i

The National Ribat University

Faculty of Pharmacy

Pharmacognostical Investigation of

Cucurbita maxima, Cucurbitaceae (Pumpkin)

Fruits, Leaves, seeds, flowers

A Thesis Submitted For Fulfilment of The

Requirement Of Master Degree In Pharmacognosy

By:

Hoyam Hashim Siddig Ahmed

B.pharm-2007

The National Ribat University-Faculty of Pharmacy

Supervisor:

Dr. AbdAllah M. Alhassan.

2016

ii

بسم هللا الرحمن الرحيم

"واذا مرضت فهو يشفين"

صدق هللا العظيم

(80األية -) الشعراء

DEDICATION

iii

This thesis is dedicated to my beloved parents,

for the sacrifices that they have made,

the unflagging love, and support,

that they have provided throughout

my life that has permitted me

to achieve my lifelong goals.

I would like to dedicate this thesis to my brother

, my sisters

To my husband for his support,

And to my beloved baby Morwan.

Hoyam Hashim

ACKNOWLEDGMENTS

iv

This work was made possible with the help of Allah. No doubt I will

not forget my supporters in this work.

To who encouraged me, special thanks and gratitude to my

supervisor, Dr. AbdAllah M. Alhassan for his supervision, patient

guidance, and constructive criticism on my research and most of his

professionalism and attention to detail, which conclude the level of

this research.

My great and honour thanks for the Research Institute of Medicinal

and Aromatic Plants, for their helpness to do this thesis. My thanks

extended to The Central Petroleum Laboratories for GC-Mass

Analysis.

My thanks for the team of Phytochemistry laboratory from faculty of

Pharmacy-The National Ribat University, Ustaz. Abdallah Ebrahim

for his help throughout this study; Cordial thanks to my husband Ehab

siddig Mohammed.

Finally, I would like to thank everyone who participated

enthusiastically in helping me to carry out my research successful.

Hoyam Hashim

TABLES OF CONTENTS

v

Chapter Particulars Page No

Dedication ii

Acknowledgement iii

Table of contents iv

List of tables vii

List of figures viii

Arabic abstract x

English abstract xi

Chapter one: INTRODUCTION & LITERATURE REVIEW

Chapter

one:

1. Introduction & Literature Review 1

1.1 Introduction 2

1.1.1 Overview 2

1.1.2 Traditional Medicine 2

1.1.3 Islamic Medicine 2

1.1.4 Ethnopharmacology 4

1.1.5 Importance of Phytochemistry in Therapy 4

1.1.6 Terms & definitions 5

1.2 Objectives 6-7

1.3 Literature Review 8

1.3.1 Introduction 8

1.3.1.1 Description 8

1.3.1.2 Pumpkin in Quran & Sunnah 10

1.3.1.3 Habitation &Distribution 10

1.3.1.4 Cultivation 10

1.3.2.1 Pumpkin in Traditional Medicine 11-19

Chapter two: MATERIALS AND METHODOLOGY

vi

Chapter

two:

2. Materials and Methodology 20

2.1 Materials 21

2.1.1The plant 21

2.1.1.2 Collection and Identification of tested

plant

21

2.1.3 Instruments and chemicals 22-24

2.1.3.3 Reagents and solution 24-25

2.1.4 Micro-organisms 25

2.2 Method 26

2.2.1.1 Extraction methods 26-27

2.2.1.2 Phytochemical screening tests 27-30

2.2.2 Antibacterial experiment 31-32

2.2.3 The separation 32

2.2.3.1 Separation by thin layer chromatography

TLC

32-33

2.2.3.2 Separation and Identification by Gas

chromatography-mass spectrometry GC-Ms

33-34

Chapter three: RESULTS

Chapter

three:

3. Results 35

3.1 Phytochemical studies results 36-39

3.2 The antibacterial activity results 40-48

3.3 The separation results

3.3.1 Thin layer chromatography

49-57

3.3.2 Gas chromatography- mass spectrometry

(GC.MS) Results

58-79

Chapter four: DISCUSSION

vii

Chapter five: CONCLUSION AND RECOMMENDATIONS

REFERENCES:

Appendix:

List of Tables

Chapter four 4.1 Discussion 81

4.2 correlations between the identified/

detected phytochemicals and current historical

uses

83-95

Chapter five 5.1 Conclusion 97-98

5.2 Recommendations 99

References 101-113

appendix 114

viii

No Name Page

1.1 Structure of cucurbitacins A, B, C, D 15

2.1 Instruments used during course of this study 22

2.2 Chemicals 23-24

2.3 Micro-organisms 25

3.1 The yields percentages of ethanol extracts of each part of

pumpkin

36

3.2 the weights and yields percentages of the different three

extracts of each four parts of pumpkin (Continuous

extraction- 150gms)

37

3.3 Phytochemical screening tests results 39

3.4 Antibacterial Activity results of Pumpkin seeds, leaves,

flowers and fruit extracts against four standard bacteria

43

3.5 The Chromatogram and Rf values of Pumpkin Seeds

extracts

50

3.6 The Chromatogram and Rf values of Pumpkin Flowers

extracts

51

3.7 The Chromatogram and Rf values of Pumpkin Leaves

extracts

54

3.8 The Chromatogram and Rf values of Pumpkin Fruit extracts 55

3.9 The name, formula, RT, MW and area% of phytochemicals

identified in pumpkin flowers by GC.MS

58

3.10 The structure of identified phytochemicals in pumpkin

flowers

59

3.11 The name, formula, RT, MW and area% of phytochemicals

identified in pumpkin fruit by GC.MS

61.63

3.12 The structure of identified phytochemicals in pumpkin fruit 65-67

3.13 The name, formula, RT, MW and area% of phytochemicals

identified in pumpkin leaves by GC.MS

68-69

3.14 The structure of identified phytochemicals in pumpkin

leaves

71-72

3.15 The name, formula, RT, MW and area% of phytochemicals

identified in pumpkin seeds by GC.MS

73-75

3.16 The structure of identified phytochemicals in pumpkin

seeds

76-79

List of Figures:

ix

No Name Page

1.1 Pumpkin Plant 8

1.2 Pumpkin Fruit 8

1.3 Pumpkin Seeds 9

1.4 Pumpkin Leaves 9

1.5 Pumpkin Flowers 9

1.6 Basic structure of cucurbitacins 14

1.7 Structure of Beta-carotene 16

1.8 Structure of Alpa-Tocopherol Vit E 16

1.9 Structure of Vit A 18

1.10 Structure of Vit C 18

1.11 Structure of Vit B5 19

3.1 Charts showing comparison between the diameters of the

growth inhibition zones of three successive extracts of

pumpkin seeds against the four tested bacteria

43

3.2 Charts showing comparison between the diameters of the

growth inhibition zones of three successive extracts of

pumpkin leaves against the four tested bacteria

44

3.3 Charts showing comparison between the diameters of the

growth inhibition zones of three successive extracts of

pumpkin fruit against the four tested bacteria

44

3.4 Charts showing comparison between the diameters of the

growth inhibition zones of three successive extracts of

pumpkin flowers against the four tested bacteria

44

3.5 The growth inhibition zones of pumpkin seeds, leaves,

fruit and flowers petroleum ether extracts on E. Coli

45

3.6 The growth inhibition zones of pumpkin seeds, leaves,

fruit and flowers (petroleum ether, chloroform, ethanol)

extracts on S. aureus

46

3.7 The growth inhibition zones of pumpkin seeds, leaves,

fruit and flowers (petroleum ether, chloroform, ethanol)

extracts on Ps

47

3.8 The growth inhibition zones of pumpkin seeds, leaves,

fruit and flowers (petroleum ether, chloroform, ethanol)

extracts on B. subtilis

48

3.9 TLC of pumpkin seeds extracts (p. Ether, chloroform,

ethanol) respectively from left to right

52

x

3.10 TLC of pumpkin flowers extracts (p. Ether, chloroform,

ethanol) respectively from left to right

53

3.11

TLC of pumpkin leaves extracts (p. Ether, chloroform,

ethanol) respectively from left to right

56

3.12 TLC of pumpkin fruit extracts (p. Ether, chloroform,

ethanol) respectively from left to right

57

3.13 GC.MS chromatogram of the general ethanol extract of

pumpkin flowers

60

3.14 GC.MS chromatogram of the general ethanol extract of

pumpkin fruit

64

3.15 GC.MS chromatogram of the general ethanol extract of

pumpkin leaves

70

3.16 GC.MS chromatogram of the general ethanol extract of

pumpkin seeds

75

4.1 Chemical structure of squalene, β-carotene, vit K1, A, E 86

4.2 Chemical structure of Phytol, vit K1, E

87

xi

الملخص العربى:

تعمال التقليدى ولبيان المعجزة الربانيه من انبات نبات القرع سهذه الدراسة اجريت لتأكيد او دحض اال

سيدنا يونس عليه السالم من خالل:علي

اربعة اجزاء من القرع وهى البذور, االوراق, الثمار والزهور. دراسة -

ت الدراسة وجود تاجراء المسح الكيميائى النباتى لثالث مستخلصات من كل جزء من القرع, حيث اثب -

ى كل الجزاء االربعه.اليكوسيدات فمواد اساسيه مثل االستيرويد, الفالفونيات, الصابونيات والق

تم تقييم الفعالية لكل المستخلصات الثاللثة من االجزاء االربعه لمعرفة فعاليتها ضد اربعه انواع من -

البكتيريا, نوعان من موجبة الغرام )العصوية الرقيقة والعنقودية الذهبية( ونوعان من البكتيريا سالبة

باستخدام طريق االنتشار المعياري. حيث اظهرت النتائج اريه(الغرام )االشريكيه القولونيه والزائفة الزنج

فعاليه مهمه لكل المستخلصات ضد انواع البكتيريا المستحدمه في البحث.

لى الغاز ضع المستحلص االيثانولى العام للبذور والزهورو االوراق والثمار للتحليل االاخ -

تم التعرف عل تسعة مركبات فى الزهور, واثنان كروماتوغرافى لمعرفة المركبات الموجوده فيها , حيث

وعشرون فى الثمار وسته وثالثون فى البذور وتسعة عشر مركب فى االوراق.

تم البحث عن االثار العالجيه والحيويه لكل المركبات السبعه والثمانون التى تم التعرف عليها فى -

الناحية البذور واالوراق والثمار والزهورمنفوائد جزاء االربعه من نبات القرع , حيث اثبتت الدراسهاال

العالجية، ، وفي موضوعنا هذا تبين لنا أن اليقطين يتميز بصفات وخصائص تجعله من أولويات النباتات

.التي تصلح ألن تكون مجاورة ليونس عليه السالم

التوصيات : نوصى باجرا دراسات متقدمه وضرورة عمل الرنين المغنطيسى وتحليل باالشعه تحت

مكونات الكيمائيه لكل جزء الحمراءلكل االجزاء وكذلك البد من دراسه اكلينيكيه لتوضيح العالقه بين ال

وخواصهاالدوائيه

xii

English abstract:

The present study was carried to confirm or refute on scientific basis, the

traditional uses of pumpkin plant in the treatment of many diseases via:

- First, basic information about pumpkin (seeds, leaves, fruits, flowers) was

collected from internet, books and the chemical materials, reagents and

equipments needed for laboratory experiments were prepared.

- Four parts of pumpkin were collected; fruits and seeds were obtained from the

local market of Khartoum state while leaves and flowers were collected after

planting of pumpkin in Khartoum university farm for three months.

- The collected plant materials were dried in shade and powdered to be ready for

next steps.

- The active constituents of different parts were extracted in soxhletˊs apparatus

using petroleum ether, chloroform and ethanol in successive manner.

–The twelve extracts were dried and reserved in refrigerator for another

experiments (petroleum ether extract, chloroform extract and ethanol extract

from each four parts of pumpkin plant).

- TLC technique was used to separate and know active constituents in each

extracts by using different types of solvents in different percents like hexane:

chloroform: toluene (10:25:75) and benzene: ethyl acetate (95:5) and other

types to the best one.

- Phytochemical screening tests were carried out for all extracts and the

presence of different phytochemical groups like tannins, glycosides, alkaloids

and other materials by using special tests were detected in the four parts of

pumpkin.

- Assessment of antimicrobial activity of the 12 extracts was carried out against

two standard gram positive bacteria (Bacillus subtilis, Staphylococcus aureus)

and two gram negative bacteria (E coli, Ps aeruginosa) and the results showed

a significant activity of all four parts of pumpkin against the tested bacteria..

- Finally, ethanol extract of seeds, flowers, fruit and leaves were prepared for

GC-MS analysis to determine the chemical structure and name of active

substances that can be found in each extracts of four part of pumpkin plant to be

matched with the reported folkloric uses of pumpkin, the result showed the

presence of nine phytocomponents in flower, twenty two in fruit, thirty six in

seeds and nineteen in leaves. The biological and therapeutic effects of these

eighty seven phytocomponents were collected and the results proved that

pumpkin seeds, leaves, Flowers and fruit to hav a big usefulness in curing

diseases and provide an important source of medicine (the medicinal importance

xiii

of pumpkin plant due to the presence of chemical substances that produce a

distinct physiological action on the human body).

This study describes the miracle event with our prophet Yunus (peace be upon

him pbuh) from the chemical and the pharmaceutical point of view.

Recommendation: Advanced studies on all parts of pumpkin, respectively

advance analysis such as IR and NMR are necessary. Clinical studies are

recommended to elucidate the correlation between chemical ingredients and

their pharmacological properties, and to confirm the data of traditional

medicine.

xiv

Chapter One:

Introduction &Literature Review

xv

1. Introduction & Literature Review

1.1 Introduction:

1.1.1 Overview:

Pharmacognosy is the parent of all pharmacy sciences, it has been coined by

merger of two Greek words Pharmakon (drug) and Gnosis (knowledge) i.e.,

knowledge of drugs. The nomenclature pharmacognosy was used for the first

time by C.A.Seydler, amedical student in Halle/ Saale, Germany, who

emphatically employed Analetica Pharmacognostica as the main title of his

thesis in the year 1815 (Evans, 2002).

Pharmacognosy is closely related to botany and plant chemistry and, indeed,

both originated from the earlier scientific studies on medicinal plants. As late at

the beginning of the 20th century, the subject had developed from the botanical

side and being concerned with the description and identification of drugs, both

in the whole state and in powder and with their history, commerce, collection,

preparation and storage (Evans,2002).

1.1.2 Traditional Medicine:

Plants have formed the basis of traditional medicine (TM) which has been used

for thousands of years. Traditional medicine is a comprehensive term used to

refer to all TM systems such as traditional Chinese medicine, Indian and Islamic

medicine, Ancient medicine and to various forms of medicine (WHO, 2002).

Traditional medicine deal with health practices, knowledge and beliefs

incorporating, plant, animal and mineral-based medicines, spiritual therapies,

manual techniques and exercises; applied singularly or in combination to treat

or to diagnose and prevent illnesses or maintain well-being (Karou et al., 2007).

The use of plant-based medicines continue to play an essential role in health

care, it has been estimated that approximately 80% of the population in

developing countries depend on TM for their primary health care (WHO, 2002;

Ghorbani et al., 2006; Azaizeh et al., 2010; Singh and Jain, 2011).

1.1.3 Islamic Medicine: Traditional Arabic and Islamic Medicine (TAIM) therapies have shown

remarkable success in healing acute as well as chronic diseases and have been

utilized by people in most countries (Azaizeh et al., 2010).

Many of things which humanity discovering every day in all branches of

science including Medicine were predicted by the Holly Quran over 14

centuries ago.

As such, Quran has many constructive ideas to offer in the fields of health and

medicine.

In order to find out what Islam teaches, one has to read the Qur'an and the

Hadith. Allah says in the Qur'an in Surah (Ash-Shu'ara') about healing from

diseases: (and when I sicken, then He (Allah) heals me). ( )واذا مرضت فهو يشفين 80األيةالشعراء

xvi

As far as the prophet is concerned, related to the healing of diseases, it was

reported by Jabir bin Abdullah that prophet Muhammad (Prayers and peace be

upon him (ppbuh) said: (for any disease there is a cure((Al-Albani,1990).

The Holly Quran tell us many miracle healing stories, one of these stories is the

story of Prophet Yunus Ibn Matta (pbuh). Prophet Yunus (pbuh) was sent by

Allah to a big town where the people had forgotten almighty Allah orders and

did many things which Allah had forbidden. Yunus (pbuh) told them. ―You

should worship Him alone and do well; otherwise a severe punishment will

come upon you! ‖

But Yunus (pbuh) soon discovered that the people did not want to listen to him.

He lost patience with them and left the town in anger.

Afterwards, Yunus (pbuh) decided to go across the sea, and boarded a ship for

the voyage. But when the ship was in the middle of the ocean, Yunus (pbuh)

suffered a great misfortune. He was thrown overboard and swallowed up by an

enormous fish! (Sperm Whale). Fortunately, though, the Whale had swallowed

Yunus (pbuh) in one big gulp, so he landed in its stomach unhurt. It was very

dark inside the fish's stomach, and Yunus (pbuh) grew very fearful.

In his loneliness, he started to think over what had happened in the town, and

came to realize that he should not have acted so hastily and in such a quick-

tempered manner. Instead, he should have stayed and kept on speaking to the

people and ask them to return to Allah. (Al-Dimashqi, 1990).

In this despair, Yunus (pbuh) started to pray with all his heart to Allah. He said:

(Allah, there is no Allah apart from You, you alone do I praise and honour, I

have done wrong; if you do not help me, I shall be lost forever)(Anbeia’a 87).

حانك )وذا النون اذ ذهب مغاضبا فظن ان لن نقدر عليه فنادى في الظلمات ان ال اله اال انت سب

ت من الظالمين( االنبياءانى كن

Allah heard Yunus (pbuh)'s prayer, and he caused Yunus ( pbuh) to come out of

the whale's stomach, and to be swept by the waves of the sea onto the shore.

Poor Yunus (pbuh) was in a terrible state! He lay on the shore, weak, ill and

helpless. He felt dreadfully miserable, but Allah caused a tree to grow and this

tree provided Yunus (pbuh) with shade and nourishing fruits. Before long,

Yunus (pbuh) had recovered his health and strength (Al-Dimashqi,1990).

Allah Said in Quran:

(and Yunus too was one of the messengers (139) call to mind when he fled to the

laden ship (140) cast lots, and was among the losers (141) then a fish swallowed

him, and he was blameworthy (142) had he not been one of those who glorify

Allah (143) he would certainly have remained in its belly till the day of

resurrection (144) but we threw him on a wide bare tract of land while he was

ill(145) and caused a gourd tree to grow over him (146) and we sent him forth to

nation of a hundred thousand or more (147) and they believed, so we let them

enjoy life for a while (148) (Al Saffat).

xvii

( فالتقمه 141( فساهم فكان من المدحضين)140(اذ ابق الى الفلك المشحون)139)وان يونس لمن المرسلين)

(فنبذناه بالعراء 144)( للبث فى بطنه الى يوم يبعثون143)( فلوال انه كان من المسبحين142الحوت وهو مليم)

( فامنوا فمتعناهم 147يزيدون)لف او (وارسلناه الى مائة ا146)( وانبتنا عليه شجرة من يقطين145وهو سقيم)

( الصافات 148الى حين)

1.1.4 Ethnopharmacology: Ethnopharmacology, a term only in 1967can be defined as the scientific study

of materials used by ethnic and cultural groups as medicines (El-Sakka, 2004)

and is nowadays much more broadly defined:

As the observation, identification, description and experimental investigation of

the ingredients and the effects of the ingredients and the effects of such

indigenous drugs are a truly interdisciplinary field of research which is very

important in the study of traditional medicine.

The ethnopharmacology approach is based on botany, chemistry and

pharmacology; based on these consideration, Ethnopharmacology is defined as

the interdisciplinary scientific exploration of biologically active agents

traditionally employed or observed (Heinrich and Gibbons, 2001; Raza, 2006).

Thus, it plays an immense role in the evaluation of natural products and more

particularly herbal drugs from traditional and folklore resources.

1.1.5 Importance of Phytochemistry in Therapy:

Phytochemical studies deal with revelation and understanding of

phytoconstituents, as much as possible conserving their bioactivities, and are on

how to standardize them and compared with the crude herbal methods.

Phytochemical methods mainly involve extractions, purifications and isolations

of the active compounds in plants.

All plants produce chemical compounds as part of their normal metabolic

activities; these phyto-compounds are divided into primary metabolites such as

Carbohydrates, proteins, fats and oils are utilized as food by human and

animals; and secondary metabolites which are serving specific function and can

have therapeutic actions in humans and can be refined to produced drugs.

Alkaloids, polyphenols, glycosides and terpenes are secondary metabolites,

such compounds usually exert peculiar, unique and specific active physiological

effects responsible for their therapeutic and pharmacological functions

(Vallisuta and Olimat, 2012).

About 25% of prescribed drugs are obtained from phytochemical in higher

plants; plants are safe means of obtaining drugs. About 250,000 higher plants

have promising phytochemical, half of which are located in tropical forests;

60% of these have their biological activities established, while about 15% of

xviii

them have their phytocompounds isolated and reported (Vallisuta and Olimat,

2012).

1.1.6 Terms & definition: A pumpkin is a gourd-like squash of the genus Cucurbita and the family

Cucurbitaceae (which also includes gourds). It commonly refers to cultivars of

any one of the species Cucurbita pepo, Cucurbita mixta, Cucurbita maxima,

and Cucurbita moschata, and is native to North America. They typically have a

thick, orange or yellow shell, creased from the stem to the bottom, containing

the seeds and pulp. Pumpkins are widely grown for commercial use, and are

used both in food and healing (Itis. Gov.2009).

The word pumpkin originates from the word pepon, which is Greek for ―large

melon".

The French adapted this word to pompon, which the British changed to

pumpion and later American colonists changed that to the word we use today,

"pumpkin". The origin of pumpkins is not definitively known, although they are

thought to have originated in North America. The oldest evidence, pumpkin-

related seeds dating between 7000 and 5500 BC, were found in Mexico.

Pumpkins are a squash-like fruit that range in size from less than 1 pound (0.45

kilograms) to over 1,000 pounds (450 kilograms) (The Columbia Encyclopedia.

2004).

xix

Goal:

The goal of this study is to improve the research and knowledge on scientific

signals in Quran and Sunnah in the diseases prevention and treatment (public

health).

1.2 Objectives:

1. To investigate pumpkin fruit, seeds, leaves and flowers pharmacognostically

and biologically.

2. To detect, in depth, the chemical content and the active ingredient (s) of

pumpkin flowers, fruit, seeds and leaves.

3. To separate and determine the structures of the main ingredient (s) in

pumpkin extracts using advanced instrumental analysis.

4. To re-evaluate of the four selected pumpkin parts for investment in natural

remedies.

5. To identify the relationship between the phyto-chemicals detected or

identified and the reported current and historical uses of different parts of

pumpkin in complementary and alternative medicine (CAM).

6. To discuss results of research in view of Quranic wisdom.

Hypothesis:

The presence of different natural chemical ingredients may have contributed to

the treatment of Yunus (peace be upon him) after suffering from much severe

body damage as a result of being swallowed by the Whale.

Problem statement:

There are two principal questions appear in this research; The first one is why it

was chosen by Allah pumpkin herb to be the protective, healing and food of the

prophet Yunus (pbuh)? The second question that was asked what is the secret in

pumpkin?

The scholar said that the pumpkin is a gourd and was chosen by Allah because

the pumpkin is characterized by several advantages: the leaves are characterized

by big size and multitude, so it can protect from the blazing sun in that

wilderness where no shelter from the heat of the sun. But in plant kingdom we

have several plants with the same or more magnification and multitude than

pumpkin! (Al Kortobi, 1987; Al- Dimashqi, 1988).

xx

Others said that the leaves are smooth and repellent to insects, hence it protected

the prophet Yunus from the flies (Al-Kortobi,1987;Al-Dimashqi,1988)

In fact, many Hadeeth ensure the benefit and the great value of pumpkin in

therapy. Our prophet Mohamed (ppbuh), as in Sonan Alnisaey, liked pumpkin

and said: “the tree of my brother Yunus Ibn Matta” (Al-Askalani, 1985).

As it was mentioned in Saheeh Bukhari, Anas Ibn Malek had said that, a tailor

invited our prophet to have a meal that consists of bread of barley and a soup of

pumpkin and dry meat, also was said Debaa, and so, the prophet was careful to

eat it, since I, Anas Ibn Malek, have seen him eating it, I liked it (Al- Bukhari,

1986).

نعه، لطعام ص -صلى هللا عليه وسلم-عن أنس بن مالك رضي هللا عنه يقول: "إن خياطا دعا رسول هللا

-ول هللا إلى ذلك الطعام فقرب إلى رس -صلى هللا عليه وسلم-قال أنس ابن مالك: فذهبت مع رسول هللا

تتبع الدباء من ي -صلى هللا عليه وسلم-زا ومرقا فيه دباء وقديد، فرأيت النبي خب -صلى هللا عليه وسلم

الت: ان . وعن عائشه رضى هللا عنه ق()صحيح البخارى"حوالي القصعة، قال فلم أزل أحب الدباء من يومئذ

اء فانه يشد قلب دبرسول هللا صلى هللا علي وسلم كان يقول :) يا عائشه اذا طبختم قدرا فاكثروا فيها من ال

الحزين(.

Justification:

This is one of the studies which take in consideration the scientific explanation

of wisdom in Qura’n and Sunnah from pharmaceutical point of view based on

Arabian Islamic medicine and supported by traditional medicine.

xxi

1.3 Literature Review:-

1.3.1 Introduction:-

The pumpkin is a gourd of the genus Cucurbita and the family Cucurbitaceae, it

is refers to cultivars of any one of the species Cucurbita pepo or Cucurbita

maxima (Cred, 2008).

They typically have a thick orange or yellow shell, creased from the stem to

bottom, containing the seeds and pulp.

1.3.1.1 Description:-

-Pumpkin plant: it is leafy green vegetable being an annual, herbaceous plant

cultivated for its fruits, seeds, flowers and leaves (Tyler, 1993).

Figure 1.1: Pumpkin Plant:

-Fruit: pumpkin is a squash like fruit that range in size from less than 0.45kg to

over 500kg and it varies greatly in shape ranging from oblate to oblong, they

have a moderately hard rind with thick, edible flesh below and a central cavity

containing numerous seeds(Credo,2008).

Although pumpkin fruits are usually orange or yellow, some fruits are dark

green, pale green, orange-yellow, white and grey and can be stored without

damage for a long time and that due to the thickness of it is wall (Dhiman et al,

2012).

Figure 1.2: Pumpkin Fruit:

xxii

-Seeds: pumpkin seeds, also known as pepitas are small, flat, green, edible

seeds; Most pumpkin seeds are encased in tan or soft white husk (testa)

(Watson, 2012).

Figure 1.3: Pumpkin Seeds:

-leaves: pumpkin has large, green, very soft, shade, lobed leaves with well

marked veins.

Figure 1.4: Pumpkin Leaves:

-Flowers: the big, bright, yellow colourful flowers of pumpkin are often

produced in abundance, especially the male flowers when compared to number

of the female flowers. Pumpkin is hermaphrodite has both male and female

flowers in the same plant, the female flower is distinguished by the small ovary

at the base of the plant, these flowers have extremely short life spans and only

open for as short as time as one day (Watson,2012).

xxiii

Figure 1.5: Pumpkin Flowers:

1.3.1.2 Pumpkin in Quran & Sunnah:-

Pumpkin mentioned in Quran Allah says :(( and we caused by a tree of gourd to

grow over him)) (Al-saaffat, 146), as the wisdom of the god in the pumpkin to

germinate pumpkin plant on the prophet Yunus for food and healing.

The Mufassirin (commentators) said: The gourd (al-Yaqtin) is a kind of

pumpkin. Some of them described the benefits of the pumpkin, such as: it grows

quickly, it provides shade, it has large, smooth leaves, it keeps flies away and its

fruit provides good nourishment: it can be eaten raw or cooked. It is known that

the Messenger of Allah (peace and blessings be upon him) liked this kind of

pumpkin and used to look for it on the plate of food (Tafsir Ibn Kathir).

Ibin Al-Jawziyyah for pumpkin :( it cuts thirst, relief headache, laxative, and is

one of the nicer foods, the seeds of pumpkin expel tape worm from intestine,

and good for patients with kidney problems, bowel, arthritis and rheumatism).

The prophet Mohamed (ppbuh) said :( it is strengthens the mind and the brain)

(Albukhary, 5379).

1.3.1.3 Habitation &Distribution: -

The name of pumpkin originates from the word pepon, which is the Greek for

large melon. The French adapted this word to pompon, while British changed to

pumpion and later American colonists changed that to the world we use today

"pumpkin"(Credo,2008).

The origin of pumpkin is not definitively known, although they are thought to

have originated in central Africa as far back as 7000-5500 B.C (Decker-Walter,

1997).

Pumpkin was grown all around the world for a variety of reasons ranging from

agricultural purposes such as animals feed to commercial and ornamental

purposes (New York time, Oct 2007), and it is a celebrated food of the native

America, Indian who treasured them for their dietary and medicinal properties

xxiv

and now it serve as a raw material of pharmaceutical products, Today, the

leading commercial producers of pumpkin include the USA, Mexico, India and

China (Oxford university press, 2007). In Sudan, local pumpkin (Cucurbita

maxima) has been grown widely in several major growing areas for many

decades and can be considered one of the most vegetable crops and their annual

production was 380,579 tons in 2000( ministry of agriculture,2002).

1.3.1.4 Cultivation:-

-Soil: pumpkin prefers a well drained sandy soil with high organic matter.

-Climate: pumpkin is warm-season annual preferring high temperature and they

need 90 to 120 hot days to reach maturity.

-Fertilization: pumpkin is monoecious produce both male and female flower,

the female is distinguished by small ovary at the base of the petals. Honey bees

play significant role in fertilization, if there are inadequate bees for pollination,

gardeners often to have handled pollinate, and inadequately pollinated pumpkin

usually starts growing but abort before full development (Roberts-Tammy

Aug2006).

-Harvest: the fruits are ripe when they turn to a bright orange colour, the fruits

must be picked.

Generally pumpkin does not require too much attention and care after it is

planted and it should be known that pumpkin plant grows extremely easy from

either seeds or cutting roots.

Giant pumpkins:-

The largest pumpkin is Cucurbita maxima; they were cultivated by farmers

through intermittent effort in USA since the early 19th century and by 2010

giant pumpkin crossed the 1500kg mark (Credo, 2008).

1.3.2 Current uses of pumpkin:-

1.3.2.1 Pumpkin Value in Traditional Medicine:

Plants provide an alternative strategy in search for new drugs. There is a rich

abundance of plants reputed in traditional medicine for protective and

therapeutic properties.

Traditional medicinal plants are a therapeutic resource used by population of

continent specifically for healthcare, which may also serve a starting material

for drugs (kayoed A.A.A and kayoed O.T, 2011). Popularity of pumpkin in

various systems of traditional medicine for several uses (antidiabetic,

antihypertensive, antitumor, immunomodulation, antibacterial,

xxv

antihypercholesterolemia, intestinal antiparasitia, anti-inflammation) focused

the investigators' attention on this plant (Caili et al., 2006).

Fruit pulp: sedative, emollient and refrigerant; used as poultice, applied to

burns, inflammations, boils, and burns.

Seeds: diuretic, anthelminth (for tapeworm). Because of their zinc content and

anti mitotic effect, seeds are used to arrest enlargement of prostate gland. Also

used in cystitis and minor kidney dysfunction. (Khare, 2007; Chonoko&Rufai.,

2011).The medicinal uses of pumpkin were adopted by oriental healer in the

seventeenth century which includes:

1.3.2.1.1 Pumpkin seeds may promote prostate health:

Long an ancient Egyptian and eastern European folk remedy for the prostate

problem of middle aged and older men, the pumpkin seeds have in fact been

shown to improve symptoms associated with an enlarged prostate due to Benign

Prostatic hyperplasia (BPH ) especially in it is early stages (Raver, Anne, Oct

2007). BPH is a health problem involving non-cancer enlargement of the

prostate gland and one factor that contribute to BPH is over stimulation of the

prostate cells by testosterone and it is conversion product Di-Hydro testosterone

(DHT), in multi centre controlled study involving more than 2000 subjects, a

products containing pumpkin seeds was evaluated for the treatment of BPH, the

result indicate that components in pumpkin seeds appear to be interrupt this

triggering of prostate cells multiplication by testosterone and DHT but the

mechanism is not known (Michael-Hogan, 2011).

1.3.2.1.2 Anti` inflammatory benefits to arthritis:

The healing properties of pumpkin seeds have also been recently investigated

with respect to arthritis. In animal studies, the addition of pumpkin seeds to the

diet has compared favourably with use of the non-steroidal anti- inflammatory

drug indomethacin in reducing inflammatory symptoms. Importantly, though,

pumpkin seeds did not have one extremely unwanted effect of indomethacin:

unlike the drug, pumpkin seeds do not increase the level of damaged fats (lipid

peroxides) in the linings of the joints, a side-effect that actually contributes to

the progression of arthritis, but the healing benefits of pumpkin seeds for

arthritis is a potential benefit to the human (Hyun T, Barrette E- sep 2004).

1.3.2.1.3 Protection 0f men’s bones:

In addition to maintaining prostate health, another reason for older men to use

pumpkin seeds, a regular part of their healthy way of eating is bone mineral

density. Although osteoporosis is often thought to be a disease for which

xxvi

postmenopausal women are at highest risk, it is also a potential problem for

older men. Almost 30% of hip fractures occur in men, and 1 in 8 men over age

50 will have an osteoporotic fracture. A study of almost 400 men ranging in age

from 45-92 that was published in the American Journal of Clinical Nutrition

found a clear correlation between low dietary intake of zinc rich foods

(pumpkin seeds are rich source of zinc), low blood levels of the trace mineral,

and osteoporosis at the hip and spine(Hyun T, Barrette E- sep 2004).

1.3.2.1.4 Lower cholesterol blood level:

In a study in the journal of agriculture and food chemistry (America),

researchers published the pumpkin seeds contain compound that have a

chemical structure very similar to cholesterol, and when present in the diet in

sufficient amounts, are believed to reduce blood levels of cholesterol, enhance

the immune response and decrease risk of certain cancers and cardiovascular

diseases (Phillips KM, 2005).

1.3.2.1.5 Address depression:

Pumpkin seeds are suggested to help remedy of depression, some cultures

especially China believed that having pumpkin seeds and fruit as a part of your

diet can prevent depression (Jennifer Murray, 2012).

1.3.2.1.6 Traditional Anthelminth, Taeniacide and diuretics: The seeds are regarded as very potent and effective de-worming agents that can

easily paralyze and eliminate intestinal worms from the digestive system of a

person. Both tapeworms and roundworms, which are very common helminth

endoparasites are said to be easily expelled and eliminated (Younis et al., 2000;

Stuart, 2003; Díaz et al., 2004; Caili et al.,2006 Gold,2009; Marie-Magdeleine

et al.,2009). Also it is used as a paediatric urinary aid to treat bed-wetting, and

then the seeds of pumpkin are one of the most efficient vermifuges, and are

particularly useful against taenia or tapeworms in pregnant women.

From 1863 to 1936, the United States Pharmacopoeia listed pumpkin seeds as a

treatment for intestinal parasites.

The seeds and fruits of pumpkin are mildly natural diuretic and have been used

in Central American medicine as a treatment for nephritis and other urinary

system problems.

1.3.2.1.7 Prevention of Kidneys stones: Two studies have shown that eating pumpkin seeds as a snack can help to

prevent the most common type of kidney stone, called a calcium oxalate stone.

Pumpkin (Cucurbita pepo) seeds appear to reduce levels of substances that

promote stone formation in the urine and increase levels of substances that

inhibit stone formation. The active constituents of pumpkin seeds responsible

for this action have not been identified. Approximately 5to10 grams per day of

xxvii

pumpkin seeds may be needed for kidney stone prevention (Suphakarn VS-

1987).

1.3.1.1.8 The pumpkin seeds and fruit increase power of intelligence and

mental vitality:

Where scientists have discovered medicine in Germany, a new article of

pumpkin called (Encephalic - stimulant) have great influence in stimulating the

brain and the development of convolutions of the brain and may be issued by

German scientists recently made the decision enforceable in all relevant

institutions to accustom the students and young people since the first of the age

of a lot of Pumpkin eating meals in the diet, and also recommend that all

workers in the field of mental activity of the intellectuals and scientists and

experts, teachers and students to focus on care to deal with this material useful

in their diet(Carbin BE-1990).

1.3.2.1.9 Gastro duodenal protective and anti-ulcerogenic: In a study a significant decrease in alkaline phosphatase activity and mucosal

thickness and increase in ulcer index was observed in aspirin treated stomach

and duodenum of albino rats.

However, pretreatment with (Cucurbita pepo) fruit pulp extract for 14

consecutive days showed increase in alkaline phosphatase activity and mucosal

thickness along with decrease in ulcer index( UI), suggesting gastroduodenal

protective and anti-ulcerogenic properties of (Cucurbita pepo) fruit (Sarkar &

Buha,2008 ).

1.3.2.1.10 Great on your skin:

Pumpkin fruits pulp and leaves is used as external emollient and anti

inflammatory to soften the skin dryness or roughness, pimples, spots, freckles

(when apply to face), insects stinging. Burnings: by applying the previous

treatment on a burning area, accelerate it is healing (Susan D Van-1998).

1.3.2.1.11 As a protective and anticancer agent:

There is recent evidence indicates that the pumpkin fruit, flowers, seeds and

leaves are useful in the prevention of cancer, published research journal

Albyukemiaoip 1985 study conducted at the national cancer institute in the

united states of America, pointed out the pumpkin really protective of lung

cancer in residents of new jersey in the united states (Jayaprakasam et al., 2003;

Liu et al., 2008; Liu et al., 2010).

Cucurbitacin B ( phytocomponent previously detected in pumpkin fruit), it IS

one of the most widely used for in vivo and in vitro studies on tumour inhibition

(Jayaprakasam et al., 2003; Liu et al.,2008; Liu et al.,2010). Accumulated

evidences have shown that cucurbitacin B inhibits the growth of numerous

xxviii

human cancer cell lines, including breast, prostate, lung, uterine cervix, liver,

skin, and brain cancers ( Liu et al., 2008).

Chemistry of Cucurbitacin: Four bitter principles of Cucurbitacin have been isolated from the fruits of

pumpkin, provisionally named as Cucurbitacin A (C32H46O9), B (C32H46O8), C

(C32H48O8) D (C30H44O7). Cucurbitacins are chemically classified as

triterpenoids, formally derived from Cucurbitane,

a triterpene hydrocarbon and the bio effective one is Cucurbitacin B (Hua,

2005).

Figure 1.6: Basic structure of Cucurbitacins (Dhiman et al., 2012):

Table 1.1: Structures of Cucurbitacins A, B, C and D (Dhiman et al., 2012)

and E. (Wiart, 2006):

Cucurbitacins Structure

A

xxix

B

C

D

1.3.2.1.12 Anti-oxidant activities

Pumpkin seed oil is a rich source of antioxidants (active beneficial components

that may protect important biological molecules from oxidative stress) and

polyunsaturated fatty acids, some researchers believed that there are some

benefits associated with the administration of poly-unsaturated fatty acids in

cases of prostate disorders (Levin, Rachel-2008).

Pumpkin flesh and seeds are rich in antioxidant vitamins such as β -carotene

and α-tocopherol.

Beta-carotene reduce skin damage from the sun and acts as an anti-

inflammatory agent also it is thought to slow the aging process, reduce the risk

of developing cataracts, and prevent tumour growth. The potent anti-

inflammatory action and powerful anti-oxidant action of the beta-carotene has

been effectively demonstrated in laboratory studies, β-carotene is one of the

most effective organic compounds around, helping in the effective prevention of

cholesterol oxidation under laboratory conditions, this property of the

compound is very helpful as oxidized cholesterol is the form of the sterol which

often accumulates in the walls of the blood vessels and may contributes to a

heightened risk of heart attack and stroke in the person. For this reason, the

progression of atherosclerosis and heart disease in the body may possible be

slowed down by regular high intake of the β-carotene from dietary sources

(Adaramoye, et. al, 2007).

xxx

Vitamin E (α-tocopherol) protects the cell from oxidative damage by preventing

the oxidation of unsaturated fatty acids in cell membrane (Stevenson, et al.,

2007).

Figure 1.7: Structure of β-carotene and Vitamin A (Dewick, 2009):

Vitamin A

Figure 1.8: Structure of α- tocopherol (Dewick, 2009):

1.3.2.1.13 Laxative agent :

Pumpkin fruit has a laxative action, then being useful in case of gastrointestinal

disorders such as dyspepsia and constipation, pumpkin juice is indicated for

ulcer and hyperacidity, pumpkin flesh contains abundant quantities of extremely

good dietary fibre, this high amount of fibres help in lowering the low density

lipoprotein LDL bad cholesterol levels in the blood and in regulating the blood

sugar levels (Tylor-1993).

1.3.2.1.14 Anti-diabetic agent:

Diabetes mellitus is considered as a common, growing, serious, costly, and

potentially preventable public health problem. In 2030, the number of people

with diabetes is estimated to increase from 117 million in 2000 to 366 million.

Pumpkin is widely considered to have active hypoglycemic properties (Alarcon-

Aguilar et al., 2002; Caili et al., 2006; Gary et al., 2011). In a study, the

antihyperglycemic effects of Cucurbita maxima/pepo fruit extract were

investigated on streptozotocin-induced experimental diabetes in rats. Oral

administration of the extract (300 and 600 mg/kg body weight, day) for 30 days

resulted in a significant reduction in blood glucose, glycosylated haemoglobin,

xxxi

and an increase in plasma insulin and total haemoglobin. The effect was

compared with 150 mg/kg between tolbutamide (Xia & Wang, 2006). East

China University research on type-1 diabetic rats, published in July 2007,

suggests that chemical compounds found in pumpkin fruits and seeds promote

regeneration of damaged pancreatic cells, resulting in increased bloodstream

insulin levels. According to the research team leader, pumpkin extract may be

"a very good product for pre-diabetic people, as well as those who already have

diabetes," possibly reducing or eliminating the need for insulin injections for

some type-1 diabetics and diabetes mellitus type 2 (Quanhong et al., 2005).

1.3.2.1.15 Antihypertensive agent (decrease elevated blood pressure):

Pumpkin seed oil may offer benefits of blood pressure lowering and cardiac-

protection. A study suggested that pumpkin seed oil might enhance the blood

pressure lowering effect of calcium antagonist Felodipine or angiotensin-

converting enzyme inhibitor (ACE-inhibitor) captopril. Pumpkin seed oil

(100mg/kg), and amlodipine (0.9mg/kg) were given once daily orally for 6

weeks. Pumpkin seed oil / amlodipine treatment significantly reduced the

elevation in blood pressure BP. This study has shown that pumpkin seed oil

exhibits an antihypertensive and cardioprotective effects. Thus, there is a

potential that pumpkin seed oil may benefit people at risk of high blood

pressure (Zhang X-1994).

1.3.2.1.16 Antimicrobial agent: Another beneficial effects of all pumpkin parts is their potent activity against

microbes specially bacteria (Cowan, 1999).

1.3.2.1.17 Intestinal inflammation:

Intestinal inflammation can be treated by consuming an herbal decoction made

from the pumpkin pulp (Gill, 2012).

Assistant to the process of digestion, laxative, demulcent, antitussive, diuretic,

soothing to mucous membranes, and is also used to treat haemorrhoids (Levin,

Rachel-2008).

1.3.2.1.18 A Rich Source of Healthful Minerals and Protein:

In addition to their above-listed unique health benefits, pumpkin seeds, fruit,

leaves, flowers also provide a wide range of traditional nutrients. Food Ranking

Systems qualified them as a very good source of the minerals, vitamins, and

other healthy nutrients such as magnesium, iron, manganese, copper, protein,

mono-unsaturated fat, and as previously mentioned, zinc. Snack on a quarter-

cup of pumpkin seeds will give us 57.7% of the daily value (DV) for

magnesium, 34.4% of the DV for iron, 29.7% of the DV for manganese, 19.2%

of the DV for copper, 16.9% of the DV for protein, 19.7% of the DV for mono-

unsaturated fat, and 21.4% of the DV for zinc(Credo-2008).

Nutritional composition of all pumpkin parts:

xxxii

Vitamin A: all parts of pumpkin are rich source of vit A, regular consumption of

pumpkin can promote the health of eyes and boost immune system remarkably

(Jang SM, 2001).

Figure 1.9: Structure of Vitamin A (Dewick, 2009):

Vitamin C: Vitamin C helps fight free radical, improves immunity and promotes

the production of collagen, the high content of vitamin C in pumpkin also offers

protection against various forms of cancer (Iheanchoet.al 2009; kayoed A.A.A

and kayoed O.T, 2011).

Figure 1.10: Structure of Vitamin C (Dewick, 2009):

Magnesium: both the pulp and seeds of pumpkin are rich source in magnesium,

which is an important mineral required for various biological processes includes

promoting a healthy immune system, contributing to bone strength, and

normalizing heart function and it is required for maintenance of bones and

teeth(Fan et al.,2006).

Potassium& zinc: pumpkin is also rich with potassium and zinc, studies showed

that eating a potassium-rich diet can prevent onset of cardiovascular diseases

and hypertension, potassium found in pumpkin aids in balancing fluid levels in

the body, promotes strong bones, is necessary for energy production, and helps

to control blood pressure, zinc is important for providing bone density support

for peoples at risk for osteoporosis, it also boosts the immune system and

promotes reproductive health (Trease &Evan -1989). Zinc is one further

nutrient found in pumpkin seeds that might impact prostate function. The fact

that pumpkin seeds serve as a good source of zinc may contribute to the role of

pumpkin seeds in support of the prostate, and more research is needed to

determine the circumstances under which zinc might be helpful versus harmful

(Gold, 2009).

Fibres: pumpkin flesh is very low in calories and contains abundant quantities

of extremely good dietary fibre, it is extremely effective for treating

gastrointestinal disorders such as constipation, indigestion, etc. the high amount

of fibres also help in lowering the LDL (bad) cholesterol level in the blood, in

xxxiii

regulating the blood sugar level and play a role in weight loss (Levin, Rachel-

2008).

Vitamin E: pumpkin is also a good source of vitamin E which promotes healthy

skin by protecting the body from sun damage and may reduce the risk of

Alzheimer’s disease and certain cancers (Clin, Nutr-2004).

Pantothenic acid (vitamin B5): is also found in pumpkin, Vitamin B5 helps to

balance hormone levels and manage stress. (Khare, 2007; Iheancho et al., 2009;

Hussain etal., 2010).

Figure 1.11: Structure of Vitamin B5 (Dewick, 2009):

Dark green pumpkin leaves contain a great amount of vitamin A that is vital for

a proper growth, healthy eyes and protection from disease. Moreover, pumpkin

leaves are a rich source of calcium, iron, protein, and calcium

(Chonoko&Rufai., 2011).

Pumpkin flowers have important vitamins and minerals like vitamin C, vitamin

A, iron, potassium, calcium, magnesium and also provide B vitamin`s. Pumpkin

flowers also have a nice supply of food folate, make pregnancy an excellent

excuse to eat an abundance of fried pumpkin flowers (Jerry, Hirsh-2009).

1.3.2.2 Other uses: Pumpkin is very versatile in their uses for cooking. Most parts of the pumpkin

are edible, including the fleshy shell, the seeds, the leaves, and even the flowers.

In Canada, Mexico, the United States, Europe and China, the seeds are often

roasted and eaten as a snack (Azevedo-Meleiro& Rodriguez-Amaya, 2007). In

the south-western United States and Mexico, pumpkin and squash flowers are a

popular and widely available food item. They may be used to garnish dishes,

and they may be dredged in a batter then fried in oil, Pumpkin leaves are a

popular vegetable in the Western and central regions of Kenya;, whereas the

pumpkin itself is usually boiled or steamed. The seeds are popular with children

who roast them before eating them. Canned pumpkin is often recommended by

veterinarians as a dietary supplement, raw pumpkin can be fed to poultry, as a

supplement of regular feed to help maintain egg production (Trease&Evan-

1989).

xxxiv

Chapter Two:

Materials &Methodology

xxxv

2. Materials and Methodology:

2.1 Materials:

2.1.1The plant:

The experimental plants include the seeds, fruit, leaves and flowers of pumpkin

were purchased from local market in Sudan.

Cucurbita maxima / pepo

Family: Cucurbitaceae.

2.1.1.1 Description:

-Pumpkin seeds: also known as pepitas are small, flat, whitish green in colour.

Most pumpkin seeds are encased in tan or soft white testa.

-Pumpkin leaves: pumpkin has large, green, very soft, shade and lobed leaves

with well marked veins.

-Pumpkin fruit: is squash like fruit, their weight is2Kg, orange green and

ranging from oblate to oblong in shape , they have a moderately hard rind with

thick, edible flesh below and a central cavity containing numerous seeds

(Credo, 2008).

-Pumpkin flowers: big, bright, yellow colourful flowers of pumpkin are often

produced in abundance, Pumpkin has both male and female flowers in the same

plant, the female flower is distinguished by the small ovary at the base of the

flower, these flowers have extremely short life spans and only open for as short

as a time as one day (Decker-Walter, 2007).

2.1.1.2 Collection and Identification of tested plant:

A pumpkin Fruit was purchased from the local market of Khartoum city in

February 2012; it was authenticated by the botanists in medicinal and aromatic

plants research institute and Pharmacognosy department, faculty of Pharmacy,

Khartoum University-Sudan. The seeds were removed from the ripe, cleaned,

washed, dried under shade and planted for cultivation and the ripe fruit was cut

to small pieces. The leaves and Flowers are collected each month from April to

July 2012. Collected leaves and Flowers, Fruit and Seeds were put for drying at

shade and reduced to fine powder using pestle and mortar in the laboratory as

described by (Bean, A.R, 2006).The powders of four parts were stored dry, and

used as the stock samples for further analyses.

xxxvi

2.1.2 Instruments and chemicals:

A wide range of instruments and chemical were used during the course of this

study as follow:

2.1.2.1 Instruments (table 2.1):

Device Manufacture by

-Autoclave Griffin &George. Ltd England.

-Freeze drier Alpha -1-12w. Germany.

-Glass wares Griffin &George. Ltd England.

-Hot air oven Gallenkamp England.

-Incubator Griffin &George. Ltd England.

-Rotatory evaporator BUCHI 011Switzerland.

-Rough balance SARTORIUS 2351, Germany.

-Sensitive balance HF-300G A&D company, limited Quick FIT,

EX5⁄83, England.

-UV lamb Fisher.

-Water bath BUCHI 461, Switzerland.

-Water bath Fisher.

-GC-MS Instrument Central Petroleum Laboratories-Sudan HP-

5MS Column.

xxxvii

2.1.2.2 Chemicals (table 2.2):

Chemicals Manufacture by

-Silica Gel GF 254 for TLC. Merck limited, India.

-Calcium sulphate, anhydrous. HI media laboratories limited,

Mumbai.

-Acetic anhydride. PANREAC, Spain.

-Chloroform. LAB-TECH-chemicals, India.

-Sulphuric acid. LOBA- chemie, India.

-Sodium nitrate purified LR. Rasayan laboratory, India.

-Sodium nitro-prusside. BDH chemical Ltd, England.

-Phenazone. LOBA-chemie PVT, India.

-Pyridine. Merck limited, India.

- Aqueous Hydrochloric HCL 37%. MRS-scientific, England.

-Sodium hydroxide pellets. LOBA-chemie, India.

-3, 5 Di-nitro-benzoic acid. ACROS- organic, Belgium.

-Picric acid, A.R. HI media laboratory, India.

-DL Tartaric acid. Techno-pharmchem, India.

-Mercuric chloride. Techno-pharmchem, India.

-Iodine LR. Breckland-scientific, uk.

-Potassium iodide. Techno- pharchem, India.

-Ferric chloride anhydrous. SDFINE-chem limited, India.

xxxviii

-Solution of hydrogen peroxide B.P

6%.

Bell-sons Ltd, England.

-Potassium hydroxide pellets AR. Techno- pharchem, India.

-Benzene. FINKEM-Ltd, Mumbai.

-Glacial acetic acid. E- Mark, India.

-Ethyl acetate. ROMIL-Ltd, UK.

-Magnesium metal turnings. CDH laboratory, India.

-Thymol. May and Baker Ltd, England.

-Molish’s reagent. Techno-pharmchem, India.

-Barium chloride. Merck limited, India.

-Silica gel 200-400 mesh. LOBA CHEMIE, India.

-Petroleum ether. Merck limited, India.

-Ethanol. LAB-TECH chemicals.

-Dichloromethane. Central drug house, India.

-Ammonia solution. BDH- analar, England.

-Acetone. MRS-scientific, England.

-nutrient agar. Oxide limited, England.

2.1.2.3 Reagents and solution:

1- Fehling’s reagent (cupric alkaline solution):

34.66g of copper sulphate dissolved in 200ml of distilled water and diluted to

500ml (solution A); 173g of sodium and potassium tartarate and 100g of sodium

hydroxide were dissolved in 300ml of distilled water and diluted to 500ml

(solution B), after cooling, equal volumes of solution A and B were mixed

immediately before use.

2-Mayer’s reagent:

xxxix

1.35g of mercuric chloride were dissolved in 60ml of distilled water, 5g of

potassium iodide in 10ml of distil water were added, the whole solution was

diluted to 100ml.

3-Dragendorff’s reagent:

Two stock solution were prepared, 0.6g bismuth sub-nitrate in 2ml concentrated

hydrochloric acid and 10ml of distilled water (solution A); 6g potassium iodide

in10ml water (solution B), these two stock solution A and B are mixed together

with 7ml concentrated hydrochloric acid and 15ml water and the whole diluted

with 400ml water.

4-Wagner’s reagent:

2g of iodine and 6g of potassium iodide were dissolved in 100ml of distilled

water.

5-Baljet’s reagent:

Equal volumes from 1% picric acid, 95% ethanol and 10% aqueous sodium

hydroxide were mixed together.

6-MC Farland`s reagent:

0.5g of barium chloride was dissolved in 50ml distilled water to make the

concentration of barium chloride 1% (stock solution), 0.6 ml of this stock

solution was diluted with 99.4ml of 1% sulphuric acid were added, then mixed,

the result is formation of white suspension (should be stored in the dark, in a

tightly sealed container at 20-25⁰c).

2.1.3 Micro-organisms (table 2.3):

Bacteria Type

-Bacillus subtilis. NCTC 8236 Gram +ve bacteria

-Staphylococcs aureus. ATCC 25923 Gram +ve bacteria

-Escherichia coli. ATCC 25922 Gram -ve bacteria

-Pseudomonas aeruginosa. NCTC 6750 Gram -ve bacteria

xl

NCTC: is the national collection of type culture, Colindale England.

ATCC:is the American type culture collection, Rockville, Maryland, USA.

2.2 Method:

In this study all experiments were divided into four main parts:

1- The first part concerned with plant (collection of the four parts of pumpkin,

their active constituents extraction and phyto-chemical screening tests).

2- The second part dealt with their activity against the selected microorganisms.

3- The third with separation of pumpkin seeds, leaves, flowers and fruits

extracts constituents (the identification of chemical constituents of each four

parts of pumpkin) by using thin layer TLC and Gas Chromatography-Mass

Spectrum techniques GC-Ms.

4- The fourth part dealt with the structure activity relationship and correlation

between the detected active constituents and the traditionally reported uses of

the selected four parts of pumpkin.

2.2.1 The plant:

Four experiments were performed in the tested four parts of pumpkin (seeds,

leaves, fruits, flowers):

2.2.1.1 Extraction methods:

The extraction was carried by two methods:

2.2.1.1.1 Maceration:

To prepare an alcoholic extract for the gas chromatography studies, Fifty grams

(50g) each of the powdered plant materials was soaked in 500ml of 70% ethanol

in separate conical flasks (1L) and kept for twenty four hours with shaking from

time to times, after which the mixture was filtered using whatman’s No 1filter

paper. The filtrate was evaporated by using Rotatory Evaporator Instrument

(Komolafe et, 1988), the yields percentage was calculated and the dry residues

obtained were kept in a refrigerator for further study (Fatope et al, 1993).

2.2.1.1.2 Successive soxhlet’s extraction for phyto-chemical and

antimicrobial tests:

xli

One hundred fifty grams (150 g) from each of the four parts of powdered plant

materials (seeds, leaves, fruit, flowers) were extracted by using petroleum ether,

chloroform, ethanol in successive manner by applying continuous extraction

method; 150g from the powdered seeds of pumpkin were weighed using rough

balance, these powder then packed in a thimble which has been placed in the

extraction column of the soxhlet (continuous extraction apparatus). 500ml of

petroleum ether were poured in the round flask of soxhlet; excess petroleum

ether was used to soak the powder of pumpkin seeds in the thimble. The soxhlet

was settled by conducting it to a continuous water supply and a heating mantel

with it is thermostat been adjusted at 60⁰c (petroleum ether boiling point), the

procedure was continued until the solvent become clear (end point of the

extraction process), then the apparatus was put off and the extract was collected

in a pre-weighted container, then evaporated to eliminate solvent and to obtain

dry extract by using rotator evaporator. The yield percentage was calculated and

the residue obtained was covered by using aluminium sheath and kept in a

refrigerator for further use. The marc in the thimble was completely dried for

the next extraction step. To the previous thimble, the same procedure was

repeated using chloroform (medium polar solvent 70⁰c) and ethanol (high polar

solvent 78⁰c) with adjustment of heating mantle temperature according to

boiling point of each solvent, in this point continuous extraction complete by

using three successive solvents petroleum ether, chloroform and ethanol, and

three extracts from pumpkin seeds were obtained (petroleum ether extract,

chloroform extract and ethanol extract). The same procedure was applied to the

leaves, flowers, fruit and the result is obtaining of three different extract of each

part.

2.2.1.2 Phytochemical screening tests:

There are simple but standard chemical tests to detect the presence of Alkaloids,

Tannins, Saponins, Anthraquinones, Sterols, Flavanoids,...etc in a plant extracts.

According to the World Health Organization (WHO) a medicinal plant is any

plant which in one or more of its organ contains substances that can be used for

the synthesis of useful drugs (WHO, 1977). Medicinal plants contain

biologically active chemical substances such as Saponins, Tannins, Essential

oils, Flavonoids, Alkaloids and other chemical compounds (Trease & Evans,

2002) which have curative properties.

Samples of pumpkin (Seeds, leaves, Fruit and Flowers) were analyzed for

phytochemicals to get information for their active ingredients. Total Alkaloids,

Flavonoids and Cardiac glycosides were determined by using method reported

xlii

by (Ciulei, 1982). Whereas Saponin and Tannin contents were determined by

routine analytical procedures mentioned in (Sofowora, 1993) as follows:

2.2.1.2.1 Detection of Sterols and Triterpenes:

(a) Liebermann-Burchard’s test:

Each extract was treated with chloroform (5ml) and filtered.1ml of acetic

anhydride was added to the filtrates, boiled and cooled. The solutions were

transferred to a dry tube and by means of a pipette; a concentrated sulphuric

acid (1-2ml) was added at the bottom of tube, formation of brown ring at the

junction of the two liquids indicates the presence of phytosterols.

(b)Salkowiski’s test:

Each extract was treated with chloroform (1ml) and filtered. The filtrates were

treated with few drops of concentrated sulphuric acid, shaken and allowed to

stand. Appearance of golden yellow colour indicates the presence of triterpenes.

2.2.1.2.2 Detection of Tannins:

(a) Gelatin test:

Each extract was treated with chloroform and filtered. To filtrates, 1% gelatin

solution containing sodium chloride was added. Formation of white precipitate

indicates the presence of tannins.

(b)Ferric chloride test:

3-4 drops of ferric chloride were added to 2mlof each extract. Formation of a

bluish back colour indicated the presence of hydrolysable tannins and a green

black colour indicates condensed tannins.

(c)Vanillin-hydrochloric acid test:

2ml of vanillin reagent were added to extract in white dish, then evaporated to

dryness, after that one drop of concentrated hydrochloric acid was added. A

crimson- red precipitate indicates the presence of condensed tannins.

2.2.1.2.3 Detection of Cardiac Glycosides:

(a)Baljet’s test:

Each extract was dissolved in 1ml 70% ethanol and two drops of Baljet’s

reagent. A light orange to dark red colour was produced in the presence of

cardiac glycosides.

(b)Kedd’s test:

xliii

To each extract, 2ml of 3-5 dinitrobenzoic acid and 1ml of 20% sodium

hydroxide were added. Appearance of violet colour indicates the presence of

cardiac glycosides.

(c)Keller’s-Killiani test:

Extracts were dissolved in 1ml glacial acetic acid and 3m of 3.5%ferric chloride

in acetic acid, mixed well and left for one minute. 1m of concentrated sulphuric

acid was added on wall of test tube, formation of brown layer in interface

indicates the presence of cardiac glycosides.

2.2.1.2.4 Detection of Saponins:

(a) Froth test:

Extracts were diluted with distilled water to 20ml and this was shaken in a

graduated cylinder for 15 minutes. Formation of 1cm layer of foam indicates the

presence of saponins.

(b)Emulsion test:

3ml of each extract in test tube were diluted to 10ml with water, 5ml of castor

oil was added and the mixture was shaken violently. Formation of thick white

emulsion indicates the presence of saponins.

2.2.1.2.5 Detection of Anthracenoside glycoside:

(a) Modified Borntrager’s test:

Each extract was hydrolysed with diluted hydrochloric acid and then treated

with ferric chloride solution and heated in boiling water for about 5minutes, the

mixture was cooled and extracted with equal volume of benzene; the benzene

layer was separated and treated with 10% ammonia solution. Formation of rose-

pink colour in the ammonial layer indicates the presence of anthraquinone

glycosides (emodols).

(b)Legal’s test:

Each extract was boiled with aqueous sulphuric acid and then filtered, to the

filtrates 5ml of chloroform and 10% ammonia solution were added. Formation

of pink or violet colour in the ammonia layer indicates the presence of

anthraquinone glycosides.

2.2.1.2.6 Detection of Flavonoids:

(a) Cyaniding test or Shibata’s reaction:

xliv

Each extract was dissolved in 2ml of 50% methanol and heated. To them

metallic magnesium and 4-5 drops of concentrated hydrochloric acid were

added. A red or orange colour indicates the presence of flavonic aglycones.

(b)Alkaline reagent test:

Extracts were treated with 2ml of ethanol and few drops of 1% potassium

hydroxide solution. Formation of intense yellow colour, which became

colourless on addition of dilute acid, indicates the presence of flavonoids.

2.2.1.2.7 Detection of Alkaloids:

Each extract was dissolved in 1.5 ml of 2% hydrochloric acid; the solution thus

obtained was filtered and divided into equal volumes in three test tubes:

(a)Mayer’s test:

Filtrate in one of test tube was treated with Mayer’s reagent. A yellow colour

precipitate indicates the presence of alkaloids.

(b)Wagner’s test:

Filtrate was treated with Wagner’s reagent. Formation of brown/reddish

precipitate indicates the presence of alkaloids.

(c)Hager’s test:

Filtrates were treated with Hager’s reagent. Presence of alkaloids was

confirmed by the formation of yellow coloured precipitate.

2.2.1.2.8 Detection of Coumarins:

(a) Coumarin itself can be easily detected in plant material; a small amount of

moistened plant was placed in test tube and covered with filter paper moistened

with dilute potassium hydroxide solution. The test tube was heated in boiled

water bath for several minutes; the paper was removed and exposed to

ultraviolet light. If Coumarins present, a yellowish- green fluorescence spot

appear within few minutes.

(b) Each extract was dissolved in hot water. After cooling, the solution was

divided in two test tubes: one tube will contain the reference and the aqueous

solution of the second tube was made alkaline with 0.5ml of 10% ammonia. The

appearance of an intense fluorescence under UV light indicates the presence of

Coumarins.

xlv

2.2.1.2 Antibacterial Experiments:

In this part of this experiment the antibacterial activity of the crude plant

extracts (petroleum ether, chloroform and ethanol) of each parts of pumpkin

plant was studies on clinical strains of four standard bacteria made up of two

Gram-positive (Staphyloccocus aereus and Bacillus subtilis) and two Gram-

negative bacteria (Escherichia coli and Pseudomonas aeruginosa).

2.2.1.2.1 Preparation of standard bacterial suspension:

One ml aliquots of 24 hours broth of the test organisms were aseptically

distributed on to nutrient agar slopes and incubated at 37⁰c for 24 hours, the

bacterial growth was washed off with normal saline to produce a suspension

containing about 108 -109 colonies per 1 ml of bacterial suspension. The

suspension was stored in the refrigerator at 4⁰c till used. The average number of

viable organisms per ml of the stock suspension was made by means of Mc

Farland standards technique.

In microbiology, Mc Farland standards are used as a reference to adjust the

xlvi

turbidity of bacterial suspensions so that the number of bacteria will be within a

given range to standardize microbial testing (108 -109 colonies/1 ml of bacterial

suspension). 0riginal MC Farland standards were prepared by mixing specified

amount of barium chloride (0.6 ml of 1% stock solution of barium chloride) and

99.4ml of 1% sulphuric acid, mixing of the two compounds forms a barium

sulphate precipitate which cause turbidity in the solution.

This standard can be compared visually to a suspension of bacteria in sterile

saline in test tube, if bacterial suspension is too turbid, it can be diluted with

more diluents and if suspension is not turbid enough, more bacteria can be

added. All the above experiment conditions were maintained constant so that

suspension with very close viable counts would be obtained (Miles & Misra,

1938).

2.2.1.2.2 Preparation of test plant samples:

In the study of the antibacterial activities of this plant, concentrations of

512mg/ml of each extracts were used for screening. This was done by

dissolving 2.05g of the extract in 4ml of distilled water (stock solution). From

the stock of 512mg/ml, serial dilutions were made to 4mg/ml (NCCLS, 2000).

2.2.1.2.3 Testing of extracts for antibacterial activity:

The cup-plate agar diffusion method was adopted with some minor

modifications to assess the antibacterial activity of the prepared extracts

(petroleum ether, chloroform and ethanol of each parts of pumpkin). Three ml

of the standardized bacterial stock suspension108-109 colonies forming units per

ml were thoroughly mixed with 300ml of sterile molten nutrient agar which was

maintained at 45⁰c (Kavanagh, 1972).

Twenty ml aliquots of the inoculated agar were distributed into sterile petri-dish

(15 ptri-dishes in each 20ml of inoculated agar were added), the agar plates

were left to set and each of these 15 plates 4cups (5mm in diameter) were cut

using a sterile cork borer ( 4) and agar discs were removed (every 3 agar

plates were inoculated with same type bacteria).alternate cups were filled with

0.1ml sample using adjustable pipette and the plates were reserved at room

temperature for about one hour to allow the extract to diffuse into agar

(Atata,2003)(for example: three plate of nutrient agar were inoculated with

Staphylococcus aureus and then 4 cups were cut in every one of these plates.

In plate 1, the cups were filled with petroleum ether extracts of each 4 parts of

pumpkin, plate 2cups filled with chloroform extracts and the third with ethanol

extracts)then the12agar plates were incubated at 37⁰c for 24 hours. The

xlvii

antibacterial activity was determined by measuring the diameter of zones of

inhibition in mm produced after incubation and then the values were tabulated.

2.2.1.3 The separation:

In separation two techniques were applied:

2.2.1.3.1 Separation by thin layer chromatography TLC:

Thin layer chromatography (TLC) is a planar chromatographic technique

introduced in the 1950s as fast, sensitive and inexpensive analytical technique

used to obtain well define, well separated spots of components in the sample

mixture. Performing of TLC analysis consists of a number of steps were made

in turn:

2.2.1.3.1.1 Preparation of TLC silica gel plates (stationary phase):

60 g of silica gel were shaken with 120 ml distilled water for two minutes in a

stopper conical flask. The slurry was spread using spreader making 0.25mm

thick layer on 10 glass plates 20x20cm in diameter. The resultant coated plates

were allowed to dry at room temperature then activated by heating in an oven at

110⁰c for one hour. The hot plates were allowed to cool and stored.

2.2.1.3.1.2 Selection of the mobile phase:

To determine the best solvent or mixtures of solvents (Mobile system) for the

development of a TLC plate of pumpkin fruits, flowers, seeds and leaves

extracts, a process of trial and error was used( the polarity of solvents were

varied in several trial runs to adjust the separation of components) as follows:

-Toluene: petroleum ether: formic acid (90:10:1).

-Hexane: chloroform: toluene (75:25:10).

- Petroleum ether: hexane: ethyl acetate (75:20:15).

- Benzene: ethyl acetate (95:5). - Petroleum ether: toluene

(10:90).

2.2.1.3.1.3 Spotting the plates with Pumpkin extracts:

In plate 0ne, three different spots were applied (concentrated) 1.5 cm above the

plate bottom by means of capillary tube(three spots for petroleum ether,

chloroform and ethanol extracts of seeds successively from left to right), then

were left to be dried. About 1.5 cm from the top of the plate, a straight line was

drawn to determine the solvent front. The same application process was done

for fruit, leaves and flowers in different plates.

xlviii

2.2.1.3.1.4 Chromatogram development:

The spotted TLC plate was developed in a closed glass jar containing the

selected solvent system, the solvent was allowed to travel up the plate until

reach the previously drawn line (solvent front), then the plate was picked out of

the jar and left to be dried at room temperature (evaporation of solvent from

plate).

2.2.1.3.1.5 Visualization (Detection):

As the chemicals being separated may be colourless, several methods were used

to visualize or detect the spots (separated components):

1- The spots were visualized under UV light 245nm (Harbone, 1983).

2- Specific reagents were sprayed onto the plate to detect the spots (1gm

vanillin was dissolved in 100ml concentrated sulphuric acid reagent) then

heating the plate. Once the spots appeared, circled to have a permanent record

how far the compound was travelled on the plate.

2.2.1.3.1.6 Analysis:

Once the components visible as separated spots, the retardation factor (Rf value)

of each spots were determined by dividing the distance spot travelled by the

distance solvent front travelled using the initial spotting site as reference.

The separation achieved with a TLC plate can be used to estimate gas

chromatography- mass spectrometry separation.

2.2.1.3.2 Separation and Identification by Gas chromatography-mass

spectrometry GC-Ms:

Gas chromatography- mass spectrometry (GC-MS) is an analytical techniques

being originated by James and Martin in 1952s. As the name implies, it is

actually tow techniques that are combined to form a single method by which

mixtures of components in a test sample may be separated, identified and

quantified. Applications of GC-MS include drug detection, environmental

analysis and identification of unknown samples.

GC-MS analysis of ethanolic extracts of pumpkin seeds, leaves, fruit and

flowers were performed in the following steps:

2.2.1.3.2.1 Preparation of extract for GC-MS analysis:

As mentioned above, each parts of pumpkin (seeds, flowers, fruit and leaves)

were extracted by using ethanol as solvent and maceration as extraction

technique, then dried and reserved the obtained dried residue in refrigerator. For

the application of GC-MS, one gram from this dried extract was taken and re-

xlix

dissolved in 10ml of solvent called dichloromethane (1:10),then about 2 μl of

the sample solution was injected in GC-MS for analysis of different compounds.

2.2.1.3.2.2 GC-MS analysis:

GC-MS analysis of the ethanol extract of each Pumpkin part was performed

using a Perkin–Elmer GC Clarus 500 system comprising an AOC-20i auto-

sampler and a Gas Chromatograph interfaced to a Mass Spectrometer (GC-MS)

equipped with a Elite-5MS (5% diphenyl/95% dimethyl poly siloxane) fused a

capillary column (30 × 0.25 mm Inner diameter ID × 0.25 mm film

thickness). For GC-MS detection, an electron ionization system was operated in

electron impact mode with an-ionization energy of 70 eV. Helium gas

(99.999%) was used as a carrier gas at a constant flow rate of 1 ml/min, and an

injection volume of 2 μl was employed (a split ratio of 10:1). The injector

temperature was maintained at 250 °C, the ion-source temperature was 200 °C,

the oven temperature was programmed from 110 °C (isothermal for 2 min),

with an increase of 10 °C/min to 200°C, then 5 °C/min to 280°C, ending with a

9 min isothermal at 280 °C. Mass spectra were taken at 70 eV; a scan interval of

0.5 s and fragments from 45 to 450 Da. The solvent delay was 0 to 2 min, and

the total GC/MS running time was 36 min. The relative percentage amount of

each component was calculated by comparing its average peak area to the total

areas. The mass-detector used in this analysis was Turbo-Mass Gold-Perkin-

Elmer, and the software adopted to handle mass spectra and chromatograms was

a Turbo-Mass ver-5.2.

Generally, GC analysis conditions:

We were likely need to optimize GC conditions depending on their analytical

needs, equipment, and sample type. In general, a typical gas chromatography

method will include:

-Column: HP-5 MS 30m length, inner diameter 0.25mm, 0.25mm film

thickness.

- Temperature program: 110°C to 280°C at a rate of 10°C/min.

- Injector temperature 250°C; detector temperature: 280°C.

- Injection volume: 2 μl.

- Carrier gas: helium at a constant flow rate of 1 ml/min.

2.2.1.3.2.3 Identification of phyto-components:

Interpretation of mass-spectrum GC-MS was carried out using the database of

National Institute of Standard and Technology (NIST) having more than 62,000

patterns. The spectrum of the unknown components was compared with the

spectrum of known components stored in the NIST library. The name,

molecular weight, and structure of the components of the test materials were

obtained.

l

Chapter three: Results

3. Results:

li

3.1 phytochemical studies results:

3.1.1 The yield of extracts:

3.1.1.1 Extraction by maceration method:

By using maceration as extraction method, 50gms of each parts of pumpkin

(seeds, leaves, flowers and fruits) were extracted separately with ethanol as

solvent, the obtained extracts were dried and the yield percentage of each

extract recorded in the table below (table 3.1):

Table 3.1 the yield percentage of ethanol extracts of each part of pumpkin

alone (Maceration):

pumpkin Weight of extract/50gms Yield%

- Seeds 11.25gms 22.5%

- Fruit 4.8gms 9.6%

- Leaves 6.2 12.4%

- Flowers 2.7gms 5.4%

3.1.1.2 Successive continuous extraction:

3.1.1.2.1 Pumpkin seeds:

By the soxhlet apparatus, 150gms of pumpkin seeds were extracted successively

with petroleum ether, chloroform and ethanol, the obtained extracts were dried

(the same procedure was applied to other pumpkin parts, fruit, leaves and

flowers) and the yields percentages of the three extracts (petroleum ether

extract, chloroform extract and ethanol extract of each four parts) recorded in

the table below (table 3.2).

lii

Table 3.2 the weights and yields percentages of the different three extracts

of each four parts of pumpkin (Continuous extraction- 150gms):

Solvent Seeds Leaves Fruit Flowers

Wt

gm

Yield% Wt

gm

Yield% Wt

gm

Yield% Wt

gm

Yield%

Petroleum

ether

23.2

15.7%

6.7

4.5%

3.6

2.4%

4.6

3.1%

Chloroform

10.3

6.9%

4.1

2.7%

5.1

3.4%

2.1

1.3%

Ethanol

6.6

4.4%

8.3

5.6%

11.2

7.6%

2.3

1.6%

3.1.2 Phytochemical screening tests results:

3.1.2.1 Pumpkin Seeds Extracts:

3.1.2.1.1 Petroleum ether extract:

The phytochemical screening of pumpkin seeds petroleum ether extract

revealed the presence of glycosides, sterols and triterpens. The results

summarized in (table3.3).

3.1.2.1.2 Chloroform extract:

The phytochemical screening of pumpkin seeds chloroform extract revealed the

presence of flavonoids, glycosides and saponins. The results summarized in

(table3.3).

3.1.2.1.3 Ethanol extract:

The analysis of pumpkin seeds ethanol extract, showed the presence of

alkaloids, flavonoids, saponins, triterpens and coumarins. The extract devoid of

tannins, anthraquinone. The result reported in the (table 3.3).

liii

3.1.2.2 Pumpkin leaves Extracts:

3.1.2.2.1 Petroleum ether extract:

The phytochemical screening of pumpkin leaves petroleum ether extract

revealed the presence of fatty acids, glycosides, sterols and triterpens. The

results summarized in (table3.3).

3.1.2.2.2 Chloroform extract:

The phytochemical screening of pumpkin leaves chloroform extract revealed

the presence of cardiac glycosides, alkaloids and coumarins. The results

summarized in (table3.3).

3.1.2.2.3 Ethanol extract:

The analysis of pumpkin leaves ethanol extract, showed the presence of

alkaloids, flavonoids, saponins, triterpens and alkaloids. The extract devoid of

tannins. The result was reported in the (table 3.3).

3.1.2.3 Pumpkin fruit Extracts:

3.1.2.3.1 Petroleum ether extract:

Glycosides, Alkaloids as well as Coumarins detected in the petroleum ether

extract of pumpkin fruit, table (3.3).

3.1.2.3.2 Chloroform extract:

The analysis of pumpkin fruit extract, showed the presence of cardiac

glycosides, alkaloids and saponins, table (3.3).

3.1.2.3.3 Ethanol extract:

Coumarin, flavonoids, alkaloids and glycosides represent the photochemical

constituents revealed in the ethanol extract of pumpkin fruit. The extract was

found to be devoid of tannins, table (3.3).

3.1.2.4 Pumpkin flowers Extracts:

3.1.2.4.1 Petroleum ether extract:

Glycosides, sterol/ triterpenes and coumarins detected in the petroleum ether

extract of pumpkin flowers, table (3.3).

3.1.2.4.2 Chloroform extract:

The analysis of pumpkin flowers extract, showed the presence of cardiac

glycosides, alkaloids and saponins, table (3.3).

3.1.2.4.3 Ethanol extract:

Tannins, coumarin, saponin, alkaloids and glycosides represent the

photochemical constituents were revealed in the ethanol extract of pumpkin

flowers. The extract was found to be devoid of anthraquinone and flavonoids.

Table (3.3).

liv

Table 3.3 Phytochemical screening tests results:

Constituent

Results

Seeds Leaves Fruit Flowers

P.

ether

CHcl3 Ethan

ol

P.

ether

CHcl3 Ethanol P.

ether

CHcl3 Ethan

ol

P.

ether

CHcl3 Etha

nol

Sterol/

Triterpenes

+ + + + - - - - - + + -

Tannins - - - - - - - - - - - +

Cardiac

glycosides

+ + + + + + + + + + + +

Alkaloids

_ _ + _ + + + + + - + +

Anthraquin

ones

_ _ _ _ _ _ _ _ _ _ _ _

Flavanoids - + + - - + - - + - - -

Saponins - + + - - + - + - - + +

coumarins - - + - + - + - + + - +

(−): means not detected.

(+): means detected.

lv

3.2 The antibacterial activity results:

As mentioned above, the antibacterial activity of the crude plant extracts

(petroleum ether, chloroform and ethanol) of each four parts of pumpkin plant

was studies on clinical strains of four standard bacteria made up of two Gram-

positive (Staphyloccocus aereus and Bacillus subtilis) and two Gram- negative

bacteria (Escherichia coli and Pseudomonas aeruginosa), the extract consider

effective against bacteria if the inhibition zone ≥15mm in diameter. The results

obtained were documented and reported as follows:

3.2.1 Screening of Antibacterial Activity of Pumpkin Seeds extracts against

four standard bacteria:

3.2.1.1 Escherichia coli (E. coli):

The results were illustrated in Tables (3.4), indicated that the three successive

extracts from the seeds of pumpkin (petroleum ether, chloroform, ethanol)

showed a high antibacterial activity against Escherichia coli, the ethanol and

petroleum ether extracts had the more affectivity than chloroform (it had the

large inhibition zones 32-30mm respectively).

3.2.1.2 Staphylococcus aereus (S. a):

The antibacterial activity of pumpkin seeds extracts (petroleum ether,

chloroform and ethanol) against S. a were studied, it was found to be moderate

compared to the activity against E. Coli (table3.4).

3.2.1.3 Pseudomonas aeruginosa (Ps. a):

The chloroform extract has a high activity on this bacterium compared with

other extracts, the diameters of the resultant growth inhibition zones were

tabulated in table (3.4).

3.2.1.4 Bacillus subtilis (B. s):

The three successive extract of pumpkin seeds (petroleum ether, chloroform and

ethanol) had the most effectiveness against Bacillus subtilis than any type of

other tested bacteria and the chloroform had highest inhibition zone. Table

(3.4).

3.2.2 Screening of Antibacterial Activity of Pumpkin Leaves extracts

against four standard bacteria:

3.2.2.1 Escherichia coli (E. Coli):

The antibacterial activity of pumpkin leaves extracts (petroleum ether,

chloroform and ethanol) was studied; it was found that the chloroform and

petroleum ether extracts showed a significant inhibitory effect on E.coli while

lvi

ethanolic extract had no effect( 0mm inhibition zone), the diameters of resultant

growth inhibition zones were tabulated in table (3.4).

3.2.2.2 Staphylococcus aereus (S. a): The results were illustrated in Tables (3.19), indicated that the ethanol and

chloroform extracts had moderate effect on these bacterium while petroleum

ether has a little effect.

3.2.2.3 Pseudomonas aeruginosa (Ps. a):

Petroleum ether and chloroform extracts had no effect on these bacteria, while

ethanol has moderate effect. See table (3.19).

3.2.2.4 Bacillus subtilis (B. s): The chloroform extract has a significant effect on this bacterium when we

compare with other extracts (petroleum ether has no effect and ethanol has

moderate effect), the diameters of the resultant growth inhibition zones were

tabulated in table (3.19).

3.2.3 Screening of Antibacterial Activity of Pumpkin Fruit extracts against

four standard bacteria:

3.2.3.1 Escherichia coli (E. Coli):

The diameter of the inhibition zones of the three successive extract of pumpkin

fruit against E.coli were scored as 22mm, 24mm and 20mm respectively( see

table 3.4), these result indicating that the zones of inhibition were all above

15mm for all three extracts indicating their antibacterial activity.

3.2.3.2 Staphylococcus aereus (S. a):

The chloroform extract of pumpkin fruit was the most effective one against this

bacterium compared with other extracts (petroleum ether and ethanol had no

effect due to their MDIZ≤15mm), the mean diameters of the resultant growth

inhibition zones were tabulated in table (3.4).

3.2.3.3 Pseudomonas aeruginosa (Ps. a):

From the resultant growth inhibition zones tabulated in table (3.4) we see the

petroleum ether and chloroform pumpkin fruit extracts had no effect on these

bacteria while the ethanol extract had high potency (DIZ of ethanol extract was

28mm).

3.2.3.4 Bacillus subtilis (B. s):

The antibacterial sensitivity of the three extracts of pumpkin fruit (petroleum

ether, chloroform and ethanol) were assessed quantitatively by determining the

zones of inhibition as given in Table (3.4). In this study, the growth of B.s was

remarkably inhibited by the petroleum ether pumpkin fruit extract

lvii

(MDIZ₌38mm). The growth of B.s was also inhibited but to a lesser extent by

the chloroform extract while ethanol extract had no effect.

3.2.4 Screening of Antibacterial Activity of Pumpkin Flowers extracts

against four standard bacteria:

3.2.4.1 Escherichia coli (E. Coli):

The chloroform extract of pumpkin flowers was the most effective one against

this bacterium compared with other extracts, the diameters of the resultant

growth inhibition zones were tabulated in table (3.4).

3.2.4.2 Staphylococcus aereus (S. a):

From the resultant growth inhibition zones tabulated in table (3.4) the petroleum

ether and ethanol pumpkin flowers extracts had moderate effect on these

bacteria while the chloroform extract had no effect (DIZ of chloroform extract

was 10mm i.e ≤15mm).

3.2.4.3 Pseudomonas aeruginosa (Ps. a):

The chloroform extract has a significant effect on this bacterium compared with

other extracts (petroleum ether and ethanol had no effect), the diameters of the

resultant growth inhibition zones were tabulated in table (3.4).

3.2.4.4 Bacillus subtilis (B. s):

The diameter of the inhibition zones of the three successive extract of pumpkin

flowers against B.s were scored as 0mm, 28mm and 20mm respectively (table

3.4), from these result the ethanol and chloroform had moderate effect while

petroleum ether had no effect.

lviii

Table (3.4) Antibacterial Activity results of Pumpkin seeds, leaves, flowers

and fruit extracts against four standard bacteria:

Bacteria

Results (DIZ mm)

Seeds Leaves Fruit Flowers

P.

ether

CHcl3 Ethan

ol

P.

ether

CHcl3 Etha

nol

P.

ether

CHcl3 Ethan

ol

P.

ether

CHcl3 Etha

nol

Escherichia coli 30 21 32 24 28 0 22 24 20 20 28 0

Staphylococcus

aereus

22 28 26 16 20 22 0 38 10 16 10 20

Pseudomonas

aeruginosa

22 30 24 0 14 22 12 0 28 0 24 10

Bacillus subtilis

30 36 24 14 26 20 34 16 0 0 28 20

Figure 3.1 contains charts showing a comparison between the diameters of

the growth inhibition zones of the three successive extracts of pumpkin

seeds against the four tested bacteria:

lix

Figure 3.2 contains charts showing a comparison between the diameters of

the growth inhibition zones of the three successive extracts of pumpkin

leaves against the four tested bacteria:

Figure 3.3 contains charts showing a comparison between the diameters of

the growth inhibition zones of the three successive extracts of pumpkin

fruit against the four tested bacteria:

0

10

20

30

40

pet chl ethanol

DIZ(mm)

extracts of Pumpkin Seeds

E.coli

S.a

Ps.a

B.s

0

5

10

15

20

25

30

pet chl ethanol

DIZ (mm)

Extracts of Pumpkin Leaves

E.coli

S.a

Ps.a

B.s

0

10

20

30

40

pet chl ethanol

DIZ (mm)

Extracts of Pumpkin Fruit

E.coli

S.a

Ps.a

B.s

lx

Figure 3.4 contains charts showing a comparison between the mean

diameters of the growth inhibition zones of the three successive extracts of

pumpkin flowers against the four tested bacteria (see below):

E.coli: Escherichia coli. S.a:

Staphylococcus aureus.

B.s: Bacillus subtilis PS.a:

Pseudomonas aeuroginosa.

pet: petroleum ether chl:

chloroform.

DIZ: Diameter of Inhibition Zone in mm.

Figure 3.5The growth inhibition zones of Pumpkin Seeds, Leaves, Fruit

and Flowers (petroleum ether, chloroform and ethanol of each) on

Escherichia coli bacteria:

(1) Petroleum ether extracts: (2)

Chloroform extracts:

0

5

10

15

20

25

30

pet chl ethanol

DIZ(mm)

Extracts of Pumpkin Flowers

E.coli

S.a

Ps.a

B.s

lxi

(3) Ethanol extracts:

Figure 3.6 The growth inhibition zones of Pumpkin Seeds, Leaves, Fruit

and Flowers (petroleum ether, chloroform and ethanol of each) on

Staphylococcus aereus bacteria (S.a):

(1) Petroleum ether extracts: (2) Chloroform extracts:

(3) Ethanol extracts of:

lxii

Figure 3.7 The growth inhibition zones of Pumpkin Seeds, Leaves, Fruit

and Flowers (petroleum ether, chloroform and ethanol of each) on

Pseudomonas aeruginosa bacteria (P.s):

(1) Petroleum ether extracts: (2) Chloroform extracts:

lxiii

(3) Ethanol extracts of:

Figure 3.8The growth inhibition zones of Pumpkin Seeds, Leaves, Fruit

and Flowers (petroleum ether, chloroform and ethanol of each) on Bacillus

subtilis bacteria (B.s):

(1) Petroleum ether extracts: (2)

Chloroform extracts:

lxiv

(3) Ethanol extracts of:

N.B:

(1) Pumpkin seeds. (2) Pumpkin leaves.

(3) Pumpkin fruit. (4) Pumpkin flowers.

3.3 The separation results:

3.3.1 Thin layer chromatography:

As mentioned above in method, a process of trial and

error was used( the polarity of solvents were varied

in several trial runs) to adjust the separation of

components and to determine the best solvent system

for the development of a TLC plate of pumpkin fruits,

flowers, seeds and leaves extracts.

Solvent systems that were tried:

1- Toluene: petroleum ether: formic acid (90:10:1

drop). 2- Hexane: chloroform: toluene

(75:25:10). 3- Petroleum ether:

hexane: ethyl acetate (75:20:15). 4- Benzene:

ethyl acetate (95:5). 5-

Petroleum ether: toluene (10:90).

Solvent system (Petroleum ether: hexane: ethyl acetate 75:20:15) is the better

solvent system than others for the development of a TLC plate and for

lxv

separation of components of pumpkin seeds and flowers, the results were

recorded as follows:

1.1- Pumpkin Seed Extracts:

-Petroleum ether extract of pumpkin seeds gave eight fractions. Their Rf values

as well as the chromatogram were shown bellow in table (3.5) and figure (3.9).

-Chloroform extract of pumpkin seeds gave rise to seven fractions. Their Rf

values as well as the chromatogram were shown bellow in table (3.5) and figure

(3.9). -Ethanol extract of pumpkin seeds gave five fractions. Their Rf

values as well as the chromatogram were shown bellow in table (3.5) and figure

(3.9).

Table (3.5): The Chromatogram and Rf values of Pumpkin Seeds extracts

(petroleum ether: hexane: ethyl acetate (75: 20: 15) as mobile phase):

Fraction TLC separation results of pumpkin seeds (15 cm)

Petroleum ether Chloroform Ethanol

V.colour Rf values V.colour Rf values V.colour Rf values

Fraction 1 brown 0.067 brown 0.067 greenish

brown

0.067

Fraction 2 purple 0.133 green 0.167 purple 0.467

lxvi

Fraction 3 green 0.233 red 0.3 reddish

brown

0.6

Fraction 4 purple 0.367 reddish

brown

0.467 green 0.733

Fraction 5 pale

brown

0.533 purple 0.6 red 1

Fraction 6 brown 0.8 brown 0.7

Fraction 7 red 0.9 red 1

Fraction 8 reddish

brown

1

1.2- Pumpkin Flowers Extracts:

-Petroleum ether extract of pumpkin flowers gave six fractions. Their Rf values

as well as the chromatogram were shown bellow in table (3.6) and figure (3.10).

-Chloroform extract of pumpkin flowers gave rise to three fractions. Their Rf

values as well as the chromatogram were shown bellow in table (3.6) and figure

(3.10). -Ethanol extract of pumpkin flowers gave rise to six fractions.

Their Rf values as well as the chromatogram were shown bellow in table (3.6)

and figure (3.10).

Table (3.6): The Chromatogram and Rf values of Pumpkin Flowers

extracts (petroleum ether: hexane: ethyl acetate (75: 20: 15) as mobile phase):

lxvii

Fraction TLC separation results of pumpkin Flowers (15 cm)

Petroleum ether Chloroform Ethanol

V.colour Rf values V.colour Rf values V.colour Rf values

Fraction 1 brown 0.063 purple 0.063 purple 0.063

Fraction 2 yellow 0.313 brown 0.313 yellow 0.219

Fraction 3 purple 0.563 black 0.563 orange 0.344

Fraction 4 black 0.875 yellow 1 brown 0.438

Fraction 5 green 0.938 green 0.594

Fraction 6 brown 1 brown 1

Figure (3.9): TLC of Pumpkin Seeds (petroleum ether, chloroform and

ethanol) extracts respectively from left to right:

lxviii

1 2 3

-Solvent system: petroleum ether: hexane: ethyl acetate (75: 20: 15).

-Stationary phase: silica gel.

-Detector: vanillin /sulphuric acid.

1: Pumpkin Seeds petroleum ether fraction spots.

2: Pumpkin Seeds chloroform fraction spots.

3: Pumpkin Seeds ethanol fraction spots.

Figure (3.10): TLC of Pumpkin Flowers (petroleum ether, chloroform and

ethanol) extracts respectively from left to right:

lxix

1 2 3

-Solvent system: petroleum ether: hexane: ethyl acetate (75: 20: 15).

-Stationary phase: silica gel.

-Detector: vanillin /sulphuric acid.

1: Pumpkin Flowers petroleum ether fraction spot.

2: Pumpkin Flowers chloroform fraction spot.

3: Pumpkin Flowers ethanol fraction spot.

2- Solvent system (benzene: ethyl acetate 95:5) is the better solvent system than

others for the development of a TLC plate and for separation of components of

pumpkin leaves and fruit, the results were recorded as follows:

2.1- Pumpkin Leaves Extracts:

lxx

-Petroleum ether extract of pumpkin leaves gave ten fractions. Their Rf values

as well as the chromatogram were shown bellow in table (3.7) and figure (3.11).

-Chloroform extract of pumpkin leaves gave rise to ten fractions. Their Rf

values as well as the chromatogram were shown bellow in table (3.7) and figure

(3.11). -Ethanol extract of pumpkin leaves gave four fractions.

Their Rf value as well as the chromatogram were shown bellow in table (3.7)

and figure (3.11).

Table (3.7): The Chromatogram and Rf values of Pumpkin Leaves extracts

(benzene: ethyl acetate 95:5) as mobile phase):

Fraction TLC separation results of pumpkin Leaves (16 cm)

Petroleum ether Chloroform Ethanol

V.colour Rf values V.colour Rf values V.colour Rf values

Fraction 1 green 0.063 green 0.063 green 0.25

Fraction 2 brown 0.156 yellow 0.25 Dark

green

0.281

Fraction 3 pale

green

0.25 dark

green

0.281 Pale green 0.531

Fraction 4 dark

green

0.281 purple 0.313 purple 0.688

Fraction 5 purple 0.313 Yellowis

h brown

0.469

Fraction 6 dark

green

0.375 green 0.531

Fraction 7 dark blue 0.531 purple 0.688

Fraction 8 purple 0.688 dark

blue

0.813

Fraction 9 Pale blue 0.813 blue 0.875

Fraction 10 brown 1 brown 1

lxxi

2.2- Pumpkin Fruit Extracts:

-Petroleum ether extract of pumpkin fruit gave seven fractions. Their Rf values

as well as the chromatogram were shown bellow in table (3.8) and figure (3.12).

-Chloroform extract of pumpkin fruit gave rise to six fractions. Their Rf value as

well as the chromatogram were shown bellow in table (3.8) and figure (3.12).

-Ethanol extract of pumpkin fruit gave seven fractions. Their Rf value as well as

the chromatogram were shown bellow in table (3.8) and figure (3.12).

Table (3.8): The Chromatogram and Rf values of Pumpkin Fruit extracts

(benzene: ethyl acetate 95:5) as mobile phase):

Fraction TLC separation results of pumpkin Fruit (16 cm)

Petroleum ether Chloroform Ethanol

V.colour Rf

values

V.colour Rf

values

V.colour Rf values

Fraction 1 green 0.063 green 0.063 green 0.063

Fraction 2 purple 0.281 purple 0.281 purple 0.281

Fraction 3 blue 0.344 blue 0.344 blue 0.344

Fraction 4 Pale blue 0.594 yellow 0.5 yellow 0.5

Fraction 5 purple 0.906 dark

green

0.513 dark green 0.513

Fraction 6 brown 0.967 brown 0.967 brown 0.967

Fraction 7 dark blue 1 Dark

blue

1

Figure (3.11): TLC of Pumpkin Leaves (petroleum ether, chloroform and

ethanol) extracts respectively from left to right:

lxxii

1 2 3

-Solvent system: benzene: ethyl acetate (95:5).

-Stationary phase: silica gel.

-Detector: vanillin /sulphuric acid.

1: Pumpkin Leaves petroleum ether fraction spot.

2: Pumpkin Leaves s chloroform fraction spot.

3: Pumpkin Leaves ethanol fraction spot.

Figure (3.12): TLC of Pumpkin Fruit (petroleum ether, chloroform and

ethanol) extracts respectively from left to right:

lxxiii

1 2 3

-Solvent system: benzene: ethyl acetate (95:5). -

Stationary phase: silica gel.

-Detector: vanillin /sulphuric acid.

1: Pumpkin Fruit petroleum ether fraction spot.

2: Pumpkin Fruit chloroform fraction spot.

3: Pumpkin Fruit ethanol fraction spot.

3.3.2 Gas Chromatography- Mass Spectrometry (GC-MS) Results:

lxxiv

As mentioned above, the Gas chromatography- mass spectrometry analysis of

the crude pumpkin plant extracts (ethanol extracts) of each four parts of

pumpkin plant flowers, fruit, seeds and leaves was studies and interpretation of

mass spectrum Gc.ms was conducted using the data base of the national

institute standard and technology (NIST), the spectra of unknown compound

were compared with the spectrum of known compound stored in the NIST

library. The results obtained were documented and reported as follows:

3.3.2.1 GC-MS Analysis Result of Pumpkin Flowers:

This investigation was carried out to determine the possible chemical

components from pumpkin flowers by GC-MS. The results pertaining to the

GC-MS analysis of pumpkin flowers were given in table 3.9 and figure 3.13.

Table (3.9): The Name, Formula, Retention Time RT, Molecular Weight

MW and Area percent of phytochemical components identified in Pumpkin

Flowers by GC.MS:

No of

beak

Name Formula RT

(min)

MW

(g.mol)

Area%

1 Palmitic acid C16H32O2 38.96 256 21.82

2 Oxalic acid, allyl octyl ester C13H22O4 41.4 242 2.378

3 8,11,14- Eicosatrienoic acid C20H34O2 42.42 306 12.18

4 Oxalic acid, allyl tridecyl

ester

C18H32O4 46.19 312 2.946

5 Not identified - 53.01 - 8.256

6 Oxalic acid, allyl tridecyl

ester

C18H32O4 72.04 312 25.66

7 Oxalic acid, allyl hexadecyl

ester

C21H38O4 83.00 354 6.831

8 Stigmasta-7, 16- dien-3-ol C29H48O 92.30 412 14.60

9 Stigmast-7-en-3-ol C29H50O 95.57 414 4.293

Table (3-10): Structures of Identified Phytochemicals in Pumpkin Flowers:

lxxv

Nо Phytochemical name Structure

1 Palmitic acid

2 Oxalic acid allyl octyl ester

3 8,11,14- Eicosatrienoic acid

4 - 6 Oxalic acid allyl tridecyl

ester

7 Oxalic acid, allyl hexadecyl

ester

8 Stigmasta-7, 16- dien-3-ol

9 Stigmast-7-en-3-ol

lxxvi

Figure (3-13): GC.MS Chromatogram of the ethanol extract of Pumpkin

Flowers:

lxxvii

Library search report

Data path: C:/msdchem/1/data/general/

Data file: Pumpkin-Flowers.

Search libraries: C/database/NIST08.L

Integration Events: RTE Integrator-events.e

Acquired: 21 Sep 2014- 12:16

Operator: Sara

3.3.2.2 GC-MS Analysis Result of Pumpkin Fruit:

The results pertaining to the GC.MS analysis are given in figure 3-14 and table

3-11. Twenty two compounds were detected in the ethanol extract of pumpkin

lxxviii

fruit as follows:

Table (3.11): The Name, Formula, Retention Time RT, Molecular Weight

MW and Area percent of phyto-chemical components detected in Pumpkin

Fruit by GC-MS:

No of

peak

Name Formula RT

(min)

MW

(g.mol)

Area

%

1 Vit E C29H50O2 8.8 430 1.22

2 5-methyl-

5a,6,8,9,10,11,11a,12-

octahydro-5H-6,10,11,12a-

dimethanoindol[3,2-

b]quinolizine-8,13-diol

C20H26N2O2 9.4 326 1.47

3 1H-imidazole, 2-ethyl-4-

methyl

C6H1ON2 11.8 110 2.44

4 Methoxyphenamine

C11H17NO 12 179 2.93

5 1,2-dihydroxyethyl-3,4-

dihydroxyfuran-2(5H)-one

C6H8O6 12.4 176 2.20

6 5-[1-hydroxy-2-

(isopropylamino)ethyl]

benzene-1,3-diol

C11H17NO3 12.8 211 0.73

7 2-Amino-9-[3,4-dihydroxy-

5-(hydroxymethyl)oxolan-2-

yl]-3 H-purine-6-

C10H13N5O5 13 283 0.98

8 4,4’-

methylenebis(tetrahydro-

1,2H,4-thiadiazine)1,1,1’,1’-

tetraoxide

C7H16N4O4

S2

13.2 284 1.22

9 3,7,11-trimethyl-2,6,10-

dodecantrien-1-ol

C15H26O 14 222 2.93

lxxix

10 Tetradecanoic acid

C14H28O2 14.2 228 3.91

11 Squalene C30H50 14.4 410 4.63

12 3,7,11,15-tetramethyl-2-

hexadecan-1-ol

C20H40O 15.2 296 1.95

13 1H-Purine-2, 6-dione, 3,7-

dihydro-1,3-dimethyl-

C7H8N4O2 16.4 180 0.98

14 1-β-D-ribofuranosyl-1H-

1,2,4trazole-3-carboxamide

C8H12N4O5 18 244 3.17

15 6,7-dimethoxy-3-[(5R)-4-

methoxy-6-methyl-7,8-

dihydro-5H-

[1,3]dioxolo[4.5-

g]isoquinolin-5-yl]-3H-2-

benzofuran-1-one

C22H23NO7 18.6 413 7.80

16 1-(4-tert-butylphenyl)-4-[4-

[hydroxyl(diphenyl)methyl]p

iperidin-1-yl]butan-1-ol

C32H41NO2 19 471 10.02

17 2-phenylethyl hydrazine C8H12N2 19.8 130 11.93

18 2,6-di(propan-2-yl)phenyl C12H18O 20.2 178 11.20

19 4-[3-(5H-Dibenz [b,f]

azepin-5-yl)-1-

piperazinethanol

C23H29N3O 20.6 363 10.49

20 5-fluoro-1-[4-hydroxy-5-

(hydroxymethyl)tetrahydrofu

ran-2-yl]-1H-pyrimidine-2,4-

dione

C9H11FN2O5 21.2 246 7.32

21 4-chloro-N-(2-methyl-2,3-

dihydroindol-1-yl)-3-

C16H16cl

N3O3S

21.8 365 5.61

lxxx

sulfamoyl-bnzamide

22 10,13-

dimethylspiro[2,8,9,11,12,14

,15,16-octahydro-1H-

cyclopenta[a]phenanthrene-

17,5’-oxolane]2’,3-dione

C22H28O3 22 340 4.88

Figure 3-14 GC.MS Chromatogram of the ethanol extract of Pumpkin Fruit:

lxxxi

Library search report

Data path: C:/msdchem/1/data/general/

Data file: Pumpkin-Fruit

Search libraries: C/database/NIST08.L

Integration Events: RTE Integrator-events.e

Acquired: 13 Jan 2015- 10:00

Operator: Hiba

lxxxii

Table (3-12): The Structures of Identified Phytochemicals in Pumpkin

Fruit:

No of

peak

Name Structure

1 Vit E

2 5-methyl-5a,6,8,9,10,11,11a,12-

octahydro-5H-6,10,11,12a-

dimethanoindol[3,2-b]quinolizine-

8,13-diol

3 1H-imidazole, 2-ethyl-4-methyl

4 Methoxyphenamine

5 1,2-dihydroxyethyl-3,4-

dihydroxyfuran-2(5H)-one

6 5-[1-hydroxy-2-

(isopropylamino)ethyl]benzene-

1,3-diol

7 2-Amino-9-[3,4-dihydroxy-5-

(hydroxymethyl)oxolan-2-yl]-3 H-

purine-6-

lxxxiii

8 4,4’-methylenebis(tetrahydro-

1,2H,4-thiadiazine)1,1,1’,1’-

tetraoxide

9 3,7,11-trimethyl-2,6,10-

dodecantrien-1-ol

10 Tetradecanoic acid

11 Squalene

12 3,7,11,15-tetramethyl-2-hexadecan-

1-ol

13 1H-Purine-2, 6-dione, 3,7-dihydro-

1,3-dimethyl-

14 1-β-D-ribofuranosyl-1H-

1,2,4trazole-3-carboxamide

15 6,7-dimethoxy-3-[(5R)-4-methoxy-

6-methyl-7,8-dihydro-5H-

[1,3]dioxolo[4.5-g]isoquinolin-5-

yl]-3H-2-benzofuran-1-one

16 1-(4-tert-butylphenyl)-4-[4-

[hydroxyl(diphenyl)methyl]piperidi

n-1-yl]butan-1-ol

17 2-phenylethyl hydrazine

lxxxiv

18 2,6-di(propan-2-yl)phenyl

19 4-[3-(5H-Dibenz [b,f] azepin-5-yl)-

1-piperazinethanol

20 5-fluoro-1-[4-hydroxy-5-

(hydroxymethyl)tetrahydrofuran-2-

yl]-1H-pyrimidine-2,4-dione

21 4-chloro-N-(2-methyl-2,3-

dihydroindol-1-yl)-3-sulfamoyl-

benzamide

22 10,13-

dimethylspiro[2,8,9,11,12,14,15,16

-octahydro-1H-

cyclopenta[a]phenanthrene-17,5’-

oxolane]2’,3-dione

lxxxv

3.3.2.3 GC.MS Analysis Result of Pumpkin Leaves:

The GC.MS analysis has shown the presence of different phyto-constituents in

the ethanolic extract of pumpkin leaves. A total of nineteen compounds were

identified. The results pertaining to the GC.MS analysis are given in figure 3-15

and table 3-13 as follows:

Table (3.13): The Name, Formula, Retention time RT, Molecular Weight

MW and Area percent of phyto-chemical components detected in Pumpkin

Leaves by GC.MS:

No Name Formula RT

min

MW

g/mol

Area%

1 6,6-Dimethyl-10-methylene-1-oxa-

spiro[4.5]decane

C12H20O 5 180 3.61

2 Octahydrobenzo[b]pyran,4a-

acetoxy-5,5,8a-trimethyl-

C14H24O3 5.5 240 5.69

3 2(4H)Benzofuranone,5,6,7.7a-

tetrahydro-4,4,7a-trimethyl-,(R)-

C11H16O2 10.7 180 4.17

4 1-Hexadecanol,2-methyl- C17H36O 12 256 7.78

5 E-15-heptadecenal C17H32O 18.5 252 8.33

6 2-Pentadecanone,6,10,14-trimethyl- C18H36O 21 268 20.82

7 1-Heneicosyl formate C22H44O2 27 340 6.94

8 Z-(13,14-epoxy)tetradec-11-en-1-ol

acetate

C16H28O3 33 268 5.27

9 1-Hexadecanol,2-methyl- C17H36O 35 256 5

10 4,8,12,16-tetramethyl heptadecan-

4-olide

C21H40O2 41.5 324 4.03

lxxxvi

11 1-Hexadecanol,2-methyl- C17H36O 43.3 256 3.61

12 9-(2’,2’-

Dimethylpropanoylhydrazono)-3,6-

dichloro-2,7-bis-[2-(diethylamino)-

ethoxy]fluorene

C30H42CL2

N4O3

49 576 2.78

13 17- Pentatriacontene C35H70 51 490 2.23

14 Cholestane-3,6,7-triol C27H48O3 59.5 420 2.64

15 5,14,23-Octadecatrien-14,15-diol C28H52O2 6o.2 420 2.36

16 17- Pentatriacontene C35H70 62.4 490 2.25

17 1-Benzoxirene,5a[3-oxo-1-

butenyl]perhydro-2-hydroxy-

1a,5,5-trimethyl-,acetate

C15H22O4 65 266 2.64

18 Octadecane,3-ethyl-5-(2-ethyl

butyl)-

C26H54 67.8 366 3.75

19 4,4,6a,6b,8a,11,11,14b-Octamethyl-

1,4,4a,5,6a,6b,7,8,8a,9,10,11,12,12

a,14,14a,14b-octadecahydro-2H-

picen-3-one

C30H48O 74 424 3.47

lxxxvii

Figure (3-15): GC.MS Chromatogram of the ethanol extract of Pumpkin

Leaves:

lxxxviii

Library search report

Data path: C:/msdchem/1/data/general/

Data file: Pumpkin-Leaves.

Search libraries: C/database/NIST08.L

Integration Events: RTE Integrator-events.e

Aquired: 6 OCT 2014- 12:45

Operator: Maweia.

lxxxix

Table (3-14): The Structures of Identified phytochemicals in Pumpkin

Leaves:

No Name Structure

1 6,6-Dimethyl-10-methylene-1-oxa-

spiro[4.5]decane

2 Octahydrobenzo[b]pyran,4a-acetoxy-

5,5,8a-trimethyl-

3 2(4H)Benzofuranone,5,6,7.7a-

tetrahydro-4,4,7a-trimethyl-,(R)-

4-9-

11

1-Hexadecanol,2-methyl-

5 E-15-heptadecenal

6 2-Pentadecanone,6,10,14-trimethyl-

7 1-Heneicosyl formate

xc

8 Z-(13,14-epoxy)tetradec-11-en-1-ol

acetate

10 4,8,12,16-tetramethyl heptadecan-4-

olide

12 9-(2’,2’-Dimethylpropanoilhydrazono)-

3,6-dichloro-2,7-bis-[2-(diethyl

amino)-ethoxy]fluorene

13 17- Pentatriacontene

14 Cholestane-3,5,6-triol

15 5,14,23-Octadecatrien-14,15-diol

17 1-Benzoxirene,5a[3-oxo-1-

butenyl]perhydro-2-hydroxy-1a,5,5-

trimethyl-,acetate

18 Octadecane,3-ethyl-5-(2-ethyl butyl)-

xci

19 4,4,6a,6b,8a,11,11,14b-Octamethyl-

1,4,4a,5,6a,6b,7,8,8a,9,10,11,12,12a,14

,14a,14b-octadecahydro-2H-picen-3-

one

3.3.2.4 GC.MS Analysis Result of Pumpkin Seeds:

The GC.MS analysis has shown the presence of different phyto-constituents in

the ethanolic extract of pumpkin seeds. A total of thirty six compounds were

identified. The results pertaining to the GC.MS analysis are given in figure 3-16

and table 3-15 as follows:

Table (3.15): The Name, Formula, Retention time RT, Molecular Weight

MW and Area percent of phyto-chemical components found in Pumpkin

Seeds by GC.MS:

No Name Formula RT

(min)

MW

g/mol

Area%

1 Hexadecanoic acid, methyl ester C17H34O2 5.3 270 1.19

2 Heptanoic acid C7H14O2 5.5 130 0.77

3 Octanoic acid C8H16O2 6.3 144 2.80

4 2-t-Butyl-5-propyl-[1,3]Dioxolan-

4-one

C10H8O3 6.5 186 4.50

5 Hexanoic acid C6H12O2 8.5 116 0.76

6 2H-pyran,3,4-dihydro- C5H8O 9.5 84 1.68

7 Benz[c]acridine,5,10-dimethyl- C19H15N 10 257 0.98

8 9-methyl-z-10-tetradecen-1-ol

acetate

C17H32O2 10.5 268 4.72

9 1-Hexadecanethiol C16H34S 11 258 0.43

xcii

10 6-Amino-2,3diphenyl-1H-

pyrrolo[2,3- b] pyridine

C19H15N3 12.5 285 0.86

11 1-(+)-Ascorbic acid-2,6-

dihexadecanoate

C38H68O8 13 652 4.30

12 Heptadecane C17H36 14 240 1.08

13 1,2-Bezenedicarboxylic acid,

diisooctyl ester

C24H38O4 14.5 390 5.36

14 15-Hydroxy pentadecanoic acid C15H30O3 16 258 6.21

15 Cyclotetradecane C14H28 16.5 196 0.76

16 Dodecane,1,1-oxybis C24H50O 17 354 1.40

17 Tridecanoic acid C13H26O2 17.5 214 1.25

18 9-Tricosene,(z)- C23H46 18 322 0.76

19 Octadecane,1-iodo- C18H37I 18.5 380 5.77

20 Octacosyl acetate C30H60O2 20.5 452 0.44

21 I-propyl hexadecanoate C19H38O2 21 298 1.41

22 Oxacyclotridecan-2-one C12H22O2 22.5 198 6

23 Octacosane C28H58 24 394 1.07

24 Cobaltocene,1,1’-diphenyl- C22H18

Co-6

24.5 341 2.14

25 Purin-2,6-dione,1,3,9-trimethyl-8-

[2-nitrophenethenyl]-

C16H15N5O4 25.5 341 5.90

26 1- Chloroeicosane C29H41cl 27.5 317 0.75

27 9,12-Octadecadienoic acid C18H32O2 28 280 1.62

28 Delta-7-avenasterol C29H48O 29 412 5.30

29 Propanedioic acid, propyl C6H1004 30.5 146 1.51

xciii

30 Hexadecane, 2-methyl C17H36 31 240 0.87

31 Cyclooctanone,3,7-dimthyl C10H18O 32 154 4.93

32 1-Docosanethiol C22H46S 33.5 342 1.10

33 Palmitic anhydride C32H62O3 34 494 1.73

34 1,2-Benisothiazole, 3-(hexahydro-

1H-azepin-1-yl)-,1,1-dioxide

C13H16N2O2

S

34.5 264 12.37

35 Diethylenediamine C4H10N2 35 86 5.55

36 2-Piperidinone, N-[4-bromo-n-

butyl]-

C9H16BrN

O

36.5 234 0.63

Figure 3-16: GC.MS Chromatogram of the ethanol extract of Pumpkin

Seeds:

xciv

Library search report

Data path: C:/msdchem/1/data/general/

Data file: Pumpkin-Seeds.

Search libraries: C/database/NIST08.L

Integration Events: RTE Integrator-events.e

Aquired: 15 March 2014- 13:00

Operator: Hiba

Table (3-16): Structures of Identified phytochemicals in Pumpkin Seeds:

No Name Structure

xcv

1 Hexadecanoic acid, methyl ester

2 Heptanoic acid

3 Octanoic acid

4 2-t-Butyl-5-propyl-[1,3]Dioxolan-4-

one

5 Hexanoic acid

6 2H-pyran,3,4-dihydro-

7 Benz[c]acridine,5,10-dimethyl-

8 9-methyl-z-10-tetradecen-1-ol acetate

9 1-Hexadecanethiol

10 6-Amino-2,3diphenyl-1H-pyrrolo[2,3-

b] pyridine

xcvi

11 1-(+)-Ascorbic acid-2,6-

dihexadecanoate

12 Heptadecane

13 1,2-Bezenedicarboxylic acid,

diisooctyl ester

14 15-Hydroxypentadecanoic acid

15 Cyclotetradecane

16 Dodecane,1,1’-oxybis

17 Tridecanoic acid

18 9-Tricosene,(z)-

19 Octadecane,1-iodo-

20 Octacosyl acetate

21 I-propyl hexadecanoate

xcvii

22 Oxacyclotridecan-2-one

23 Octacosane

24 Cobaltocene,1,1’-diphenyl-

25 Purin-2,6-dione,1,3,9-trimethyl-8-[2-

nitrophenethenyl]-

26 1- Chloroeicosane

27 9,12-Octadecadienoic acid

28 Delta-7-avenasterol

29 Propanedioic acid, propyl

30 Hexadecane, 2-methyl

xcviii

31 Cyclooctanone,3,7-dimthyl

32 1-Docosanethiol

33 Palmitic anhydride

34 1,2-Benisothiazole, 3-(hexahydro-1H-

azepin-1-yl)-,1,1-dioxide

35 Diethylenediamine

36 2-Piperidinone, N-[4-bromo-n-butyl]-

xcix

Chapter four:

Discussion

c

4.1 Discussion:

Miracles from Quran represent a real challenge in discovering of chemical

ingredients and its relationship with remedies. Selection of pumpkins within

billion of plants did not come by chance but it was an indirect message from

God to search in-depth in the mysteries and secrets of this great plant. Modern

science demonstrate, by modern techniques, and through this work, eighty six

natural chemical compounds have been detected in the investigated four parts of

pumpkin plant (seeds 36, fruit 22, flowers 9 and leaves 19), they need more

efforts and deep analysis and interpretation to elucidate them in the plant.

Through the presence of Eighty six of natural chemical compounds can clearly

see the extent of physical and psychological illness suffered by Younis (peace

be upon him) and the most prominent role of pumpkin leaves in his recovery.

This chapter will talk about the interpretation and importance of these

compounds as well as the qualitative analysis.

4.1 Qualitative:

4.1.1Extraction:

1- Pumpkin seeds:

Pumpkin seeds were extracted with continuous soxhlet extraction method by

petroleum ether, chloroform and ethanol respectively. The petroleum ether

extract gave the predominant yield percentage, these indicates the presence of

high contents of non polar metabolites such as fatty acids and other lipids such

as sterols and triterpens.

2- Pumpkin leaves:

The same procedure of extraction was applied to the leaves and the yields

percentages of petroleum ether and ethanol are very close, these indicate the

equilibrium presence of polar metabolites like Tannins and glycosides and non

polar one.

3- Pumpkin fruit:

The same procedure of extraction was applied to the fruit and the ethanol

extract gave the high percentage of yield, these indicate the presence of high

contents of polar metabolites like Tannins and glycosides.

4- Pumpkin flower:

The same procedure of extraction was applied to the flowers and the petroleum

ether extract gave the predominant yield percentage this indicates the presence

of high contents of non polar metabolites such as fatty acids and lipids.

The three successive continuous soxhlet extractives yields of pumpkin flowers,

seeds, fruit and leaves were used for:

A- phytochemical screening tests:

ci

A.1- seeds:

The preliminary phytochemical investigations of three successive extracts

(petroleum ether, chloroform and ethanol) of the pumpkin seeds were carried

out. Glycosides, sterols and triterpens were detected in all three fractions of

seeds. Flavanoids and saponins were detected in the chloroform fraction while

alkaloid, saponins and coumarins were detected in ethanolic fraction.

A.2- leaves:

The qualitative phytochemical analysis of pumpkin leaves when fractionated

successively by the three mentioned solvents to detect its major constituents

showed the presence of flavonoids, alkaloids and saponin in ethanol extract with

glycosides, while sterol and triterpens were detected only in petroleum ether

fraction. Chloroform fraction showed the presence of alkaloidsand coumarins.

All three fractions were devoid of tannins and anthraquinone.

A.3- Fruit:

Alkaloids and glycosides were detected in all three fractions, in addition to

coumarins in ether and ethanol only while flavonoids were found to be present

in the ethanolic fraction.

A.4- Flowers:

It was found to be the only part of pumpkin in which tannins was detected in its

ethanol fraction. Alkaloids and saponins detected in both chloroform and

ethanol while coumarins in ether and ethanol fractions.

B- Antibacterial Experiments:

The three successive extracts of all four parts of pumpkin were subjected to

preliminary antimicrobial screening against two standard gram positive bacteria

(Staphyloccocus aereus and Bacillus subtilis) and two Gram- negative bacteria

(Escherichia coli and Pseudomonas aeruginosa).

B.1- Pumpkin seeds:

All extracts were exhibited a significant antibacterial activity against the all

tested bacteria.

B.2- Leaves:

Pseudomonas aeruginosa growth was inhibited only by ethanol fraction; the

petroleum ether fraction has a significant effect on Escherichia coli and

Staphyloccocus aereus while it has no activity against other tested bacteria.

Chloroform has no activity against Pseudomonas aeruginosa and it has a

significant activity against other tested bacteria.

B.3- Fruit:

cii

Ethanol fraction has activity against gram positives with no activity on gram

negatives, chloroform had a significant activity against Staphyloccocus aereus

more than other tested bacteria. Ether has no activity against Staphyloccocus

aereus and Pseudomonas aeruginosa with a high activity on Bacillus subtilis

and Escherichia coli.

B.4- Flowers:

Pseudomonas aeruginosa growth was inhibited only by chloroform fraction;

ether fraction has activity against E.coli and Staphyloccocus aereus.

Chloroform has no activity against Staphyloccocus aereus.

4.1.2 TLC separation:

TLC separation technique is done to adjust the separation of components and to

determine the best solvent system for the development of a TLC plate of

pumpkin fruits, flowers, seeds and leaves extracts.

Solvent system (Petroleum ether: hexane: ethyl acetate 75:20:15) is the better

solvent system than others for the development of a TLC plate and for

separation of components of pumpkin seeds and flowers, while Solvent system

(benzene: ethyl acetate 95:5) is the better solvent system than others for the

development of a TLC plate and for separation of components of pumpkin

leaves and fruit.

4.2 GC/MS:

Correlations between the identified/detected phytochemicals in pumpkin

Flowers, Fruit, Leaves and Seeds by GC.MS and current uses of it:

4.2.1 Pumpkin flowers: The phytochemical compounds in the flowers of pumpkin extracted with

ethanol screened by GC-MS method, 9 bioactive phytochemical compounds

were identified.

These different active phytochemicals have been found to possess a wide range

of activities, which may help in the protection against numerous diseases:

Activity of the identified phytochemicals in pumpkin flowers: 1- Palmitic acid:

According to a Korean study published in a 2010 edition of the "Journal of

Medicinal Food," palmitic acid posses antioxidant properties and can help to

prevent atherosclerosis (hypo-cholesterolemic). Also it has antitumor activity

with selective cytotoxicity to cancer cells and not cytotoxic to the normal

human cells, suggesting that palmitic acid may be a lead compound of

anticancer drugs (Harada H, et al, 1997). In moisturizers, palmitic acid is a very

good emollient just as many other naturally-occurring fatty acids, it helps

reinforce skin's healthy barrier function for a smoother surface good for skin

ciii

(decrease aging by as much as 56%) also it has ability to fight skin cancer.

Palmitic acid is known to has potential antibacterial and antifungal activities

(McGaw. L.J, 2002).

2- Oxalic acid esters:

Is an important element to stimulate and maintain the peristaltic motion in our

gut (Clark H. T, 2011).

3- 8, 11, 14- Eicosatrienoic acid:

It is omega- 3- poly unsaturated fatty acid, potent antioxidant, has cardio-

benefit, lower cholesterol, reduce depression, lower blood pressure, alleviate

insulin resistance, improve brain function and boost children concentration

levels. Also known to have anti- inflammatory properties and essential for

normal growth and development (Baggott and James, 1997).

5- Stigmasta-7, 16- dien-3-ol and Stigmast-7-en-3-ol:

It is B- sistosterol like cholesterol structure use for insulin resistance diabetes

(potent anti-diabetic agent i.e stimulates glucose transport) and induces anti-

proliferation in human leukaemia (H.X. Wang,T.B.Ng, 1999).

4.2.2 Pumpkin fruit:

This GC.MS analysis was carried out to determine the possible chemical

components from pumpkin fruit ethanol extract. This analysis revealed the

presence of 22 phytochemicals and their biological activities is reported bellow

Activity of the identified phyto-components in pumpkin fruit:

1- Vit E:

The primary function of vitamin E is antioxidant. Vitamin E deficiency is

almost entirely restricted to premature infants. When observed in adults, it is

usually associated with defective lipid absorption or transport (Champe etal.,

2008; Ritter et al., 2008). Free radicals cause membrane and epithelial injury

and have been implicated in the pathophysiology of numerous diseases,

including cancer and atheroma. Epidemiological studies suggested that reduced

vitamin E intake is associated with increased atherogenesis (Ritter et al., 2008).

2-5-methyl-5a,6,8,9,10,11,11a,12-octahydro-5H-6,10,11,12a-dimethanoindol

[3,2-b]quinolizine-8,13-diol:

Also known as Ajmaline, it is an indol alkaloid and a highly effective anti-

arrhythmic drug. It has been used for the treatment of various types of both

atrial and ventricular tachyarrhythmia in clinical practice for over forty years. In

the medical treatment of sustained ventricular tachycardia, and also in the

treatment of patients with Wolf-Parkinson-White syndrome with paroxysmal

civ

atrial fibrillation (Bahnikova et al.,2002; Bébarová et al.,2005;Batchvarov et

al.,2009).

3- 1H-Imidazole, 2-ethyl-4-methyl- or (Imidazole, 2-ethyl-4 methyl):

Imidazole is a planer five-member heterocyclic ring with 3C and 2N atoms and

in ring N is present in 1st and 3rd positions. The imidazole ring is a constituent

of several important natural products, including purine, histamine, histidine and

nucleic acid.

On the basis of various literature surveys Imidazole derivatives shows various

pharmacological activities include:

Anti fungal, Anti-bacterial activity, Anti inflammatory activity, analgesic

activity, Anti tubercular activity, Anti depressant activity, Anti cancer activity,

Anti viral activity and Antileishmanial activity (Shalini et al., 2010).

4- Methoxyphenamine:

It is a sympathomimetic agent which causes bronchodilatation and is used in

commercial preparations as a nasal decongestant (Lau et al., 1990). It is

frequently used in alleviating the bronchial asthma and symptoms of Chronic

obstructive pulmonary diseases COPD including cough, sputum and asthma.

However, there are few studies of its anti-inflammatory effect on COPD (Yue-

Hong et al., 2003).

5- 1,2-dihydroxyethyl-3,4-dihydroxyfuran-2(5H)-one:

1,2-dihydroxyethyl-3,4-dihydroxyfuran-2(5H)-one (VitC/Ascorbic acid) is

required for the maintenance of normal connective tissue, as well as for wound

healing. Vitamin C facilitates the absorption of dietary iron from the intestine

(Champe et al., 2008).

A deficiency of ascorbic acid results in scurvy, vitamin C is one of a group of

nutrients that includes vitamin E and β- carotene, which are

Known as antioxidants, consumption of diets rich in these compounds is

associated with a decreased incidence of some chronic diseases, such as

coronary heart disease and certain cancers (Champe et al., 2008).

6- 5-[1-hydroxy-2-(isopropylamino)ethyl]benzene-1,3-diol: It was used in the treatment of bronchial asthma and it is a central nervous

system stimulant (Nadendla, 2005).

7- 2-Amino-9-[3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-3 H-purine-6-

:

2-Amino-9-[3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-3H-purine-6

(Guanosine), it is similar to acyclovir. Acyclovir (Zovirax) is a guanine

nucleoside analogue, most effective against herpes simplex virus

(Criage&stitzel, 1997).

8- 4,4’-methylene bis(tetrahydro-1,2H,4-thiadiazine)1,1,1’,1’-tetraoxide:

Taurolidine (TN), or 4,4’-methylene bis(tetrahydro-1,2H,4-

thiadiazine)1,1,1’,1’-tetraoxide is an antibacterial drug originally synthesised in

cv

1970 with activity against a broad spectrum of microorganisms and has been

used as a safe lavage antibiotic to prevent bacterial infection in patients after

abdominal surgery. Over recent years, in vitro evidence has highlighted the role

of TN as a pro-apoptotic and antiangiogenic agent. Some of these in vitro

effects were also observed in brain tumour cells, whereas normal cells were

unaffected. Moreover, intra-peritoneal administration of TN in experimental rats

inhibited the growth of injected ovarian and colon cancer cells. Safety and

efficacy of intra-cavitary administration of TN, along with a prolonged i.v.

administration has been proposed to treat human tumours (Walters et al., 2007;

Aceto et al., 2009; Chromik et al., 2010).

Taurolidine also appears to have immunoregulatory properties, blunting

lipopolysaccharide induced tumour necrosis and also reducing adherence of

bacteria to human epithelial cells in vitro (Torres-Viera et al., 2000).

9- 3,7,11-trimethyl-2,6,10-dodecantrien-1-ol:

Farnesol (FOH) or 3,7,11-trimethyl-2,6,10-dodecantrien-1-ol is a naturally

available sesquiterpene alcohol and a key intermediate in de novo synthesis of

cholesterol in all mammalian cells. It acts to suppress Candida albicans

development and this isoprenoid alcohol has been demonstrated to inhibit the

growth of some bacteria, such as Staphylococcus aureus and some fungal

species and recently reports have shown that it can intensify the effect of

antimicrobial agents on Staphylococcus aureus (S. aureus) and Escherichia

coli (E. coli) (Jing et al., 2010).

10- Tetradecanoic acid (Myristic acid):

Myristic acid usually accounts for small amounts of total fatty acids (FA) in

animal tissues but is abundant in milk fat. Epidemiological and clinical studies

have shown that dietary fats containing high levels of saturated fatty acids

induce an increase in plasma total and low density lipoprotein (LDL)

cholesterol concentrations in humans, among saturated FA, myristic acid

consistently seems to increase animal and human blood cholesterol

concentrations less than other FA (Rioux et al., 2005).

11- Squalene:

Squalene is a triterpene that is an intermediate in the cholesterol biosynthesis

pathway, and it is the main component of skin surface polyunsaturated lipids,

shows some advantages for the skin as an emollient and antioxidant and for

hydration and its antitumor activities. It is also used as a material in topically

applied vehicles.

Squalene might be a useful addition to potentiate the effects of some

cholesterol-lowering drugs, the primary therapeutic use of squalene currently is

an adjunctive therapy in a variety of cancers (Smith TJ, RT al, 2000).

cvi

Substances related to squalene, including β-carotene, coenzyme Q10

(ubiquinone) and vitamins A, E and K (Zih-R., 2009).

Fig 4-1: Chemical structures of (A) squalene, (B) β-carotene, (C) coenzyme

Q10, and (D) vitamins A, (E) E, and (F) K1.( Zih-Rou et al.,2009):

12- 3,7,11,15-tetramethyl-2-hexadecan-1-ol:

(2E,7R,11R)-3,7,11,15-tetramethyl-2-hexadecan-1-ol (phytol)is one of the

simplest and most important of the diterpenes is phytol, a reduced form of

geranylgeraniol. A phytyl substituent is also found in vitamin K1

(phylloquinone), a naphthoquinone derivative found in plants, though other

members of the vitamin K group (menaquinones) from bacteria (Dewick, 2002).

Fig4-2: phytol, vitamin K1and vitamin E (Dewick, 2002):

13- 1H-Purine-2, 6-dione, 3,7-dihydro-1,3-dimethyl- :

cvii

1H-Purine-2, 6-dione, 3,7-dihydro-1,3-dimethyl- or Theophylline remains one

of the most widely prescribed drugs for the treatment of asthma and Chronic

Obstruction Pulmonary Disease (COPD) world-wide, since it is inexpensive and

widely available also has anti-inflammatory effects (Barnes, 2005; Barnes,

2010).

14- 1-β-D-ribofuranosyl-1H-1,2,4trazole-3-carboxamide:

Ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) shows broad-

spectrum antiviral activity against a variety of viruses and is used in

combination with interferon-α to treat hepatitis C virus infection (Crotty et al.,

2000).

15- 6,7-dimethoxy-3-[(5R)-4-methoxy-6-methyl-7,8-dihydro-

5H[1,3]dioxolo[4.5-g]isoquinolin-5-yl]-3H-2-benzofuran-1-one:

Noscapine or (3S)-6,7-dimethoxy-3-[(5R)-4-methoxy-6-methyl-7,8-dihydro-5H

[1,3]dioxolo[4.5-g]isoquinolin-5-yl]-3H-2-benzofuran-1-one (opioid alkaloid) is

an antitussive agent(very safe cough suppressant), acting on central nervous

system site (Matchett et al., 2001; Nadendla, 2005) It also possess weak

bronchodilator properties. However, unlike codeine and other narcotics,

noscapine lacks addictive effect. Other clinical applications of this drug are

based on its anti-stroke and anti-cancer activities. Some studies have shown

anxiolytic effects of noscapine in mice.

Noscapine effectively inhibits the progression of various cancer types both in

vitro and in vivo with no obvious side effects like lymphoma, breast cancer,

melanoma, ovarian carcinoma, colon cancer, lung cancer, bladder tumours and

prostate cancer (Barken et al., 2008).

16- 1-(4-tert-butylphenyl)-4-[4-[hydroxyl(diphenyl)methyl] piperidin-1-yl]

butan-1-ol:

Terfenadine or 1-(4-tert-butylphenyl)-4-[4-

[hydroxyl(diphenyl)methyl]piperidin-1-yl]butan-1-ol is an antihistaminic

formerly used for the treatment of allergic conditions (Zünkler et al., 2000).

17- 2-phenylethyl hydrazine:

Phenelzine is 2-phenylethyl hydrazine. It is the hydrazine analogue of phenyl

ethylamine, a substrate of MAO. Phenelzine and several other MAOIs, such as

isocarboxazide, are structurally related to amphetamine and were synthesized in

an attempt to enhance central stimulant properties. (Nadendla, 2005).

18- 2,6-di(propan-2-yl)phenyl:

Propofol or 2,6-di(propan-2-yl)phenyl is a liquid short-acting general

anaesthetic. Also it is an intravenous sedative– hypnotic agent that is commonly

administered for sedation of patients in the intensive care unit (ICU) who are

being treated with tracheal intubation and mechanical ventilation (Barr et al.,

2001; Kam & Cardone, 2007).

19- 4-[3-(5H-Dibenz [b,f] azepin-5-yl)-1-piperazinethanol:

cviii

Opipramol, 4-[3-(5H-Dibenz [b,f] azepin-5-yl)-1-piperazinethanol, is a tricyclic

compound for therapy of anxious-depressive states and general anxiety

disorders.

It is clinically used in Germany and a few European countries with increasing

tendency to be used as a substitute for benzodiazepines for long-term

application (Möller et al., 2001; Turhan& Uslu, 2008).

20- 5-fluoro-1-[4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-1H-

pyrimidine-2,4-dione:

Floxuridine or 5-fluoro-1-[4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-

1H-pyrimidine-2,4-dione is a fluorinated pyrimidine that is primarily used for

the treatment of metastatic carcinoma of the colon. Floxuridine and other

pyrimidine analogues have been extensively used in the treatment of a variety of

cancers for the past 40 years (Ning etal., 2007).

21- 4-chloro-N-(2-methyl-2,3-dihydroindol-1-yl)-3-sulfamoyl-benzamide:

Indapamide, 4-chloro-N-(2-methyl-2,3-dihydroindol-1-yl)-3-sulfamoyl-

benzamide is a thiazide-type diuretic, a widely used as antihypertensive agent

(Sanam et al., 2012).

22- Canrenone:

Canrenone, a cardiovascular drug, a sort of steroid, is spironolactone’s major

metabolite and has been widely used as a nonselective aldosterone receptor

antagonist clinically to treat heart failure, high blood pressure, oedema, liver

ascites, and other cardiovascular diseases (Da-Ming et al., 2011).

4.2.3 Pumpkin leaves:

The Gc.ms analysis has shown the presence of different phyto-constituents in

the ethanolic extract of pumpkin leaves. A total of nineteen compounds were

identified:

Activity of the identified phyto-components in pumpkin leaves:

(1). 6,6-Dimethyl-10-methylene-1-oxa-spiro[4.5]decane:

6, 6-Dimethyl-10-methylene-1-oxa-spiro[4.5]decane (Fumagillin) was first

isolated in 1949 from the microbial Aspergillus fumigatus and used as

antimicrobial agent, it is also used in the treatment of cancer and as amebicide

(Clark, AM, 1996).

(2). Octahydrobenzo[b]pyran,4a-acetoxy-5,5,8a-trimethyl-:

It is an oedema modifier, use in the treatment of asthma and lymph oedema

(John-Wiely, 2011) and when fused with 1,2-dioxepane molecule it form

artemrther which is an antimalarial agent used to treat acute uncomplicated

malaria (Clark, AM, 1996).

(3). 2(4H) Benzofuranone,5,6,7,7a-tetrahydro-4,4,7a-trimethyl-,(R)- :

cix

2(4H) Benzofuranone,5,6,7,7a-tetrahydro-4,4,7a-trimethyl-,(R)-

(EPLERENONE) is an oral medication used alone or in combination with

other medicines to treat high blood pressure. It works by blocking a chemical

(aldosterone) in the body which in turn lowers the amount of sodium and water.

Lowering high blood pressure helps to prevent strokes, heart attacks and kidney

problems. It is also used to treat congestive heart failure following a heart attack

(Nweze et al., 2004). It has anxiolytic effects in animal studies, and good oral

bioavailability.

Generally Benzofuranone is a bioactive compound possessing the properties

such as analgesic, antidiabetic, antibacterial, and antifungal, also was

investigated and shown to have antispasmodic action (Nweze et al., 2004).

(4/9/11). 1-Hexadecanol,2-methyl-:

Also known as Cetyl alcohol has been used as human sedatives, and used

chiefly as an emollient and can function as antioxidants (Smolinske- Susan C,

1992).

(5). E-15-heptadecenal:

Previous investigations reported that the compounds such as 1-Octadecene and

15-Heptadecenal present in higher plants are responsible for their anticancer,

antioxidant and antimicrobial activities (SudhaT et al, 2013).

(6). 2-Pentadecanone,6,10,14-trimethyl-:

Recently been demonstrated that aliphatic ketones like 2-Pentadecanone, 6, 10,

14-trimethyl are effective hypocholesterolemic agents and do not have the

estrogenic and anti-fertility characteristics which are associated with the cyclo-

ketones (R.T Lalonde, 1971).

(7). 1-Heneicosyl formate:

Used in the treatment of skin ulcers and has anti –inflammatory property (G.

Amresh, 2007).

(8). Z-(13,14-epoxy)tetradec-11-en-1-ol acetate:

Usually used as antimicrobial in cosmetics (Michael ash, 2004).

(10). 4,8,12,16-tetramethyl heptadecan-4-olide:

Used to treat and prevent cardiovascular diseases, dyslipidemia, glucose

metabolism disorder, hypertension, renal diseases, cancer and inflammation

(Gupta-AK, 2004).

cx

(12). 9-(2, 2-Dimethylpropanoilhydrazone)-3,6-dichloro-2,7-bis-[2 (diethyl

amino]fluorene:

Is polycyclic aromatic hydrocarbon compound, recent researches been

demonstrated that 9- methylated fluorene derivatives have high ability to inhibit

the initiation and promotion of tumour especially skin and liver tumour, and

have a high antibacterial activity especially against gram positive bacteria

(Lavoie EJ, et al, 1981).

(13/16). 17- Pentatriacontene:

Due to their high hydrophobic nature, it applies on freshly cutted skin, cover it

and accelerate their healing (Jain, SK, 1976).

(14). Cholestane-3,6,7-triol:

Cholestane-3, 6, 7-triol is an oxygenated derivative of cholesterol (oxysterol)

suppresses proliferation, migration, and invasion of human prostate cancer cells,

oxysterols are more polar and more readily diffusible through cell membranes

and the blood –brain barriers at faster rate than cholesterol itself, and possesses

higher pro-inflammatory and cytotoxic effects than cholesterol in certain cells

and tissues. Oxysterols have been shown to suppress the proliferation and

survival of human cancer cells in vitro. The cytotoxicity of oxysterols may

cause growth inhibition and cell death in cancer cells if applied locally to

tumours (N. Habib, 2009). Observed that triol treatment inhibited tumour

growth, reduced cell proliferation and altered cytoskeletal morphology of

human prostate cancer cells (N. Habib, 2009).

(15). 5.14,23-Octadecatrien-14,15-diol:

Has anti-inflammatory, anti-arthritic, anti-histaminic property (Sermakkani M et

al, 2012).

(18). Octadecane,3-ethyl-5-(2-ethyl butyl)- :

In vitro antifungal potentials and thus has a potential therapeutic use (Nweze et

al., 2004).

(19). 4,4,6a,6b,8a,11,11,14b-Octamethyl

1,4,4a,5,6a,6b,7,8,8a,9,10,11,12,12a,14, 14a,14b-octadecahydro-2H-picen-3-

one:

Known as olean-12-en-3-one, is a triterpene has antibacterial, antioxidant,

antitumor and cancer preventive properties (Duan DD et al, 2011).

4.2.4 Pumpkin seeds:

cxi

The phytochemical compounds in the seeds of pumpkin extracted with ethanol

screened by GC-MS method, 36 bioactive phytochemical compounds were

identified. These different active phytochemicals have been found to possess a

wide range of activities, which may help in the protection against numerous

diseases.

Activity of the identified phyto-components in pumpkin seeds:

(1). Hexadecanoic acid, methyl ester:

Is a saturated fatty acid ester which has been reported to show antioxidant,

hypocholesterolemic and nematocide activities (Sermalkani M et al, 2012).

(2). Heptanoic acid:

Also called Enanthic acid, it is an oily liquid organic compound used to esterify

steroids in the preparation of drugs such as testosterone (Merck index, 11th

edition.4581).

(3). Octanoic acid:

This saturated fatty acid, also known as Caprylic acid, is also present in butter

and palm oil. According to the Health Sciences Institute, caprylic acid is known

to have numerous health benefits. One of the most well known health benefits

associated with the ingestion of caprylic acid, is its ability to treat the

overgrowth of yeast-like fungus that can live and grow in the intestines. In

addition to treating bacterial issues, caprylic acid may also have a beneficial

effect on high blood pressure. Other benefits may include helpful relief from

those who suffer from Chron's disease, an inflammatory intestinal condition

(Physicians Desk Reference; 2001). According to a study

conducted by a Japanese team of researchers at the graduate school of medicine

at Chiba University, it can even help balance your blood sugar, improve

digestion, balance your hormones, and keep your heart healthy, in one early

experiment some evidence showed that medium-chain triglycerides (MCT), like

caprylic acid, can help to treat children with epilepsy and can control seizures,

However, there have been no follow-ups to confirm these findings (Physicians

Desk Reference; 2001).There is also some evidence to suggest that caprylic acid

can delay the progression of Alzheimer’s disease, according to the Alzheimer’s

Association of America, but larger trials are needed to confirm this. It is also

used as disinfectant in health care facilities, schools/colleges, animal

care/veterinary facilities (P.J.; Mallard, W.G).

(4). 2-t-Butyl-5-propyl-[1,3]Dioxolan-4-one:

Is a new drug with potent spasmolytic activity (Morsdorf K, et al.

Pharmacology, 1970).

(5). Hexanoic acid:

Generally short chain fatty acids like hexanoic acid (C6) exhibit antimicrobial

activity especially for oral microorganisms (Merk, 1989). Studies have also

cxii

shown that hexanoic acid is useful for neurodegenerative disorders like

Alzheimer’s disease, Parkinson’s disease and epilepsy, and it is an important

ingredient in parenteral nutritional emulsions for the treatment of a variety of

malabsorption condition (Journal of lipid research sept, 1967).

(6). 2H-pyran,3,4-dihydro-:

Also known as dihydropyran, it is a heterocyclic compound. Has especially high

cytotoxicity against tumour cells with less toxic effect on human normal non

cancer cells, also it is antioxidant in vitro (Journal of bioactive and compatible

compounds Oct, 1990). It is also antiviral agent and useful in the treatment of

influenza (Abbott Lab, 1999).

(7). Benz[c]acridine,5,10-dimethyl-:

Benz[c]acridine alone showed both in vitro and in vivo antimicrobial,

amebicidal activities and carcinogenic for skin but when the structure of

Benz[c]acridine substituted at position 5 and 10 by hydrocarbon groups this will

diverse carcinogenic activity displayed by Benz[c]acridine and at the same time

their antimicrobial and amebicidal activities seemed to be enhanced (Molnar J,

et al. anticancer, 1993).

(8). 9-methyl-z-10-tetradecen-1-ol acetate:

Identified originally as the sex pheromone with no biological activity reported

(Journal of chemical Ecology, Sep 1979).

(9 -32). 1- Hexadecanethiol & 1- Docosanethiol:

Is an organosulfur compounds that contain a sulphur-hydrogen bond analogous

to an o-H, markedly increase the activity of L.Ornithine decarboxylase invitro

which is an essential enzyme for normal cell growth, production of the

polyamines necessary to stabilize newly synthesized DNA and reduce the cell

apoptosis in embryo (G Gerner EW, Meyskons FL, 2004).

There is evidence that plant containing organosulfur compounds has a

protective against cancer in humans (Fukushima S, et al. J, 1997).

(10). 6-Amino-2,3diphenyl-1H-pyrrolo[2,3- b] pyridine:

Is an indole compound, pyridine nucleus is present in several naturally

occurring alkaloids and biologically active molecules. Pyridine based

heterocyclic like 6-Amino-2,3diphenyl-1H-pyrrolo[2,3- b] pyridine is found to

has wide range of activity, it exhibit anti-inflammatory, analgesic and

antipyretic activities also it is a potent inhibitor of Mitotic Kinase Mono-polar

Spindle1(MPS1) which is one of top 25 genes over expressed in many human

cancers cells. Cancers cells are particularly dependent upon MPS1 for their

survival making it a target of significant interest in oncology, then 6-Amino-

2,3diphenyl-1H-pyrrolo[2,3- b] pyridine is a potent anticancer compound due to

their high and selective MPS1inhibitory action (Weiss E, Winey M, 1996).

(11). 1-(+)-Ascorbic acid-2,6- dihexadecanoate:

cxiii

L-Ascorbic acid-2, 6- dihexadecanoate is a potent hyaluronidase inhibitor (an

enzyme that degrades hyaluronan which is an important component of the

extracellular matrix. The mammalian hyaluronidase is considered to be involved

in many (patho)-physiological processes like in fertilization and tumour growth,

such roles for hyaluronidase suggest that inhibitors could be useful as

pharmacological tools (Cherr, G.N. Yudin, A. I, J.W, 2001).

Although L- Ascorbic acid alone has been reported to be a week inhibitor of

hyaluronidase but an additional hydrocarbon might increase inhibitory activity,

this indicating that L-Ascorbic acid-2, 6- dihexadecanoate is potent inhibitor.

Moreover hyaluronidase inhibitors could be useful as drugs in the treatment of

Arthroses also combined with antibiotic in an antibacterial therapy. L-Ascorbic

acid-2, 6- dihexadecanoate has been reported to inhibit streptococcus

hyaluronidase, staphylococcus hyaluronidase (Li, S. Tylor, K. B, M J, 2001).

(12). Heptadecane:

Is an alkane hydrocarbon, volatile compound, it exhibit potent anti-oxidative

effect (ameliorate several oxidative stress related diseases) and anti-

inflammatory agent (Kim DH, Park MH, 2013).

(13). 1,2-Bezenedicarboxylic acid, diisooctyl ester:

Diisooctyl phthalate is a natural diterpene compound isolated for the first time

from the unripe fruit of plants, show potent antifungal activity against six

pathogenic fungi (Asian Journal of microbiology, 2006). Also it has

antibacterial effect (Rao P, PS, Karmarkan, s.m, 1986). This compound was

identified as inhibiting melanogenesis, compound with a hypo pigmenting

capability used in cosmetics (DT Nguyen, DH Nguyen, HL Lyun, 2007).

Diisooctyl phthalate also showed significant activity against Salmonella

(Gibbons JA, Alexander M, 1989).

(15). Cyclotetradecane:

Is a new potential copper chelating agent for neuroprotective in Alzheimer’s

disease which is associated with copper metabolism in adrenal cortex (Mort V,

et al, 2006).

(17). Tridecanoic acid:

Also known as a tridecylic acid; among all saturated fatty acid, tridecanoic acid

has the highest antioxidant and anti-inflammatory activities (J. Agric, 2002).

(20). Octacosyl acetate:

Octacosyl acetate or ferulate markedly have anti-inflammatory, antifungal and

antiviral activities. Also it is reducing anorexia, fatigue and stimulating or

improving heart response (Eugene Sebastian J, 2013).

cxiv

(21). I-propyl hexadecanoate:

Also called isopropyl palmitate, is the ester of isopropyl alcohol and palmitic

acid. It is an emollient, moisturizer and thickening agent (JE Ramirez, M,

2015). Also has antimicrobial activity (Cardoso, V.M, 2006).

(22). Oxacyclotridecan-2-one:

Is cyclolactone compound; 30 years ago, research revealed that cyclic lactones’

compounds have anthelmintics (especially control nematodes),

antileishmaniasis and antibacterial activity. Other example of cyclolactone

compounds is all macrolides antibiotics (Brown DD, et al. Vet, 2012).

(23). Octacosane:

This saturated hydrocarbon with branched chain structure has mosquitocidal

activity (J. Ethnopharmacol, 2004).

(24). Cobaltoecne,1,1’-diphenyl-:

Known as phenylcyclopentadienyl-cobalt, is an organometallic compounds used

as a radiopharmaceutical to treat small cancers also in vitro it has protozoocidal

activity (Loiseau PM, et al, 1988).

(25). Purin-2,6-dione,1,3,9-trimethyl-8-[2-nitrophenethenyl]-:

Previous studies in a group of methylated-purin-2,6-dione proved that when

these compounds substituted in a7 or 8 position by nitro or aminoalkyl group

like Purin-2,6-dione,1,3,9-trimethyl-8-[2-nitrophenethenyl]- this will increase

their activity as antidepressant and anxiolytic (Chlon-Rzepa G, t al. Pol J, 2001).

(27). 9, 12-Octadecadienoic acid:

Is polyunsaturated omega 6 fatty acid, cannot be synthesize by human body and

must be consumed for proper health. It reduces skin scaling, prevents hair loss

and stimulates wounds healing (Ruthing DJ, Meckling KA, 1999). Also has

antibacterial (especially gram positive) and antioxidant activities (F Dilika, P. D

Bremner, 2000).

(28). Delta-7-avenasterol:

Is unsaturated plant sterol, chemically similar to animal cholesterol then it has

hypocholesterolemic effect. Research has indicated that delta -7-sterol competes

with dihydrotestosterone DHT at the receptor sites in prostate, DHT is strongly

responsible for enlarging the prostate and causing benign prostatic hyperplasia

BPH but when this compound is present in diet it seems to help minimize the

harmful effects of DHT on prostate i.e prevent BPH and prostate cancer (Health

& Energy Food.com, 2015).

cxv

(29). Propanedioic acid, propyl:

Is a dicarboxylic acid propyl also known as propylmalonic acid, it has

antioxidizing activity (Dorovskikh VA,et al, 1992).

(31). Cyclooctanone,3,7-dimthyl:

Cycloalkanone like Cyclooctanone,3,7-dimthyl significantly lower cholesterol,

triglycerides levels, and also has ability to substrate growth of E. coli strain

(G.S. Abermethyl, C, Piantados, 1974).

(34). 1,2-Benisothiazole, 3-(hexahydro-1H-azepin-1-yl)-,1,1-dioxide:

In vitro, all benzisothiazole derivatives proved to have antipyretic and analgesic

activities with potency 3-fold higher than that of acetaminophen (Wang Z-Y,

2015), also it is known to have fungicidal and bactericidal effects (JP, 1973).

Additional investigation performed on this compound and found that it has

antiplatelets and spasmolytic activity (Paola Vicini, 2000).

Benzisothiazole derivatives are cholinesterase inhibitors and are useful in

enhancing memory in patients suffering from dementia and Alzheimer’s disease

(conditions of degenerative cholinergic neurons) (Becker et al, 1988).

(35). Diethylenediamine:

Known as Piperazine, is a cheap and readily available anthelmintic agent with

very wide therapeutic index. It is used in the therapy of ascariasis (round worm)

and oxyuriasis (thread worm) infestations. Also it was recently approved as an

oral anticancer drug for clinical use in Japan and it has shown potent

antiproliferative activity against colon, prostate, breast, lung and leukaemia

tumours(Yarim et al., 2012).

(36). 2-Piperidinone, N-[4-bromo-n-butyl]-:

N- Substituted lactam ring compounds as 2-Piperidinone, N-[4-bromo-n-butyl] -

have anticonvulsant and neuro-protective activity (Wayne J. Brouillette and

Garyl. Grunewald, 1983).

Others compounds had no biological activities reported.....

cxvi

Chapter Five:

CONCLUSION & RECOMMENDATIONS

cxvii

5.1 Conclusion:

This study, it aims at phytochemicals analysis of pumpkin’s Flowers, Seeds,

Leaves, Fruit and the revelation of the secrets of a Qur’an verses regarding the

protection of Yunus (pbuh) by the gourd herb.

We try to explain the event with the prophet Yunus (pbuh) from chemically and

pharmaceutical point of view. Any person in the state of the prophet Yunus

(pbuh) might have the following predicted diseases: inflammations, infections,

allergy, psychological disorders (depression), severe pain, respiratory disorders,

electrolytes imbalance, cardiovascular disorders, lipid disorders, liver

dysfunction and renal failure.

Qualitative analysis of pumpkin’s Flowers, Seeds, Fruit and Leaves

extracts confirm presence of: a. Triterpen and sterols.

b. Saponins

c. Flavonoids.

d. Alkaloids.

e. Tannins.

f. Cardiac glycosides.

g. Coumarins.

GC.MS confirms that all four parts of pumpkin contain numerous compounds

with several reported important biological activity such as:

- Protect from the formation of cancers cells (Palmitic acid, Cholestane-3,6,7-

triol).

- Encephalic stimulant (Eicosatrionic acid, Phenelazine).

-Protect from Alzheimer’s disease (2-Piperidinone, N-[4-bromo-n-butyl]-,

Cyclotetradecane, Octanoic acid).

- Reduce cholesterol level in the blood i.e protect from cardiovascular diseases

(Stigmasta-7,16- dien-3-ol, Carenone)

- Antimicrobial and antiviral activity (Farnesol).

- Antidepressant activity and anticonvulsant (Opipramol, 2-Piperidinone, N-[4-

bromo-n-butyl]-).

cxviii

- Anti-hypertensive and antidiabetic properties (Indapamide, Stigmasta-7,16-

dien-3-ol and Stigmast-7-en-3-ol).

- Antioxidant(Vit E, Vit C, Eicosatrionic acid).

- Nutrients and contains a lot of vitamins (Vit E, Vit C).

-Treat skin problems (Palmitic acid, 1-Hexadecanol,2-methyl, 9, 12-

Octadecadienoic acid, 17- Pentatriacontene ).

- Spasmolytic and anithelmintic (Oxacyclotridecan-2-one, Piperazine).

-Treat and protect from benign prostatic hyperplasia (Delta-7-avenasterol,

Cholestane-3,6,7-triol).

- Reduce anorexia and fatigue (Octacosyl acetate).

- Photoprotective (carotenoid pigments (β-carotene)).

- Antileishmaniasis (Oxacyclotridecan-2-one, 1-H-Imidazole,2-ethyl-4methyl-).

- Cardiotonic (Carenone, Eicosatrionic acid, Octacosyl acetate).

- Useful in renal problems (4,8,12,16-tetramethyl heptadecan-4-o-lide).

- Analgesic (1,2-Benisothiazole,3-(hexahydro-1-H-azepin-1-yl)-).

As a consequence of all these bioactive eighty seven compounds and their

important biological activities, Allah Almighty had chosen the pumpkin tree for

the prophet Yunus (pbuh) because of its benefits and usefulness in the large

scale in recovering health and strength, and because of the presence of several

beneficial active ingredients. However, isolation of individual phytochemicals

and subjecting it to biological activity will definitely give fruitful results.

The study revealed that cucurbita studied was a potential food (all parts of

pumpkin plant can be eaten) and a multiple components medicine of benefits to

the weak patients.

cxix

5.2 Recommendations:

1. The pharmacognostical and phytochemical evaluation of pumpkin used in

traditional medicine must be carried out to determine the identity and to control

the quality and purity of plant material.

2. Efforts must be made to quantitatively examine the active principles of

pumpkin, to standardize the product in order to obtain the most beneficial

effects.

3. Clinical studies are recommended to elucidate the correlation between

chemical ingredients and their pharmacological properties, and to confirm the

data of traditional medicine.

4. More studies are required on the pharmacokinetics of pumpkin and its

constituents and on their toxicological effects after their consumption over a

long period of time.

5. Fact of obtaining new drugs are possible, thus, the pharmaceuticals factories

are invited to take the leaves, seeds, fruit and flowers of pumpkin in

consideration for obtaining a new drugs or as healthy food.

6. Orientation of the researchers to pay attention to the plant specially that

mentioned in Holly Quran and prophet Sunnah and subjected to scientific

investigation to detect and know their beneficial effects.

7. To raise public and people’s awareness to traditional medicine, especially in

plants mentioned in Quran.

cxx

References

cxxi

6- References:

Abbott Lab, 1999 (Inhibitors of neuroaminidases Dihydropyran).

Abermethyl G S, C, Piantados, J. Med.chem, 1974. 17(2) pp154-157.

Aceto N., Bertino P., Barbone D., Tassi G., Manzo L., Portae C., Mutti L.,

Gaudino G.(2009). Taurolidine and oxidative stress: a rationale for local

treatment of mesothelioma. Eur. Respir. J. ; 34: 1399–1407.

Adaramoye A., Achem J., Akintayo O., Fafunso A. (2007).Hypolipidemic

effect of Telfairia occidentalis (fluted pumpkin) in rats fed a cholesterol-rich

diet. J Med Food. ; 10(2):330-336.

Alarcon-Aguilar J., Hernandez-Galicia E., Campos-Sepulveda E., Xolalpa-

Molina S., Rivas-Vilchis F., Vazquez-Carrillo I., Roman-Ramos R.(2002).

Evaluation of the hypoglycemic effect of Cucurbita ficifolia Bouché

(Cucurbitaceae) in different

experimental models. J Ethnopharmacol.; 82(2-3):185-189.

Al Khalifa K.(2003). Curcurbita species for prevention, treatment and food.

JScientific Miracles; 14(8):1-4.

Al Qura’n alkareem.

Azaizeh H., Saad B., Cooper E., Said O.(2010). Traditional Arabic and Islamic

Medicine, a Re-emerging Health Aid. eCAM.;7(4):419–424.

cxxii

Azevedo-Meleiro H , Rodriguez-Amaya B.(2007). Qualitative and quantitative

differences in carotenoid composition among Cucurbita moschata, Cucurbita

maxima, and Cucurbita pepo.J Agric Food Chem.; 16;55(10):4027-4033.

Baggott and James, 1997. The divinyl methane pattern in fatty acids, Salt Lake

city, UT: Knowledgeweaves.

Bahnikova M., Matejovič P., Pásek M., Šimurdová M. , Šimurda J. (2002).

Ajmalineinduced block of sodium current in rat ventricular myocytes. Scripta

Medica (BRNO).;75 (4): 169–178.

Barken I., Geller J., Rogosnitzky M.(2008). Noscapine Inhibits Human Prostate

Cancer Progression and Metastasis in a Mouse Model. Anticancer Rese.;28:

3701-3704 .

Barnes P.(2005). Theophylline in Chronic Obstructive Pulmonary Disease. New

Horizons. Proc. Am. Thorac. Soc.; 2: 334–339.

Barnes P.(2010). Review: Theophylline. Pharmaceuticals; 3: 725-747.

Barr J., Egan D., Sandoval N., Zomorodi K., Cohane C., Gambus P. Shafer

S.(2001). Propofol Dosing Regimens for ICU Sedation Based upon an

Integrated Pharmacokinetic– Pharmacodynamic Model. Anesthesiology;

95:324–333.

Batchvarov V., Christov I., Bortolan G., Govindan M., Camm A., Behr

E.(2009). Automatic Assessment of Right Ventricular Repolarisation

Dispersion during Diagnostic Ajmaline Test for Suspected Brugada Syndrome.

Computers in Cardiology 36:297−300.

Bean, A.R, 2006. Collecting and preserving plant specimens, a manual. ISBN 1-

920928-06-5.

Bébarová M., Matejovič P., Pásek M., Šimurdová M. , Šimurda J.(2005). Effect

of Ajmaline on Transient Outward Current in Rat Ventricular Myocytes. Gen.

Physiol. Biophys.; 24(1): 27—45.

Becker et al, 1988. Drug development research, 12, 163-195.

Block J., Beale J.(2004). Wilson and Gisvold’s Text book of Organic Medicinal

and Pharmaceutical Chemistry.11th edition. Lippincott Williams

&Wilkins.United state of America.pp: 867-885.

Brown DD, et al. Vet, 2012 pharmacological characterization of cycliclactone.

cxxiii

Caili F, Huan S, Quanhong L.(2006). A review on pharmacological activities

and utilization technologies of pumpkin. Plant Foods Hum Nutr.;61(2):73-80.

Carbin BE, Eliasson R. Treatment by Curbicin in benign prostatic hyperplasia

(BPH). Swed J Biol Med 1989;2:7–9 [in Swedish].

Carbin BE, Larsson B, Lindahl O. Treatment of benign prostatic hyperplasia

with phytosterols. Br J Urol 1990;66:639–41.

Cardone D, Kam P (2007). Review Article: Propofol infusion syndrome.

Anaesth.; 62: 690–701.

Cardoso,V.M. Solano, A.G, 2006. JP, 1973 - Journal of pharmaceutical and

biomedical analysis, unitd states patient specification 7,230-137.

Castells E., Delgado E. , Escoda C.(2008). Use of amitriptyline for the treatment

of chronic tension-type headache. Review of the literature. Med. Oral Patol.

Oral Cir Bucal.;13(9):567-572.

Champe P., Harvey R., Ferrier D.(2008). Biochemestry 4th edition.Lippincott

Williams &Wilkins.United state of America.pp: 377-378, 391.

Cherr, G.N. Yudin, A. I, J.W, 2001. Matrix Biol 20, 515-585 cross Ref medline.

Chlon-Rzepa G, t al. Pol J. Pharmacol Rep, 2001.

Chonoko G., Rufai B. (2011). Phytochemical screening and antibacterial

activity of Cucurbita pepo (Pumpkin) against staphylococcus aueus and

salmonella typhi. Bajopas. ;4(1): 145 – 147.

Chromik A., Hahn S., Daigeler A., Flier A., Bulut D., May C., Harati K.,

Roschinsky J., Sülberg D., Weyhe D., Mittelkötter U., Uhl W.(2010). Gene

expression analysis of cell death induction by Taurolidine in different malignant

cell lines. BMC. Cancer;10:595- 608.

Ciulei Ioan(1982).Methodology for Analysis of Vegetable Drugs, UNIDO,

Romania.

Clark AM, 1996. The natural products as a source for new drugs. Pharm.Res 13.

Clark H . T, Davis A.W 2011- oxalic acid anhydrous org.synth:421, coll. Vol.1.

Clin Nutr, Hyun T, Barrett-Connor E, Milne D. Zinc intakes and plasma

concentrations in men with osteoporosis: the Rancho Bernardo Study, Sept.

2004:80(3):715-721. 2004. PMID:15321813.

cxxiv

Cowan M.M, 1999. Plants products as antimicrobial agents. Clin. Microbiol.

Rev; vol 12, pp 564-582.

Credo Reference. 19 Feb. 2008 Pumpkin." The Columbia Encyclopedia, 2004.

http://www.credoreference.com/entry/4294972

Criage C., Stitzel R.(1997). Modern Pharmacology with clinical application. 5th

edition. Lippincott Williams &Wilkins.pp:569.

Crotty S., Maag D., Arnold J. , Zhong W., Lau J., Hong Z., Andino R.,

Cameron C.(2000). The broad-spectrum antiviral ribonucleoside ribavirin is an

RNA virus mutagen. Nature America. Inc.;6(12):1375-1379.

Da-Ming H., Tian-Zhen Z., Feng-Jie C., Wen-Jing S., Li-Ming Z., Meng-Yi Y.,

Ya-Juan W.(2011). Research Article: Simultaneous Identification and

Quantification of Canrenone and 11-α-Hydroxy-Canrenone by LC-MS and

HPLC-UVD. J. of Biomedicine and Biotechnology. 1-7.

Decker-Walters, Robinson, R.W, D.S. (1997). "What are Cucurbits". In

Cucurbits, R.W. Robinson & D.S. Decker-Walters, CAB International, New

York. pp. 1-22.

Devapriam J., Gibbons S., Pannu H., Bhaumik S.(2008).Use of

Levomepromazine in the management of aggression in adults with intellectual

disability. British Journal of Developmental Disabilities;54( 106) : 11-17.

Dewick P.(2002). Medicinal Natural Products ABiosynthetic Approach. 2nd

edition. John Wiley & Sons Ltd.pp:158.

Dewick P.(2009). Medicinal Natural Products ABiosynthetic Approach. 3rd

edition.

Dhiman K., Gupta A., Sharma D., Gill N., Goyal A. (2012). AReview of the

Medicinally important Plants of the Family Cucurbitaceae. Asian Journal of

Clinical Nutrition;4(1):16-26.

Díaz D., Lloja L., Carbajal V .(2004). Preclinical studies of cucurbita maxima

(pumpkin seeds) a traditional intestinal antiparasitic in rural urban areas. Rev.

Gastroenterol Peru.;24(4):323-327. [Article in Spanish].

Dorovskikh VA,et al. Eksp Klin Farmakol, Jun 1992.

Dreikorn K., Berges R., Pientka L., Jonas U.( 2002). Phytotherapy of benign

prostatic hyperplasia. Current evidence-based evaluation. Urologe

A.;41(5):447-451. [Article in German].

cxxv

DT Nguyen, DH Nguyen, HL Lyun, 2007. Journal of microbiology and

biochemistry 17 (10):1585-1590.

Duan DD, BU,CY.Cheng, J, Wang, YN,shi,GI,2011. Isolation and identification

of acaricidal compounds, journal of economic entomology,104(2),375-378.

El –Sakka M. (2004).Manual of Pharmacognosy. pp. 9-10,35,43.

Eugene Sebastian J, 2013. Journal of applied spectroscopy. Quantitative

estimation of octacosyl salts.

Evans W.C. (2002). Trease & Evans Pharmacognosy. 15th Edition. Saunders

Company. pp.3-4.

Fatope M.O, Al-Burtomani S.K, Ochei J.O, Abdulnor A.O. Phytochemistry

1993.62-1251.

F Dilika, P. D Bremner, J. J .M Meyer, 2000. Fitoterapia vol.71(4):450-452.

Fukushima S, et al. J, cell Biochem suppl, 1997. Cancer prevention by

organosulfur compounds.

Gary A., Imrana S., Wanga S., Mohammada A., Kokb S., Grayc D., Channellc

G.,. Morrisd G., Hardingd S.(2011). Review, The hypoglycaemic effect of

pumpkins as antidiabetic and functional medicines. Food Research International

; 44( 4): 862-867.

G.Amresh, G.D.Reddy,Ch.V.Rao and P.N.Singh, J.Ethanopharmacol, 110 .526

(2007).

Gerald Litwack; 2008 "Human Biochemistry and Disease".

G.Gerner EW, Meyskons FL, 2004 ״Polyamines and cancers״ ;old molecules,

new understanding, Nat- Rev- canor 4(10)-78-792.

Ghorbani A., Naghibi F., Mosaddegh M. (2006). Ethnobotany,

Ethnopharmacology and Drug Discovery. IJPS.; 2(2): 109-118.

Gibbons JA, Alexander M, 1989. Toxical chem. 8(4):283-292.

Gill S., Bali M.( 2012 ). Evaluation of Antioxidant, Antiulcer Activity of 9-

beta-methyl- 19-norlanosta-5-ene Type Glycosides fromCucumis sativus Seed.

Research J. Of Medicinal Plant.; 6( 4 ): 309-317 .

Gold C.(2009). Amazing pumpkin seed. (campbellmgold .com /archive.../

pumpkin seeds.pdf).

cxxvi

G.S. Abermethyl, C, Piantados, J. Med.chem, 1974. 17(2) pp154-157.

Gupta-AK, reviews on inadian medicinal plants, volume 1, Indian council of

medicinal research, new delhi. 2004. P.no:236.

Harada H, et al. Biol pharm Bull, 1997. Selective antitumor activity in vitro

from marine alge from japan coasts.

Harbone.J.B, 1984. Phytochemical methods 2nd ed-42, 196-197.

Health & Energy Food.com, 2015.

Heinrich M., Gibbons S. (2001). Ethnopharmacology in drug discovery: an

analysis of its role and potential contribution. JPP.; 53: 425–432.

Hua C., Rui N., Geoffrey A.(2005). Cordell and Samuel X. Qiu Cucurbitacins

and cucurbitane glycosides: structures and biological activities. Natural Product

Reports; 22 :386-399.

Hussain J., Ur rehman N., Khan A., Hamayun M., Hussain S., Shinwari

Z.(2010).

Proximate and essential nutrients evaluation of selected vegetable species from

kohat region, Pakestan. Pak. J. Bot.; 42(4): 2847-2855.

H.X.Wang, T.B.Ng. Natural productswith hypoglycaemic, hypotensive,

hypocholestrolemic and antiatherosclerotic activities. Life Sci, vol 65, 1999,

pp266302677

Hyun T., Barrett-Connor E., Milne D.(2004). Zinc intakes and plasma

concentrations in men with osteoporosis: the Rancho Bernardo Study. Am. J

.Clin Nutr .;80:715–721.

Iheancho K., Udeuani M., Angelac C.( 2009).Nutritional Compostion of Some

Leafy Vegetable Cosumed in Imo state, Nigeria. J.Appl.Sci.Environ.Mange.;

13(3)35-38.

Itis.gov.2009 ״ Integrated Taxonomic Information System״

http://www.itis.gov.Retrieved 2009-12-06.

J,Agric, food chem., 2002, 50(8),pp2231-2234.antioxidant and cyclooxgenase

activities of fatty acids found in food.

Jain, SK, Rao, RR. Fields and Herbarium methods, 1976.

cxxvii

Jangi S., Ruiz-Larrea M., Nicolau-Galme´s F., Andollo N., Arroyo-Berdugo Y.,

Ortega- Martı´nez I., Dı´az-Pe´rez J., Boyano M.(2008). Terfenadine induced

apoptosis in human melanoma cells is mediated through Ca+2 homeostasis

modulation and tyrosine kinase activity, independently of H1 histamine

receptors. Carcino.; 29 (3 ): 500–509.

Jang SM, Park NY, Lee JB, Ahn H. The comparison of food constituents in

different parts of pumpkin. J Korea Soc food Sci Nutr, 2001;30;1038-1040.

Jayaprakasam B., Seeram P., Nair G.(2003).Anticancer and antiinflammatory

activities of cucurbitacins from Cucurbita andreana. Cancer Lett.;189(1):11-16.

Jennifer Murray, Suite 101. Heath benefits of pumpkin seeds. Retrieved on Oct

2012. http//suite101.com/article/health-benefits-of-pumpkin-a1531140.

J.Ethnopharmacol Jan,90(1):89-9.

J.E Ramiraz, M Vishnupad. Anhydrous foaming compositions of detergents and

high levels of glycerine and emollients such as oils and esters US 5,254,334.

Jerry Hirsh, "Pumpkin pie may be scarce on Thanksgiving Day". LA Times

(2009).

http://www.tennessean.com/article/20091119/BUSINESS01/911190366/Pumpk

in-pie-may-be-scarce-on-Thanksgiving-Day

Jing J., Ji-Yu Z., Na G., Hui S., Lei L., Jun-Chao L., Xue-Lin W., Yang L.,

Ming-Yuan L., Xiu-Ping W., Lu Y.(2010). Farnesol, a Potential Efflux Pump

Inhibitor in Mycobacterium smegmatis. Molecu.; 15: 7750-7762.

Journal of bioactive and compatible compounds Oct, 1990. Vol.5no.4,420-429.

Journal of chemical Ecology, Sep 1979. V 5. pp 721-725.

Journal of lipid research sept, 1967.

Kam P., Cardone D.(2007). Review Article: Propofol infusion syndrome.

Anaesth.; 62: 690–701.

Karou D., Nadembega W., Ouattara L., Ilboudo D., Canini A., Nikiéma J.,

Simpore J., Colizzi V., Traore A.(2007). African Ethnopharmacology and New

Drug Discovery. Medicinal and Aromatic Plant Science and Biotechnology;

1(1).

Kavanagh F, (1972). Analytical microbiology; vol 11, Academic press, New

York, p 11.

cxxviii

kayoed A.A.A, kayoed O.T.( 2011).Some Medical values of Telfairia

occidentalis: AReview. Amirican Journal of Biochemistry and Molecular

Biology;1(1), 30-38.

Kemeny N., Gonen M., Sullivan D., Schwartz L., Benedetti F., Saltz L.,

Stockman J., Fong Y., Jarnagin W., Bertino J., Tong W., Paty P.(2001). Phase I

Study of Hepatic Arterial Infusion of Floxuridine and Dexamethasone With

Systemic Irinotecan for Unresectable Hepatic Metastases From Colorectal

Cancer. J. Clin. Oncol.; 19:2687-2695.

Khare C.(2004). Indian Herbal Remedies: Rational Wester Therapy, Ayurvedic

and Other Traditional Botany.Springer-Verlag Berlin Heidelberg.pp.110.

Kim DH, Park MH, Choi YJ. Chung KW, Jang EJ, et al. 2013 Molecular study

of dietary heptadecane for the anti-inflammatory modulation of NF.KB in the

aged kidney .PLOS one 8(3):e59316.doi:10.1371/journal, pone 0059316.

Kim Y., Kim J., Kim N., Choi C.,Lee H.(2012). Comparison of the chemical

compositions and nutritive values of various pumpkin (Cucurbitaceae) species

and parts, Nutr Res Pract.; 6(1): 21–27.

Komolafe, O.O, Anyabuike, C.P and Obaseki, A.O, (1988). The possible role of

mixed function oxidase in the hepatotoxicity of Azadirachta indica. Fitoterapia,

LIX(2), 109-113.

Lavoie EJ,et al,1981. Mutagenecity of methylated fluorenes.

Levin, Rachel (2008-09-17). "The Power of Pumpkin in All Its Parts". feature

article. The Food Paper.

http://www.gayot.com/lifestyle/health/features/pumpkin-health-benefits.html.

Retrieved 2008-10-14.

Li, S. Tylor, K. B,Kelly, S. J and Jedrzejas, M. J, 2001. J. Biol. Chem. 276,

15125-15130.

Liu P., Luo L., Guo J., Liu H., Wang B., Deng B., Long C.(2010). Farnesol

induces apoptosis and oxidative stress in the fungal pathogen Penicillium

expansum. Mycologia.;102(2): 311–318.

Liu T. , Zhang M. , Zhang H., Sun C. , Yang X., Deng Y., Ji W.(2008).

Combined antitumor activity of cucurbitacin B and docetaxel in laryngeal

cancer. Eur. J. Of Pharmacol.; 587: 78–84.

Loiseau PM, et al. Int J Parasitol. 1988.

cxxix

Marie-Magdeleine C., Hoste H., Mahieu M., Varo H., Archimede H.( 2009). In

vitro effects of Cucurbita moschata seed extracts on Haemonchus contortus. Vet

Parasitol.;161(1-2):99-105.

McGaw, L.J.; Jäger, A.K.; Van Staden, J. (2002). Isolation of antibacterial fatty

acids from Schotia brachypetala. Fitoter., 73, 431-433.

Merck index, 11th:A n enclopedia of chemical, drugs and biological, Merk,

1989, ISBN.

Michael ash, 2004- Hand book of green chemical.

Michael Hogan. 2011. Thanksgiving. Eds. Cutler Cleveland & Peter Saundry.

Encyclopedia of Earth. National Council for Science and the Environment.

Washington DC

Michael, Orsolek D., George L. Greaser, and Jayson K. Harper. "Pumpkin

Production." Agricultural Alternatives (2000). Penn State College of

Agricultural Sciences Feb 2008.

http://web.archive.org/web/20080625165325/http://agalternatives.psu.edu/crops

/pumpkin/pumpkin.pdf.

Miles A.A & Misra S.S 1938. The estimation of bactericidal of blood

.J.HYG.38.732.

Möller J., Volz P., Reimann W., Stoll D.(2001). Opipramol for the treatment of

generalized anxiety disorder: a placebo-controlled trial including an alprazolam-

treated group. J. Clin.Psychopharmacol.; 21(1):59-65.

Molnar J, et al. anticancer, 1993. Divrse biological carcinogenic activity

displayed by benz[c]acridine.

Morsdorf K, et al. Phamacology, 1970.

Mort V, et al. Bioorganic Med Chem Lett, 2006.

Nadendla R.(2005). Principles of Organic Medicinal Chemistry. New Age

International (P) Ltd. New Delhi.

Ning L., Min-Hua Z., Xiu-Mei L., Ding M. (2007). Regioselective synthesis of

3-Ocaproyl- floxuridine catalyzed by Pseudomonas cepacia lipase. J. of Molecu.

Cata. B. Enzymatic; 47:6–12.

N. Habib, H.Daaboul journal of clinical oncology, 2009 ASCO.

cxxx

Nweze et al., 2004; Reuben et al., 2008; Vital et al.,. 2010.

Paola Vicini, Lordana Amoretti, V, 2000. Bioorganic and medicinal chemistry.

P.J.; Mallard, W.G. (eds.) ^ Jump up to: a b c d n-Decanoic acid in Linstrom,

NIST Chemistry WebBook, NIST Standard Reference Database Number 69.

National Institute of Standards and Technology, Gaithersburg MD.

http://webbook.nist.gov.

Phillips KM, Ruggio DM, Ashraf-Khorassani M. Phytosterol composition of

nuts and seeds commonly consumed in the United States. J Agric Food Chem.

2005 Nov 30;53(24):9436-45. 2005. PMID:16302759.

Quanhong L, Caili F, Yukui R, Guanghui H, Tongyi C.(2005). Effects of

protein-bound polysaccharide isolated from pumpkin on insulin in diabetic rats.

Plant Foods Hum Nutr.;60(1):13-16

Rao P, PS, Karmarkan, s.m, 1986. Antibacterial substances from brown alge

29;pp 503-507.

Raver, Anne. "In the Pumpkin Patch, an Orange Thumb." New York Times, 18

Oct. 2007, p. F6.

Raza M. (2006). A role for physicians in ethnopharmacology and drug

discovery. J. Of Ethnopharmacol.; 104: 297–301

Rioux V., Catheline D., Bouriel M., Legrand P.(2005). Dietary myristic acid at

physiologically relevant levels increases the tissue content of C20:5 n-3 and

C20:3 n-6 in the rat. Reprod. Nutr. Dev.; 45: 599–612.

Ritter J., Lewis L., Mant T., Ferro A.(2008). AText book of Clinical

Pharmacology and Ttherapeutics. 5th edition. Hodder Arnold, an imprint of

Hodden Education, part of Hachette Livre UK.pp: 267-268.

Roberts, Tammy. "The Many Uses of Pumpkin." Food & Fitness, 7 Aug. 2006.

10 Feb 2008.

http://web.archive.org/web/20060923090620/http://missourifamilies.org/feature

s/nutritionarticles/nut107.htm>

Rodriguez-Amaya B, Azevedo-Meleiro H (2007). Qualitative and quantitative

differences in carotenoid composition among Cucurbita moschata, Cucurbita

maxima, and Cucurbita pepo.J Agric Food Chem.; 16;55(10):4027-4033.

R.T Lalonde, C.F.Wong and H.G.Howell, J.Org. chem., 36, 3703(1971).

cxxxi

Ruthing DJ, Meckling KA, 1999. Both 3 and 6 fatty acid stimulate wound

healing .Journal of nutrition 129(10):1791-8.

Sanam S., Halder S., Shuma M., Kabir A., Rashid H., Rouf A . (2012). Design

and in vitro evaluation of indapamide sulfated release tablet using methocel

K15 mcr and methocel K100M lvcr. IJPSR.; 3(4): 1011-1017 .

Sarkar S, Buha D.(2008). Effect of ripe fruit pulp extract of Cucurbita pepo

Linn. In aspirin induced gastric and duodenal ulcer in rats. Indian J Exp Biol.

;46(9):639-645.

Sermakkani M et al- Thagopandan.V.2012.Asian journal of pharmaceutical and

clinical researchs,5(2),90,94.

Selective antitumor activity in vitro frome marine alge from japan coasts

Harada H, et al. Biol pharm Bull, 1997

Shalini K., Sharma P., Kumar N.(2010). Imidazole and its biological activities:

A review. Der. Chemica. Sin.; 1 (3): 36-47.

Singh R., Jain D. (2011). Evaluation of Antimicrobial Activity of Alcoholic and

Aqueous Extracts of Five Plants used in Traditional Medicine in North India.

Int.J. PharmTech Res.;3(1):376-380.

Smith TJ, et al.Expert opin investing drugs.2000. Squalene:potential

chemoprotective agent.

Smolinske, Susan C (1992). Handbook of Food, Drug, and Cosmetic

Excipients. CRC Press. pp. 75–76. ISBN 0-8493-3585-X.

Sofowora .A, 1993. Screening plants for bioactive agents in medicinal plants

and traditional medicinal in Africa, second ed ;spectrum books Ltd, pp:134-156.

Stevenson G., Eller J., Wang L., Jane L., Wang T., Inglett E. (2007). Oil and

tocopherol content and composition of pumpkin seed oil in 12 cultivars. J.

Agric. Food Chem.;55(10):4005-4013.

Stuart A.(2003). Pumpkin seeds Cucurbita pepo.

www.herbalsafety.utep.edu/pdf.asp?ID=15)

Sudha, T, L Chidambarampillai,S ,mohan, VR, 2013. GC.MS analysis of

bioactive components- Journal of applied pharmaceutical science, 3(5), 126-

130.

cxxxii

Suphakarn VS, Yarnnon C, Ngunboonsri P. The effect of pumpkin seeds on

oxalcrystalluria and urinary compositions of children in hyperendemic area. Am

J Clin Nutr 1987;45:115–21.

Susan D. Van Arnum (1998). Vitamin A in Kirk-Othmer Encyclopedia of

Chemical Technology.

New York: John Wiley. pp. 99–107doi :10. 1002/ 0471238961

.2209200101181421.a01.

The Columbia Encyclopedia. 2004. Credo Reference. 19 Feb. 2008.

The PDR Guide to Nutritional Supplements"; Physicians Desk Reference; 2001.

The Oxford companion to American food and drink p269. Oxoford university

press, 2007. Retrieved Feb 17-2011.

Torres-Viera C., Thauvin-Eliopoulos C. , Souli M., Degirolami P., Farris M.,

Wennersten C., Sofia R., Eliopoulos G.(2000). Activities of Taurolidine In

Vitro and in Experimental Enterococcal Endocarditis. Antimicrob. Agents

Chemother.;44(6):1720-1724.

Turhan E., Uslu B.(2008). Electroanalytical Determination of Opipramol in

Pharmaceutical Preparations and Biological Fluids. Analyt. Lett.;41: 2013–

2032.

Tyler Herbst, Sharon. The New Food Lover's Companion, 3rd ed. Barron, 2001.

Pumpkin-Seed Oil. 14 Feb. 2008 [2][dead link]

Vallisuta O., Olimat S. ( 2012). Drug Discovery Research in Pharmacognosy. In

Tech . pp. 11-15

Walters D., Muff R., Langsam B., Gruber P., Born W., Fuchs B.(2007).

Taurolidine: a novel anti-neoplastic agent induces apoptosis of osteosarcoma

cell lines. Invest. NewDrug.; 25:305–312.

Wang Y., Lin k., Wang C., Liao X.(2011). Addition of Theophylline or

Increasing the Dose of Inhaled Corticosteroid in Symptomatic Asthma: A Meta-

Analysis of Randomized Controlled Trials. Yonsei. Med. J.; 52(2):268-275.

Wang Z-Y, Gult-B, Wuj-C, 2015; Asian journal of chemistry.

Watson, L., and Dallwitz, M.J. 1992 onwards. The families of flowering plants:

descriptions, illustrations, identification, and information retrieval. Version:

18th May 2012. (http://delta-intkey.com.)

cxxxiii

Wayne J. Brouillette and Garyl. Grunewald, 1983. Synthesis and anticonvulsant

activity of N-substituted lactam ring compounds (2-Piperidinone, N-[4-bromo-

n-butyl] -).

Weiss E, Winey M, 1996. Cell Bio Gene MPS1 132, 111-123. 148 - Eugene

Sebastian J, 2013. Journal of applied spectroscopy. Quantitative estimation of

octacosyl salts.

Wiart C.(2006). Medicinal Plants of Asia and the Pacific. Taylor & Francis

Group, LLC.pp.110.

World Health Organization.( 2002). WHO Traditional Medicine Strategy 2002–

2005.Geneva. (http://whqlibdoc.who.int/hq/2002/who_edm_trm_2002.1.pdf).

Xia C., Li F., Li Z., Zhang C.(2003).Purification and characterization of

Moschatin, a novel type I ribosome-inactivating protein from the mature seeds

of pumpkin (Cucurbita moschata), and preparation of its immunotoxin against

human melanoma cells. Cell Res.;13(5):369-374.

Xia T., Wang Q.( 2006 ). Antihyperglycemic effect of Cucurbita ficifolia fruit

extract in streptozotocin-induced diabetic rats. Fitoterapia.;77(7-8):530-533.

Yarim M., Koksal M. , Durmaz I., Atalay R.(2012). Cancer Cell Cytotoxicities

of 1-(4- Substitutedbenzoyl)-4-(4 chlorobenzhydryl)piperazine Derivatives. Int.

J. Mol. Sci.; 13:8071-8085.

Younis M., Ghirmay S., Al-Shihry S. (2000 ).African Cucurbita pepo L.:

properties of seed and variability in fatty acid composition of seed

oil.Phytochemistry. ;54(1):71-75.

Yue-Hong W., Chun-Xue B., Qun-Ying H.(2003). Anti inflammatory effect of

methoxy phenamine compound in rat model of chronic obstructive pulmonary

diseases. Acta. Pharmacol. Sin.; 24(12): 1324-1327.

Zhang X, Ouyang JZ, Zhang YS, et al. Effect of the extracts of pumpkin seeds

on the urodynamics of rabbits: an experimental study. J Tongji Med Univ

1994;14:2358.

Zhou J., Gupta K., Aggarwal S., Aneja R., Chanda R., Panda D., Joshi

H.(2003). Brominated Derivatives of Noscapine Are Potent Microtubule-

interfering Agents That Perturb Mitosis and Inhibit Cell Proliferation. Mol.

Pharmacol.; 63:799–807.

cxxxiv

Zih-Rou H. , Yin-Ku L. Jia-You F.(2009). Review: Biological and

Pharmacological Activities of Squalene and Related Compounds: Potential Uses

in Cosmetic Dermatology. Molecu.;14:540-554.

Zünkler B., Kü hne S., Rustenbeck I., Ott T.(2000). Mechanism of terfenadine

block of ATP-sensitive K+ channels. B. J.of Pharma.; 130: 1571 – 1574.

المراجع العربية:

.القران الكريم

.1988الطبعة االولى -ىبى الفداء الحافظ بن كثير الدمشقالبداية والنهاية أل

.(5379)-1986 صحيح البخارى لمحمد بن اسماعيل البخارى

1990ه لمحمد ناصر الدين االلبانىصحيح الجامع الصغير وزيادت

1985-نىفتح البارى شرح صحيح البخارىالبن حجر العسقال

1987محمد بن احمد االنصاري القرطبي عبدهللا بيالجامع الحكام القران ال

cxxxv

Appendix