary and medicinal purposes as a natural remedy for number of

illnesses and conditions.

Abu Huraira (may God be pleased with him) narrated that God’s Messenger (pbuh)

said, “Use this black seed regularly because it contains a cure for every disease

except death.” N. sativa seeds are claimed to have bronchodilatory, hypotensive,

antibacterial, antifungal, analgesic, anti-inflammatory and immunopotentiating

properties (Schmall, 2007).The seed extracts from this plant are used in traditional

medicine (Hakims or tabibs) in the treatment of several medical disorders including

dyslipidemia, obesity and hypertension (Le et al,.2004).

Nigella sativa seed is herbaceous annual plant belonging to the Ranunculaceae

family; it is cultivated in several countries in the Mediterranean region and Asia. In

South Asia, it is called kalonji, its Arabic name is habbat el Baraka or habbah

saouda and in English it is known as black cumin. The seeds of N. sativa have been

employed for thousands years as a spice and food preservative (Aboutabl et al.,

1986; Hanafy and Hatem, 1991). Additionally, it used in the traditional medicine

applications (Abdulelah and Zainal-Abidin, 2007).

The seeds of Nigella sativa (N. sativa) has been showed to contain >30% of fixed

oil and 0.4 – 0.45% wt/wt of volatile oil, protein, alkaloid, flavonoid and saponins

4

(Winkler et al., 2005). The volatile oil has been showed to contain 18.4 – 24%

thymoquinone (2-isopropyl-5-methyl-1, 4-benzoquinone) and 46% monoterpenes

such as p-cymene and &-piene (El-Tahair et al., 1993). The biological activity of

the seeds has been showed to be due to thymoquinone, which is also present in the

fixed oil (Ghosheh et al., 1999).

N. sativa has been known for it is hypotensive (El-Tahir et al., 1993), exhibited

hepatoprotective effect against liver damage (Al-Gharably et al., 1997; Nagi et al.,

1999; El-Dakhakhny et al., 2002), antimicrobial (El-Alfy et al.,1975), antibacterial

(El-Kamali et al.,1998), antioxidant (Burits and Mucar,2000), anti-inflammatory

(Houghton et al.,1995) and antitumoral (Worthen et al.,1998). Besides, the

essential oil was showed to have anthelmentic activity (Agarwal et al., 1979) and

the seeds were effective against cestodes and nematodes (Akhtar and Rifaat, 1991).

N. sativa seeds have also been reported to have hypoglycemic activities (Al-Hader

et al., 1993; El-Dakhakhny et al., 2002), which are principally related to the lipidic

compounds of the seeds.

1.1.1 Scientific classification

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Order: Ranunculales

Family: Ranunculaceae

Genus: Nigella

Species: N. sativa

Binomial name: Nigella sativa L.

1.1.2 Botanical description

5

N. sativa is an annual herb 35-50 cm tall, branching at the top, stems green, round,

hairy, 2-5 mm diameter, internodes 2-5 cm long, flowers regular bisexual terminal

on branches, white or greenish white about 3 cm diameter, long stalked, pedicels

1.5-5.5 cm long, becoming longer as the fruit matures (El-Dakhakhny et al., 2000).

N. sativa reproduces with itself and forms a fruit capsule which consists of many

white trigonal seeds. Once the fruit capsule has matured, it opens up and the seeds

contained within are exposed to the air, becoming black in color (Schleicher and

Saleh, 1998).

1.1.3 Chemical composition

The multiple uses of N. sativa seeds in the folk medicine encouraged many

researches to identification and isolation to the major constituent of it and to

understand the pharmacological action. The seeds of N. sativa are the source of the

active ingredients of this plant (thymoquinone). The oil and seed constituents have

showed potential medicinal properties in traditional medicine (Salem, 2005). N.

Sativa seeds contain 36%-38% fixed oils, proteins, alkaloids, saponins and 0.4%-

2.5% essential oil (Lautenbacher, 1997).

The fixed oil is composed mainly of unsaturated fatty acids, including the unusual

C20:2 arachidic and eicosadienoic acids (Houghton et al., 1995). The essential oil

was analyzed by Burits and Bucar (2000). Many components were characterized,

but the major ones were thymoquinone (27.8%- 57.0%), ñ-cymene (7.1%-15.5%),

carvacrol (5.8%-11.6%), t-anethole (0.25%-2.3%), 4-terpineol (2.0%-6.6%) and

longifoline (1.0%-8.0%). Thymoquinone readily dimerizes to form

dithymoquinone (El-Dakhakhny, 1965). Four alkaloids have been reported as

constituents of N. sativa seeds. Two, nigellicine and nigellidine have an indazole

nucleus, whereas nigellimine and its N-oxide are isoquinolines (Atta-ur-Rahman et

al., 1985; 1992; 1995).

6

In spite of there were no lead, cadmium and arsenic were found in the seeds. Zinc,

calcium, magnesium, manganese and copper were found at lower level while

potassium, phosphorus, sodium and iron are predominant elements. Linoleic and

oleic acids were the major unsaturated fatty acids while palmitic acid was the main

saturated one. Glutamic acid, arginine and aspartic acid were the main amino acids

present while cystine and methionine were the minor amino acids (Al-Jassir, 1992).

1.1.4 Distribution

N. sativa is native to southwest Asia, also it grows in Mediterranean countries and

it is also cultivated in the south of Algerian (Winkler et al., 2005). It is also found

in southern Europe and northern Africa.

1.1.5 Pharmacological properties

This plant has been a focus of much research, particularly during the past two

decades. It has several traditional uses and consequently has been extensively

studied for its chemical constituents and biological activities on various body

systems in vivo or in vitro. A lot of animal studies have already been done to

identify the various activities of N. sativa oil on different components of the

metabolic syndrome, for example blood glucose (Bamosa, 1997).

The N. sativa seeds, seeds extract and thymoquinone have long been used in the

Middle and Far East as a traditional medicine for a wide range of illnesses

including bronchial asthma, headache, dysentery, infections, obesity, back pain,

hypertension and gastrointestinal problems (Schleicher and Saleh, 1998; Al-

Rowais, 2002). Until now it have antihypertensive (Rashid et al., 1987),

antibacterial (Hanafy and Hatem, 1991; Morsi, 2000), antiviral (Salem and

Hossain, 2000), antidiabetic (Uddin et al., 2002; Uddin et al., 2005) and lipid

lowering (Shaha et al., 2004). Renoprotective (Ragheb et al., 2009) potentialities

have been obtained through research.

7

Various therapeutic effects, such as antiepileptic effects in children with refractory

seizures (Akhondian et al., 2007), anti-inflammatory and analgesic actions (Khanna

et al., 1993; Mutabagani and El-Mahdi, 1997; Abdel-Fattah et al., 2000) and

antihistaminic (El-Dakhakhny et al., 2000; Kanter, 2006) have been described for

N. sativa. Additionally, it has been shown that N. sativa has protective effect

against ischemia reperfusion injury to liver and various organs (El-Abhar et al.,

2003). Its use in skin condition as eczema has also been recognized worldwide

(Goreja, 2003).

Besides these effects, N. sativa also demonstrates anti- parasitic effects. For

instance, its administration decreases the number of eggs as well as worms in

schistosomiasis, which tends to affect hepatic and intestinal tissues (El Shenawy et

al., 2008). Intraperitoneal and oral administration of ethanol, chloroform and

aqueous seed extracts of N. sativa, showed antimalarial activity against

Plasmodium berghei in mice (Abdulelah and Zainal-Abidin, 2007). In fact, N.

sativa attenuates the damage to β-cells of the pancreas following exposure to metal

and toxic elements (Kanter et al., 2005). Similarly, N. sativa administration

attenuates the ulcerative effects of ethanol on gastric mucosa by decreasing the

glutathione-S transferase levels in gastric mucosa (Kanter et al., 2005). N. sativa oil

was found to be effective as an add-on therapy in patients of insulin resistance

syndrome (Najmi et al., 2008).

It has been reported that thymoquinone prevents oxidative injury in various in vitro

and in vivo studies in rats (Daba and Abdel-Rahman1998; Mansour et al., 2001). It

has been suggested that thymoquinone may act as an antioxidant agent and

prevents membrane lipid peroxidation in tissues (Mansour et al., 2002). Moreover,

it has been demonstrated that N. sativa can significantly prevent hepatotoxicity (Al-

Gharably et al., 1997; Nagi et al., 1999) and might have protective effects against

nephrotoxicity induced by either disease or chemicals (Abdel -Fattah et al., 2000).

8

The protection was suggested to be related to the ability of thymoquinone to inhibit

lipid peroxidation. Also black seed oil was shown to be an effective adjuvant for

the treatment of patients with allergic diseases (Kalus et al., 2003). In another

clinical study, significant benefits of N. sativa extract in the treatment of acute

tonsillopharyngitis was shown (Dirjomuljono et al., 2008).

Furthermore, black seed preparations may have a cancer chemo preventive

potential and may reduce the toxicity of standard antineoplastic drugs (Salomi et

al., 1991). In fact, topical application of a black seed extract inhibited the two stage

initiation-promotion of skin carcinogenesis in mice by croton oil (Salomi et al.,

1991). N. sativa decreases DNA damage and thereby prevents initiation of

carcinogenesis in colonic tissue secondary to exposure to toxic agents such as a

zoxymethane (Al-Johar et al., 2008). In fact, sustained delivery of thymoquinone is

almost as effective in causing apoptosis of colon cancer cells as sustained delivery

of 5-fluorouracil (Norwood et al., 2007).

1.1.6 Toxicological properties

The seeds extract and it is constituents appear to have a low level of toxicity. The

administration of N. sativa seed extract (50 mg/kg) intraperitoneally to rats for 5

days did not significantly affect the activities of several enzymes and metabolites

indicative of hepatic and renal function (El-Daly, 1998). Oral administration of the

seed oil at dose up to 10 ml/kg in rats and mice did not cause any mortality or overt

toxicity during the observation period of 2 day (Khanna et al., 1993). This was

confirmed with a work of Zaoui et al. (2002) on the same dose for up to 12 weeks.

It was shown that oral administration of N. sativa oil did not cause any mortality or

significant alteration of the key hepatic enzymes in rats.

1.2 Effects of Nigella sativa on glucose metabolism

1.2.1 Hypoglycemic effect

9

Hypoglycemia is a syndrome characterized by a reduction in plasma glucose

concentration to a level that may induce symptoms of low blood sugar.

Hypoglycemia typically arises from abnormalities in the mechanisms involved in

glucose homeostasis.

The effect of N. sativa on some of the complications of experimental diabetic

rabbits has been investigated by a number of workers (e.g. Al-Hader et al., 1993;

El-Zawahrawy and Al-Zahraa, 1998; Meral et al., 2001). Al-Hader et al. (1993)

reported that intraperitoneal administration of the volatile oil of N. sativa seeds (50

mg/kg) significantly reduced the fasting blood glucose concentration in the normal

and hypoglycemic rabbits. Insulin concentration was unaffected by the treatment.

In rats, an aqueous extract of the seeds of N. sativa was administered orally under

light ether anesthesia for 7 and 14 days. Blood glucose, insulin level and key

hepatic enzyme concentrations in serum a histopathological change in the liver in

both treatment groups was done by El- Daly, 1994. A significant elevation of

certain key hepatic enzymes and varying degrees of histopathological changes in

the liver were documented following oral administration of boiling water extracts

of seed of N. sativa. An increase in gamma-glutamyl transpeptidase in the absence

of hepatocyte degeneration were observed following oral administration of N.

sativa extract and this suggests that these enzymes may have been released due to

hepatocellular damage caused at the molecular level.

Prolonged exposure (9 days) of beta-cell aggregates to 20 mmol/L glucose was

used by Ling and Pipeleers 1996 to examine the effects of chronically elevated

glucose levels on the survival and function of purified rat beta-cells. It did not lead

to cell losses, but reduced the amount of insulin secreted in response to glucose.

This decrease was not caused by cellular desensitization but resulted from the

lower cellular insulin content after a prolonged imbalance between stimulated rates

of insulin synthesis and release. It is concluded that chronic exposure of rat beta-

10

cells to elevated glucose levels induces a prolonged state of beta-cell activation and

glucose hypersensitivity rather than glucotoxicity or glucose desensitization.

Many studies have also examined the antidiabetic effects of N. sativa in normal and

in diabetic animal models. Crud aqueous extract of N. sativa restore glucose

homeostasis (Labhal et al., 1997). N. sativa was also shown to enhance liver cell

insulin sensitivity (Le et al., 2004). The effect of different N. sativa seed extract

(acidic, neutral and basic) on insulin secretion was studied by Rchid et al. (2004).

The insulin secretory effects of these extracts were evaluated individually at

concentrations 0.01, 0.1, 1 and 5 mg/ml, in vitro in isolated rat pancreatic islets in

the presence of 8.3 mmol/l glucose. It was postulated that the antidiabetic

properties of N. sativa seeds may be, at least partly, mediated by stimulated insulin

release, and that the basic sub fraction largely contributes to this stimulatory effect

(Rchid et al., 2004).

Study on protective effects of the volatile oil of N. sativa seeds on insulin

immunoreactivity and ultra structural changes of pancreatic b-cells in STZ-induced

diabetic rats was done by Mehmet et al. (2010). It is suggest that N. sativa

treatment exerts a therapeutic protective effect in diabetes by decreasing

morphological changes and preserving pancreatic b-cell integrity. Consequently, N.

sativa may be clinically useful for protecting b-cells against oxidative stress.

Abdelmeguid et al. (2010) investigate the effect of N. sativa extract, oil, and

thymoquinone on serum insulin and glucose concentration in streptozotocin

diabetic rats. Which have been significantly decreased the elevated serum glucose

level and significantly increased the serum insulin levels. In addition, the

hypoglycemic effect observed could be due to amelioration of beta cells thus

leading to the increased insulin levels.

1.2.2 Effects of N. sativa seeds on Oral Glucose Tolerance Test

11

The oral glucose tolerance test (OGTT) measures the body's ability to use a type of

sugar, called glucose, which is the body's main source of energy. OGTT, a test of

immense value and sentiment, in favor of using fasting plasma glucose

concentration alone was seen as a practical attempt to simplify and facilitate the

diagnosis of diabetes.

N. sativa have gotten a great therapeutic benefit in diabetic individuals and those

with glucose intolerance, as it accentuates glucose-induced secretion of insulin,

besides having a negative impact on glucose absorption from the intestinal mucosa

(Rchid et al., 2004; Meddah et al., 2009). However to date, little attention has been

paid to the effects of N. sativa on intestinal glucose absorption. One of this was

done by Meddah et al., 2009 on the effects of the crud aqueous extract of N. sativa

seeds on intestinal glucose absorption in vitro using a short-circuit current

technique and in vivo using an oral glucose tolerance test. It showed directly

inhibits the electrogenic intestinal absorption of glucose in vitro. On the other

hands there was improvement of glucose tolerance and body weight in rats after

chronic oral administration in vivo.

1.2.3 Effects of N. sativa seeds on glucose-6-phosphate dehydrogenase activity

G6PDH is the key rate-limiting enzyme of the pentose phosphate pathway in

glucose metabolism. One of the important roles of this pathway is the generation of

NADPH in the cytoplasmic fraction of the cell. The reducing power of NADPH is

utilized for various synthetic processes, especially for synthesis of fatty acids. Thus

G6PDH participates in the regulation of both lipogenesis and glucose metabolism

(Kelley and Kletzien, 1984).

Chandrasekaran and Leelavinothan (2009) reported that an oral administration of

Thymoquinone (major constituent of N. sativa seeds) for 6 weeks, dose

dependently improved the glycemic status in streptozocin-nicotinamide induced

diabetic rats. The levels of insulin, Hb increased with significant decrease in

12

glucose and HbA1c levels. The altered activities of carbohydrate metabolic enzymes

were restored to near normal. In diabetic state the activities of hexokinase and

glucose-6-phosphate dehydrogenase are decreased as a result of total absence or

insufficiency of insulin. No significant changes were noticed in normal rats treated

with thymoquinone. These result show that thymoquinone at 80 mg/kg is

associated with beneficial changes in hepatic enzyme activities and there by exert

potential antihyperglycemic effects.

1.3 Effects of N. sativa seeds on Hemoglobin level

The effect of N. sativa seeds fixed oil in rats has been investigated by Zaoui et al.

(2002) by monitoring blood homeostasis and body weight as well as toxicity. Daily

treatment by an oral dose of 1ml/kg body weight of N. sativa seeds fixed oil for 12

weeks .Significantly increased level of Hematocrit and Hemoglobin was found by

6.4% and 17.4% respectively.

Treatment of rats with the seed extract for up to 12 weeks has been reported to

induce changes in the hemogram that include an increase in both the packed cell

volume (PCV) and hemoglobin (Hb), and a decrease in plasma concentrations of

cholesterol, triglycerides and glucose (Ali and Gerald, 2003).

1.4 The effect of N. Sativa seeds on serum Alanine transaminase (ALT)

The Alanine transaminase (ALT) enzyme is found principally in the liver with only

small amounts being present in other organs. When there is liver cell damage, the

serum or plasma levels of the enzyme are raised (Cheesbrough, 1989). Al-Jishi and

Abuo Hozaifa, (2003), investigated the effects of N. sativa powdered seeds on

blood coagulation and some liver function tests in male rats. The result showed an

increase in ALT activity. In other study an aqueous extract of the N. sativa seeds

was administered orally for 7 and 14 days. The result showed a significant increase

in serum ALT concentration (El-Daly, 1994). Also serum ALT concentration was

13

significantly increased when aqueous extracts of the seeds of N. sativa were

administered orally to male rats (Tennekoon, et al., 1991).

1.5 The effects of N. sativa seeds on body weight

The animal body weight is an important indicator of general health. N. sativa diets

did not adversely affect growth (Al-Homidan et al., 2002). Another three studies

with regard to the effect of N. sativa (10 and 20 g/kg) on growth were carried out

with broilers over periods of 35 days. The body weight did not show any

differences between control and N. sativa treated groups at the end of the

experimental period (Brake, 2004).

1.6 Glibenclamide

Glibenclamide, also known as glyburide, is an anti-diabetic drug in a class of

medications known as sulfonylureas. It is sold in doses of 1.25 mg, 2.5 mg and

5 mg, under the trade names Diabeta, Glynase and Micronase in the United States

and Daonil, Semi-Daonil and Euglucon in the United Kingdom. It is also sold in

combination with metformin under the trade name Glucovance.

1.6.1 Uses of Glibenclamide

It is used in the treatment of type II diabetes. Since 2007, it is one of only two oral

anti-diabetics in the World Health Organization Model List of Essential Medicines.

Sulfonylureas have been used for type 2 diabetes for over 50 year and are still the

leading class of oral anti hyperglycemic agents. Their popularity is based on

familiarity and habit, of course, but also their easy of administration (as once-daily

tablets, in many cases), reliable effectiveness for recently diagnosed patients, lack

of symptomatic side effects other than hypoglycemia, and low cost (Riddle, 2003).

1.6.2 Mechanism of action

The drug works by blocking ATP-sensitive potassium channels in pancreatic beta

cells (Schmid-Antomarchi et al., 1987; Serrano-Martín et al., 2006). This inhibition

causes cell membrane depolarization, which opens voltage-dependent calcium

14

channels, which causes an increase in intracellular calcium in the beta cell, leads to

stimulates insulin release.

Six patients with type 2 diabetes underwent detailed metabolic studies before and

after a minimum of 3 month's Glibenclamide therapy. Treatment was associated

with a small but significant increase in body weight. Despite improvements in

almost all the measured parameters of glucose homeostasis include plasma glucose,

glycosylated hemoglobin (HbA1), hepatic glucose production and insulin-mediated

glucose disposal (Baynes et al., 1993).

15

CHAPTER TWO

MATERIALS AND METHODS

2.1 Experimental details

These experiments were carried out to investigate the hypoglycemic effect of

Nigella sativa seeds and how glucose metabolism is modulated in Wister albino

female rats fed the black seeds.

2.1.1 Experimental animals

Thirty female Wister albino rats weighing 65g to 115g were used in this

experimental study. They were randomly picked up from the experimental breeding

station at Department of Preventive Medicine, Faculty of Veterinary Medicine. The

rats were initially fed a standard basal laboratory rat diet for 7 days to be

acclimatized to the laboratory environment. All animals were maintained under

standard conditions (temperature 27-30°C, 12 h natural light and 12 h darkness);

they were housed identically in stainless cages and were supplied with water and

rodent chow ad libitum.

2.1.2 The basal rat diet

The rats were given a basal rat diet of the following composition:

Wheat flour 690g

Meat powder 165g

Oil 120g

Sodium chloride 4g

16

Nigella sativa seeds were obtained from the South western Ethiopia. The viability

of N. sativa seeds was confirmed by germination test according to Ista, (1976). The

rats were divided into five groups. For each group the calculated treatment was

mixed with 2g of the basal diet for each rat and fed to the animals first. Then the

rest of the measured daily requirements for each rat, 6g from the basal rat diet, were

supplied to the experimental animals.

2.1.3 Equipments

• Test tubes.

• Racks.

• Gloves.

• Capillary tubes.

• Pipettes.

• Tips.

• Centrifuge.

• Spectrophotometer.

• Florid Oxalate Blood Collection tube.

• EDTA Blood Collection tubes.

• Plane Blood Collection tubes.

• Eppendorf tubes.

2.2 Methods

2.2.1 Germination test

N. sativa seeds were obtained from Omdurman Market. The viability of N. sativa

seeds was confirmed by germination test according to Ista (International Seed

Testing Agency), 1976. Germination test was performed as follows:

17

1- 100 seeds were selected randomly and divided into four groups, 25 seeds in

each one.

2- Each group of the seeds was placed in a dish containing a wet filter paper and

incubated in incubator at 20oc for seven days.

3- On day four, the normal seedling that developed was count.

4- On day seven, the final count of the germination seeds was done.

5- All four set of germinated seeds should have a germination rate of at least

85%.

6- A germination rate of 85% is 85 normal seedlings of 100 seeds.

The seeds tested showed germination rate more than 85% and considered viable.

The seeds identification was done by Mr. Hamza Tag-Elsir Osman, Department

of Botany and Agricultural Biotechnology, Faculty of Agriculture, University of

Khartoum.

2.2.2 Experimental procedure

The Wister albino rats were divided into five groups A, B, C, D, and E, each

containing six rats. They were fed 8g normal basal rat’s diet per day for each rat.

The control group (A) was fed normal basal rats diet, while the treated groups (B,

C, D and E) were fed normal rat’s diet plus the following treatments calculated as

part from the 8 g of the basal diet for each rat.

Group (B): received Glibenclamide (Produced by CP Pharmaceuticals Ltd

Wrexham UK) 10mg/kg body weight.

Group (C): received N. sativa seeds 50mg/day for each rat (El-Daly, 1998).

Group (D): received N. sativa seeds 50mg/day for each rat + glucose 2g/kg body

weight.

Group (E): received glucose 2g/kg body weight.

Blood samples were collected from at the zero, 7th, 14th, 21th and 28th days.

18

2.2.3 Blood samplings

After 1 h of feeding 2g of experimental diet mixed with treatment, at zero time then

after 1, 2, 3 and 4 weeks after treatment, 1 ml of blood sample was collected from

the orbital venous plexus by plain capillary tubes into fluorinated test tubes ( to

avoid glycolysis by RBCs) for blood glucose determination. Plasma separated

spontaneously after centrifugation at 5000 rpm for 10 min, and then blood glucose

concentration was measured. Also at 14th and 28th days of experiment, blood

samples were collected in plain containers for insulin concentration. Serum was

separated and stored in clean labeled aliquots at -20oc until analyzed. For Alanine

aminotransferase (ALT) activity blood samples were collected at the 30th day of the

experiment from the orbital plexus by plain capillary tubes in fluoride containers.

Plasma was stored in clean labeled aliquots at -20oc until analyzed for ALT

activity. At day 30, Blood samples were also collected in EDTA tubes for

Hemoglobin determination and glucose-6-phosphate dehydrogenase activity

(G6PD). Plasma was separated by centrifugation at 5000 rpm for 10 min for G6PD

determination.

2.2.4 The body weight

Rats were weighed at the beginning and the end of the experiment.

2.2.5 Histopathological examinations

At the end of the experimental period, the rats were slaughtered and specimens of

liver and pancreas were taken immediately and fixed in 10% formaline for

histopathology.

2.3 Analytical methods

2.3.1 Determination of plasma glucose

19

Glucose concentration was determined according to the method described by

Trinder, (1969) and Teuscher et al., (1971).

Principle

The oxidation of glucose is catalysed by glucose oxidase (GOD). The resultant

hydrogen peroxide (H2O2) is oxidatively coupled with 4-aminophenazone and

phenol in the presence of peroxidase (POD) to yield a red quinoneimine dye, the

concentration of which at 546 nm is proportional to the concentration of glucose.

Alpha-D-glucose Mutarotase beta-D-glucose.

Beta-D-glucose + H2O + O2 GOD D-gluconic acid + H2O2

H2O2 + 4-aminophenazone + phenol POD quinoneimine + 4H2O

Reagent composition

1- Phosphate buffer (PH 7.5) 0.1mol/l

4- Aminoantipyrine 0.25mmol/l

Phenol 0.75mmol/l

Glucose oxidase 15KU/I

Peroxidase 1.5KU/I

Mutarotase 2KU/I

2- Glucose standard 100mg/dl or 5.5mmol/l

Procedure

1- Three test tubes were labeled as blank, standard and test.

2- 1 ml of working reagent was pipette into each one. 0.01 ml distelled water

was pipetted into the blank tube, 0.01ml of glucose standard was pipetted

into the standard tube, and 0.01ml plasma was pipetted into test tube.

3- Tubes were mixed and incubated for 10 min at 20 – 25oc.

20

4- 1ml content of each tube was delivered into cuvette and the absorbance of

the sample and standard were read against the blank at 500 nm using

spectrophotometer.

Calculations

Glucose (mg/dl) = A sample x concentration of standard

A standard

2.3.2 Determination of glucose-6-phosphate dehydrogenase activity (G6PD)

The determination of G6PD activity was done according to the method of Rodak,

(1995).

Principle

G6PD in the RBCS is released by a lysing agent present in the reagent. The G6PD

released catalyzes the oxidation of glucose-6-phosphate with the reduction of

NADP to NADPH. The rate of reduction of NADP to NADPH is measured as

increase in absorbance, which is proportional to the G6PD activity in the sample.

Glucose-6-phosphate + NADP G6PDH Gluconate-6-phosphate+NADPH+ H+

Procedure

1- 1ml of G6PD working reagent was pipetted into cuvette.

2- 0.01ml of whole blood with EDTA was added. The cuvette content was

mixed well and incubated for 5 min at 30oc.

3- 2ml of starter reagent was added, mixed well and incubated for another

5min at 30oc.

4- The initial absorbance A was read and the absorbance reading was

repeated after every 1, 2 and 3min.

Calculations

A/min = A2 – A1

5

21

G6PD activity (U/g Hb) = A x 4778

Hb (g/dl)

2.3.3 Determination of insulin

The determination of serum insulin was done according to Midgley et al., (1969)

using insulin Radioimmunoassay kit (RIA) IMK-414 (Beijing China, CIAE 1995).

Principle

The radioimmunoassay method in this test depends upon the competition between

iodine-125 labeled insulin and insulin in the sample (or in standard) for the limited

number of binding site on insulin specific antibody. After incubation for fixed time,

separation of bound from free is achieved by the PEG-accelerated double-antibody

procedure. The tube is then counted in a gamma counter, the counts being inversely

related to the amount of insulin present in the sample. By measuring the proportion

of iodine-125 labeled insulin bound in the presence of varying known amount of

insulin standards, the concentration of insulin in known samples can be interplated.

Assay protocol

1- Assay tubes were labeled and arranged in the assay rack.

2- 100 µl, 200 µl aliquots of buffer were pipetted into insulin standard (0), NSB

tube respectively.

3- 100 µl of insulin antibody solution were pipette into all tubes except total and

NSB tubes.

4- 100 µl of iodine – 125 labeled insulin solutions were pipetted to all tubes.

5- The tubes were mixed thoroughly and incubated at 37oc for 2 hours, the total

count tubes were been set aside at this stage.

6- 500 µl of separating agent solution were pipetted to all tubes except total

count tubes. Then tubes were mixed thoroughly and placed at 37oc for 15

min.

22

7- All tubes were centrifuged at 1500xg for 15min except total count tubes, and

the supernatants were decanted carefully.

8- All tubes were counted in a gamma counter.

Calculations

NSB% = NSB – Back grand cpm x 100

Total counts cpm – Back grand cpm

Maximum binding Bo% = zero standard cpm – NSB cpm x 100

Total counts cpm – Back grand cpm

B % = standard (or sample) cpm – NSB cpm x 100

Bo zero standard cpm – NSB cpm

2.3.4 Determination of Alanine aminotransferase (ALT)

The determination of serum ALT activity was done according to the method of

Gella et al. (1985) and Young, (1997).

Principle

Alanine aminotransferase (ALT or GPT) catalyzes the transfer of the amino group

from alanine to 2-oxoglutarate, forming pyruvate and glutamate. The catalytic

concentration is determined from the rate of decrease of NADH, measured at 340

nm; by means of the lactate dehydrogenase (LDH) coupled reaction.

Alanine + 2-oxoglutarate ALT pyruvate + Glutamate

Pyruvate + NADH + H+ LDH Lactate + NAD+

Reagents composition

Reagent (A): Tris 150 mmol/l, L-alanine 750 mmol/l, Lactate

dehydrogenase >1350U/L, PH 7.3.

Reagent (B): NADH 1.3 mmol/l, 2-oxoglutarate 75 mmol/l, sodium

hydroxide 148 mmol/l, sodium azide 9.5 g/l.

Procedure

23

1- Working reagent and the instrument were brought to reaction

temperature.

2- 1.0 ml of working reagent and 100 µl of sample were pepitted into

a cuvette at 30oc, mixed and inserted into the photometer.

3- Initial absorbance was recorded after 1 min and absorbance was

recorded at 1 min intervals thereafter for 3 min.

4- The difference between the consecutive absorbance and the average

absorbance difference per min ( A/min) were calculated.

Calculations

ALT concentration (U/L) = A/ min x Vt x 106

E x I x Vs

Whereas:

Vt: equal 1.1 at 30oc.

E: the molar absorbance of NADH at 340 nm is 6300.

I: 1 cm light bath.

Vs: equal 0.1 at 30oc.

2.3.5 Hemoglobin estimation

Haemoglobin (Hb) concentration was determined by cyanmethaemoglobin method

as describe by Van Kampen and Zijlstra (1961).

Principle

Ferrous ions of Hb are oxidized to the ferric state by potassium ferricyanide to

form methemoglobin, which reacts with cyanide to form cyanmethemoglobin that

can be measured colorimetrically.

Reagents

Cyanide reagent (Drabkin’s solution)

24

The reagent was prepared by dissolving 0.2g potassium cyanide, 0.05g of

ferricyanide and 0.14g of potassium hydrogen diorthophosphate in 1 litre of

distilled water.

Standard Hb solution

One ml of human Hb standard (Biosystem-Spain), with a concentration of 14.6 g/dl

was used as standard.

Procedure

1- Dry clean test tubes were prepared for sample and standard.

2- To each tube, 4 ml of cyanide reagent were added. Then 0.02 ml of blood

sample and Hb standard solution were added to the samples and standard

tube, respectively.

3- The tubes were allowed to stand for 15 min, and then the optical density

(O.D) was read at 540 nm in the colorimeter using cyanide reagent as blank.

Calculation

Hb concentration (g/dl) = O.D sample × 14.6

O.D standard

2.4 Oral glucose tolerance test (OGTT)

Oral glucose tolerance test was performed according to the method of Du Vigneand

and Karr, (1925).

Rats were fasted for 16 hour before being subjected to OGTT by intragastric

gavages with glucose solution to achieve a glucose load of 2 g/kg body weight.

Blood samples were collected from the orbital venous plexus by plain capillary

tubes at 0, 30, 60, 90 and 120 min and blood glucose was determined.

25

2.5 Histopathological methods

Rats were killed at the end of the experiment. The liver and pancreas were

harvested, cut into a small pieces and were fixed in 10% formalin. They were

embedded in paraffin and were cut into 5 µm thickness in microtome. These

sections were collected in slides and then stained with Hematoxylin and Eosin

before observation under light microscopy (Steward, 1960).

2.6 Statistical analysis

Data collected in various experiments were subjected to appropriate general linear

model (GLM) procedure of the statistical analysis using the SAS package. The SAS

was used to perform analysis of variance (ANOVA) and mean separations were

performed using Ryan-Einot-Gabriel-Welsch Multiple F test (REGWQ) (Day and

Quinn, 1989).

26

CHAPTER THREE

RESULTS

The present study was carried out to investigate the hypoglycemic effects of

Nigella sativa seeds and to show how glucose metabolism is modulated in normal

Wister albino female rats. The results were included estimation of serum glucose,

serum insulin and Oral Glucose Tolerance Test. Activities of glucose-6-phosphate

dehydrogenase and Alanine aminotransferase enzymes were assayed. Hemoglobin

concentration and animal body weight were also measured. In addition to the

histopathological changes of the liver and pancreas were examined.

3.1 The effects of feeding N. sativa seeds on blood glucose concentration

The effect of the treatments on glucose concentration is presented in table (1) and

(2).

The Glibenclamide treated group (B) demonstrated a highly significant decrease

(p<0.001) in the serum glucose level compared to the control group (A) at 7 and 21

days of the experiment. A significant (p<0.01) decrease was also seen at 14 and 28

days compared to the control group. The total mean of glucose level of the different

days is significantly (p<0.001) decreased compared to the total mean of the control

group. The N. sativa treated group (C) showed a significant (p<0.01) decrease in

plasma glucose level compared to the control group at all intervals. The total mean

of glucose of the different days showed a significant decrease in glucose level in

group (C) compared to the control group. N. sativa + glucose treated group (D)

revealed a significant decrease (p<0.001) in serum glucose level compared to the

control group after 7 days of treatment, but no significant difference was observed

after 14, 21 and 28 days of treatment compared to the control group. The total mean

of glucose of the different days showed a significant decrease in glucose level in

group (D) compared to the control group. On the other hand treatment with glucose

27

in group (E) showed a significant (p<0.001) decreased in serum glucose level after

7 days of treatment and showed no significant difference after 14 to 28 days of

treatment compared to the control group. The total mean of glucose at different

days showed no significant decrease in glucose level in group (E) compared to the

control group (table 1 and 2).

Table (1): The changes in the serum glucose concentration (mg/dl) among the

different groups of rats at different time intervals

Group

Time

Group A

Group B

Group C

Group D

Group E

0 day 67.750±4.49a 65.340±7.61a 52.350±6.16a 57.950±5.09a 67.983±2.00a

7 days 93.940±3.28a 26.367±4.81c 50.250±9.50b 50.880±3.98b 51.975±3.03b

14 days 47.740±4.53a 21.950±2.75b 26.050±3.68b 48.325±5.49a 48.767±8.12a

21 days 73.425±6.05a 21.220±3.51c 47.080±7.08b 67.420±3.46a 81.800±4.38a

28 days 35.675±2.77a 20.340±1.96c 27.300±3.47b 33.920±4.22a 34.175±2.94a

Mean ± SE within the same row with different superscript small letters are

significantly different at (p<0.01) based on ANOVA.

Group A: control group (received normal diet).

Group B: Glibenclamide treated group.

Group C: N. sativa treated group.

Group D: N. sativa + glucose treated group.

Group E: glucose treated group.

28

3.2 Blood insulin level

The effect of the four treatments on the level of the insulin is presented in table (2)

and fig. (1).

There is no significant different in the level of serum insulin in groups B, C, D and

E after 14 days treatment compared to the control group A. whereas, there is a

significant (p<0.01) increase in the level of serum insulin in groups B, C, D and E

after 28 days treatment compared to the control group A. In groups B, C and E

there is a significant increase in insulin level after 28 days compared to the level

after 14 days of treatment. There is insignificant increase in insulin level at 28 days

of treatment in group D compared to day 14.

Table (2): The changes in the serum insulin concentration (µIU/ml) in the

treated and control groups of rats

Group

Time

Group A

Group B

Group C

Group D

Group E

14 days 5.875±0.66a 5.775±1.22a 5.520±0.82a 7.525±1.32a 9.725±0.84a

28 days 5.833±1.97a 10.460±1.24 a b 10.480±1.47a b 10.660±1.74a b 15.700.43b








29

Fig. (1): The effect of N. sativa seeds on serum insulin level (µIU/ml) day 14th

and 28th

Bars having different small superscript letters are significantly different at (p<0.01)

based on ANOVA.






Ta Ta Ta

Ta

Ta

Ta

Tab Tab Tab

Tb

0

2

4

6

8

10

12

14

16

18

A B C D E

Insulin

(mIU/m

l)

Groups

day14th

day 28th

30

3.3 Oral glucose tolerance test (OGTT)

Results of OGTT carried out for the control and different experimental groups after

the end of the experimental period are showed in fig. (2).

After the application of the oral dose of glucose in group (A) the control the blood

glucose increased significantly (p<0.05) after 30 min but for groups (D and E) the

significant increase (P<0.05) was seen after 60 min (with very high score in group

E) and for group (B and C) was seen after 90 min. The blood glucose level returned

to the fasting levels after 2 h for all groups except groups (B and C) where it was

still significantly (P<0.05) higher than the fasting level.

Fig. (2): The effect of N. sativa seeds on oral glucose tolerance test

Values are mean ± SE of each group. (p<0.05) based on ANOVA.






0

50

100

150

200

250

300

350

400

450

o min 30min 60min 90mi n 120min

bloo

d glucose (m

g/dl)

time (min)

group A

group B

group C

group D

group E

31

3.4 Glucose-6-phosphate dehydrogenase activity (G6PD)

The effect of feeding N. sativa to normal albino rats for four weeks is presented in

table (4).

The activity of G6PD showed no significant change in groups B, C, D and E. only

numerically lower levels were observed in groups B and C and higher levels were

observed in groups D and E compared to the control group (A) after 28 days of the

experiment.

3.5 Alanine aminotransferase activity (ALT)

The levels of ALT activity in the different treated groups are presented in table (4).

The serum ALT activity at the end of the experiment is significantly (p<0.001)

decreased in group B compared to the control group (A). Whereas a significant

increase in serum ALT activity was seen in group C compared to the control group.

ALT activity is not significantly different in group D compared to the control

group. In addition, there is a significant (p<0.001) decrease in ALT activity in

group E compared to the control group (fig.3)

32

Fig. (3): The effects of N. sativa seeds on Alanine aminotransferase activity

(U/L) in the different treated groups of rats

Bars having different small superscript letters are significantly different at

(p<0.001) based on ANOVA.






3.6 The effects of N. sativa seeds on Hemoglobin concentration

The levels of Hemoglobin concentration in the different groups is presented in table

(2).

There is no significantly different between group B, C, D, and E and control group

(A) in hemoglobin concentration after 28 days of the experiments.

Ta

Tb

Tc

Ta

Tb

0

10

20

30

40

50

60

A B C D E

ALT activity (U/L)

Groups

33

3.7 The effects of N. sativa seeds on body weight

Changes in body weight in the different groups of rats are presented in table (3) and

(4).

There is no significant different between group B, C, D, and E and control group

(A) with respect to body weight at 0 and 30 days of the experiment.

Table (3): The effects of seeds on body weight (g)

Group

Time

Group A Group B Group C Group D Group E

0 day 93.4±4.86a 94.2±5.49 a 92.5±4.83 a 89.0±6.56 a 95.3±5.75 a

30 days 106.2±4.04 a 101.8±7.37 a 103.5±5.86 a 107.4±7.61 a 116.3±4.21 a








34

Table (4): Biochemical and Hemoglobin concentration changes in rats

Groups

Parameters

Group A Group B Group C Group D Group E

G6PD

(U/g Hb)

9.88±1.34a 6.40±1.71 a 7.28±0.93 a 12.44±2.61 a 11.40±3.54 a

ALT (U/L)

41.33±8.27 a 23.83±1.47b 53.83±2.33c 38.70±2.29 a 28.80±1.82 b

TMG (mg/dl)

63.89±4.5a 30.53±4.3c 41.30±3.9b 52.08±2.9b 57.21±3.9a

TMI (µIU/ml) 5.85±0.8 a 8.38±1.4 a b 8.0±1.1 a 9.27±1.2 a b 12.71±1.4 b

Hb (g/dl)

11.92±0.36a 8.80±0.67 a 11.43±1.15 a 11.78±1.15 a 10.50±1.05 a

TMBW (g)

99.80 ± 3.7 a 98.0±4.5 a

98.0 ±3.9 a 98.20±5.6 a 105.75 ±5.1 a








Abbreviations: G6PD, clucose-6-phosphate dehydrogenase; ALT, alanine

aminotransferase; TMG, total mean of glucose; TMI, total mean of insulin; Hb,

hemoglobin; TMBW, total mean of body weight.

35

3.8 Histopathological findings

The histological evaluation of pancreas in group B, C, D and E revealed normal

and healthy microscopic images fully comparable to control group A. The

histology of liver was normal in group C and D compared to the control group A

(fig. 4, 6 and 7). There is a focal infiltration of mononuclear cells especially around

blood vessels in group B (fig. 5). There is a focal infiltration of mononuclear cells

especially around blood vessels, some hepatocytes exhibit fatty degeneration and

focal areas of necrosis in glucose treated group E (fig. 8).

36

Fig. (4): liver from rats received normal basal rat’s diet. Notice no changes. (H

and E X 400).

37

Fig. (5): liver from rats received Glibenclamide (10mg/kg body weight) on

feed. Notice a focal infiltration of mononuclear cells especially around vessels.

(H and E X 400).

38

Fig. (6): liver from rats received N. sativa (50mg/day) on feed. Notice no

changes. (H and E X 400).

39

Fig. (7): liver from rats received N. sativa (50mg/day) +glucose (2g/kg body

weight) on feed. Notice no changes. (H and E X 400).

40

Fig. (8): liver from rats received 2g/kg body weight glucose on feed. Notice that

some hepatocytes exhibit fatty degeneration and focal areas of necrosis. (H

and E X 400).

41

CHAPTER FOUR

DISCUSSION

Nigella sativa (N. sativa) is widely traditionally used as hypoglycemic plant in the

Middle East, Northern Africa and Asia for treatment of various diseases. Many

studies proved that N. sativa has hypoglycemic effects (Uddin et al., 2002; Uddin

et al., 2005). The present study deals with its hypoglycemic effect and how this can

modulate glucose metabolism. Wister albino female rats were used as experimental

animals.

Normal glucose homeostasis represents the balance between intake (glucose

absorption from the gut), tissue utilization (glycolysis, pentose phosphate pathway,

tricarboxylic acid cycle, glycogen synthesis) and endogenous production

(glycogenolysis and gluconeogenesis) (Meyer et al., 2002). By this way, the body

tries to keep a constant glucose supply for cells by maintaining a constant blood

glucose level.

4.1 The effects of feeding N. sativa seeds on serum blood glucose concentration

Glucose is the most important carbohydrate in mammalian biochemistry because

nearly all carbohydrates in food are converted to glucose for further metabolism.

Blood glucose is derived from the diet, gluconeogenesis and from glycogenolysis.

In the present study blood glucose levels were decreased significantly in group

treated with Glibenclamide treated group B, N. sativa treated group C and N. sativa

+ glucose treated group D compared to the control group A. There was

insignificant decreased in blood glucose level in glucose treated group E.

Similar observations were obtained by Al-Awadi et al. (1985), who reported that

plant mixture extract comprising of N. sativa, Myrrh, Gum olibanum, Gum

asafetida have a blood glucose lowering effect. It stated that the N. sativa induced

increases in serum insulin concentration in rats which was accompanied by a

42

decrease in blood glucose level (Al-Awadi et al., 1985). Also Zaoui et al. (2002)

suggested that the daily gavages of hexane extract of N. sativa seeds in Wister-

kyoto rats for 12 weeks had reduced blood glucose level. Also N. sativa was

studied by Alhader et al. (1993) and Sinada, (2007) showed a significant

hypoglycemic effect.

The results obtained in the present study were also agree with El-Daly, (1994) who

indicated that blood glucose level significantly decreases in a group treated with N.

sativa for 7 and 14 days. Also significant increases in serum insulin level were

observed after treatment of N. sativa and there was significant decrease in blood

glucose level in a study by Farah, (2002). Other data showed that the hypoglycemic

effect of N. sativa occurred by oral treatment (20 mg/kg) N. sativa resulted, at least

partly, from stimulatory effect on B- cell function with consequent increase in

serum insulin level. Similarly (Eskander et al., 1995) and (El-shabrawy and Nada,

1996) were reported the hypoglycemic effect of N. sativa in combination with other

herbs in alloxan-induced diabetic rats.

N. sativa seeds are known to reduce blood glucose level in different animal species

(Haddad et al., 2003). These results were in line with the Kamal, (2006), who

reported that oral administration of water extract, N. sativa (30 ml/kg body weight)

significantly decreased serum glucose level in diabetic rats from 19.8 mmol to 9.7

mmol and increased serum insulin level from 0.5 mIu/l to 0.7 mIu/l. It is also agree

with Abdelmeguid et al. (2010), as they reported that N. sativa extract, oil and

thymoquinone significantly decreased the elevated serum glucose level and

significantly increased the serum insulin levels in streptozotocain induced diabetic

rats. He suggested that the hypoglycemic effect observed could be due to

amelioration of B- cells thus leading to the increased insulin level.

The mechanism of glucose reduction was reported by Kamal, (2006) who

suggested that N. sativa seeds induce preserving pancreatic B- cell integrity, thus

43

attributing to increased glucose metabolism, by increasing the serum insulin. On

the other hand, Farah et al. (2004) suggested that the reduction of blood glucose

from 391 mg/dl before treatment to 197 mg/dl after treatment in hamster is due

significantly to the low hepatic glucose production from gluconeogenic precursors.

4.2 The effects of feeding N. sativa seeds on insulin level

Insulin is a hormone that affects the carbohydrates metabolism by enhancing the

rate of glucose metabolism, decreasing blood glucose concentration and increasing

glycogen storage in tissues.

In the present study, there was no significant difference in the insulin level in

Glibenclamide treated group B, N. sativa treated group C, N. sativa + glucose

treated group D and glucose treated group E compared to control group A at day

14th of the experiment. Whereas, there were a significant (p<0.01) increased in

insulin level in group B, C, D and E after 28 days of the experiment compared to

the control group. In group B, C and E there is a significant increase in insulin level

after 28 days compared to 14 days of treatment. Whoever, there is insignificant

increase in insulin level at 28 days of treatment in group D compared to day 14.

The findings could agree with Al-Daly, (1994), who reported significant increase in

serum insulin level after administrating aqueous extract of N. sativa seeds orally

under light ether anesthesia for 7 and 14 days to rats. Similarly, Halit et al. (2002)

reported that N. sativa treatment had increased the lowered insulin levels in rats.

Moreover, Rchid et al. (2005) and Sinada, (2007) reported that using of different

doses of N. sativa had caused significant increase in serum insulin levels.

Gyton, (1966), presented the feedback mechanism between glucose and insulin,

when glucose concentration becomes elevated, the excess glucose acts directly on

the islets of langerhans to increase their secretion of insulin. Almost immediately,

more insulin become available to cause glucose transport into most cells of the

body, and this reduces the blood glucose level back towards normal. Goodner and

44

Porte (1972) reported that when beta cells are stimulated the insulin is discharged;

this is then followed by a refractory period in which responses to other wise potent

stimuli then promotes a second sustained secretion.

The increase in serum insulin level was observed by Farah, (2002), who suggested

that, the increase in serum insulin levels resulted from stimulatory effect of N.

sativa on B- cell function, which indicated that N. sativa has insulinotropic

properties. In contrast, Al-Hader et al. (1993) reported that the volatile oil of N.

sativa had produced a significant hypoglycemic effect on normal and alloxan-

induced diabetic rabbits without changes in insulin levels.

4.3 The effects of feeding N. sativa seeds on oral glucose tolerance test

After the oral dose of glucose in normal control group the blood glucose

significantly (p<0.05) increase at 30 and 60 min and then decreased at 90 min and

reached the fasting levels at 2 h. on the other hand blood glucose in Glibenclamide

treated group a significant (p<0.05) increase after 30, 60 and 90 min and

insignificant decreased after 2 h. Treatment with N. sativa showed insignificant

increase in serum glucose level after 30 min, a significant (p<0.05) increase in the

serum glucose level after 60 min and showed a significant (p<0.05) decrease in the

serum glucose level after 90 and 120 min of oral glucose dose to near normal.

These results showed that similar behavior was seen in the groups that received

Glibenclamide or only N. sativa. Treatment with N. sativa + glucose showed

insignificant increase in serum glucose level after 30 min, a significant (p<0.05)

increase in the serum glucose level after 60 and 90 min and showed a significant

(p<0.05) decrease in the serum glucose level after 120 min of oral glucose dose.

There is no significant different in glucose concentration after 120 min compared to

0 min. Treatment with glucose showed a significant (p<0.05) increase in serum

glucose level after 30 and 60 min, a significant (p<0.05) decrease in the serum

glucose level after 90 and 120 min of oral glucose dose.

45

These results therefore confirm the reduction of intestinal transport in vivo and are

consistent with increased insulin sensitivity observed in previous studies (Le et al.,

2004). They are also in agreement with results Al-Awadi et al., (1985) who

obtained an improved OGTT response in normal and streptozotocin rats treated

with a mixture of plants containing N. sativa. Indeed, in subsequent studies, the

same group failed to observe an improvement of fasting blood glucose or OGTT

response with N. sativa alone administered again at 0.5 g/kg for 1 week (Al-Awadi

and Gumaa, 1987).

Bouchra et al., 2008 reported that an OGTT challenges were carried out acutely at

the beginning of the study, 2h after the first N. sativa gavages, and chronically at

the end of 6-week treatment regimen. N. sativa was without significant effect after

acute administration but reduced the overall OGTT response after chronic

treatment.

4.4 The effects of feeding N. sativa seeds on glucose-6-phosphate

dehydrogenase activity

Liver plays a central role in blood sugar homeostasis. In diabetic state the activities

of hexokinase and glucose-6-phosphate dehydrogenase are decreased which due to

the total absence or insufficiency of insulin (Chandrasekaran and Leelavinothan,

2009). Thymoquinone up regulates the activities of both these enzymes in hepatic

tissues through insulin release and thereby it enhances the utilization of glucose for

cellular biosynthesis, which is marked by the significant decrease in plasma

glucose level (Chandrasekaran and Leelavinothan, 2009).

The activity of G6PD is insignificantly changed in group B, C, D and E compared

to the control group (A) after 28 days of the experiment. The results were agree

with Chandrasekaran and Leelavinothan, (2009) who reported that an oral

administration of Thymoquinone (major constituent of N. sativa seeds) for 6 weeks,

46

did not affect the G6PD activity in normal rats, but it significantly decreased in

diabetic rats.

4.5 The effects of feeding N. sativa seeds on Alanine transaminase (ALT)

activity

ALT is a cytoplasmic enzyme found in very high concentration in the liver. The

level of ALT in the blood is used as indicator for hepatocytes integrity; disrupt

hepatic parenchyma cells, with necrosis or the altered membrane permeability, will

lead to the leakage of the enzyme to the blood stream (Cornelius and Kaneko,

1986). In the present study the level of serum ALT showed a significant (p<0.001)

decreased in Glibenclamide treated group and Glucose treated group compared to

the control group. However, there is a significant (p<0.001) increase in serum ALT

activity in N. sativa treated group compared to the control group. There is no

significant change in ALT activity N. sativa + glucose treated group compared to

the control group. The present study suggested that, 50 mg/day of N. sativa seeds

for four weeks may slightly affect liver integrity.

The results were in line with Tennekoon, et al. (1991) who reported the increase in

serum ALT in rats consuming aqueous extract of N. sativa (250g/1000ml distilled

water) for consecutive 14 days when compared to the control group. Also Elhag,

(1998) who reported that serum ALT activity is slightly increased in rabbits fed

10% N. sativa seeds for two weeks and maintained level for the rest of the

experimental period. The oral administration of N. sativa led to marked and

significant increases in ALT enzyme activity of serum after 7 and 14 days post-

treatment (Al-Daly, 1994).

4.6 The effects of feeding N. sativa seeds on blood hemoglobin level

There is no significant (p<0.05) difference between groups B, C, D, and E and

control group (A) in Hemoglobin concentration at 28 days of the experiments

47

Similarly Chandrasekaran and Leelavinothan, (2009) reported that oral

administration of thymoquinone in normal rat did not affect the hemoglobin level

after 45 days of experiment compared to the control group.

4.7 The effects of feeding N. sativa seeds on body weight

In the present study initial and final body weight of experimental animals were

measured .There is no significant different between all treated groups (B, C, D and

E) compared to the control group with respect to the body weight at the end of the

experimental period, only group E showed a higher body weight compared to other

groups.

Similarly Brake, (2004), reported that administration of N. sativa seeds did not

causes any differences between the control and treated animals. Also Alhomidan et

al. (2002), reported that, feeding 20 and 100g/kg body weight of N. sativa seeds to

7day old broiler chicks did not affect growth. The results were in line with

Chandrasekaran and Leelavinothan, (2009); they reported that oral administration

of thymoquinone in normal rat did not affect the body weight after 45 days of

experiment compared to the control group.

Similarly Sinada, (2007) has recently reported that the live weight gain in broiler

chicks was not affected by crushed N. sativa seeds mixed with commercial ration

compared to the control group. Zaoui et al. (2002) found that the fixed oil of N.

sativa slow growth rate of normal rats.

4.8 Histopathological findings

Histopathological examination of pancreas showed no evidence of degeneration

changes in the islets of langerhans in all treated groups for four weeks.

However, histopathological examination of livers showed no evidence of

degenerative changes in hepatocytes in animals treated with N. sativa for four

weeks and animals treated with N. sativa + glucose compared to the control group.

There is a focal infiltration of mononuclear cells especially around blood vessels in

48

group B. There is a focal infiltration of mononuclear cells especially around blood

vessels, some hepatocytes exhibited fatty degeneration and focal areas of necrosis

in group E.

Similarly Al-Daly, 1994 reported that oral administration of N. sativa extract did

not have a superimposing effect. Focal infiltration of mononuclear cells especially

around blood vessels in group B it may be due to defensive role against

degenerative changes in the hepatocytes. Arseculeratna et al., (1981, 1985) have

previously reported that some plant toxins may damage the hepatic cells at the

molecular level without causing overt histopathological changes.

Bouchra et al., 2008 reported that the histological study of kidney, pancreas and

liver samples after chronic administration of N. sativa aqueous extract over 6 weeks

showed these tissues to be normal and healthy. This is also consistent with the high

safety margin observed in rats and mice with N. sativa fixed oil (Zaoui et al.,

2002).

49

CONCLUSIONS

In the present study it was found that daily oral administration of Nigella sativa

seeds, supplemented to the basal rat diet, for four weeks resulted in a significant

reduction in plasma glucose concentration and significant increase insulin level,

Suggested that this seeds could be used as a hypoglycemic agent to reduced blood

glucose level. There was no histopathological change in N. sativa treated group but

there was a significant increase in ALT activity, it may be due to the hepatocytes

damage at the molecular level. Also the live weight gain, G6PDH activity and

hemoglobin concentration in treated groups were not affected by dietary treatment

compared to the control group.

RECOMMENDATIONS

There are several lines of research arising from this work which should be pursued.

• All key enzymes in glucose metabolism should be studied.

Enzymes of glycolysis eg. hexokinase or glucokinase, phosphofructokinase-

1 and pyruvate kinase that cause decreases in blood glucose concentration.

Glycogen synthase and glycogen phosphorylase enzymes that decreases and

increases blood glucose level respectively.

Gluconeogenesis pathway enzymes eg. glucose-6-phosphatase, fructose-1, 6-

bisphosphatase, phosphoenol pyruvate carboxykinase and pyruvate

carboxylase which increase blood glucose level.

• Results provide convincing evidence that intestinal glucose transport

inhibition represents an important component through which Nigella sativa

50

seeds can reduce blood glucose in this context. Future studies will need to

elucidate the active principles as well as molecular sites of action responsible

for the effect of Nigella sativa seed on the intestinal absorption.

51

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