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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
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بسم هللا الرحمن الرحيم
"واذا مرضت فهو يشفين"
صدق هللا العظيم
(80األية -) الشعراء
DEDICATION
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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
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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
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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
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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
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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
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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:
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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
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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
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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
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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
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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.
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Chapter One:
Introduction &Literature Review
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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
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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).
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( 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
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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).
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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).
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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.
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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:
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-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).
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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
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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,
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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
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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
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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
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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
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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).
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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,
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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:
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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
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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).
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Chapter Two:
Materials &Methodology
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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.
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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.
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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.
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-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:
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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
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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:
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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
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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:
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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:
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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.
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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
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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
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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.
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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-
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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.
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Chapter three: Results
3. Results:
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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).
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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).
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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).
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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.
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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
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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
Page 57
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.
Page 58
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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:
Page 59
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
Page 60
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
Page 61
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:
Page 62
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:
Page 63
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:
Page 64
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
Page 65
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
Page 66
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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):
Page 67
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:
Page 68
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:
Page 69
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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:
Page 70
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
Page 71
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:
Page 72
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:
Page 73
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:
Page 74
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:
Page 75
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
Page 76
lxxvi
Figure (3-13): GC.MS Chromatogram of the ethanol extract of Pumpkin
Flowers:
Page 77
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
Page 78
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
Page 79
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
Page 80
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:
Page 81
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
Page 82
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-
Page 83
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
Page 84
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
Page 85
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
Page 86
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
Page 87
lxxxvii
Figure (3-15): GC.MS Chromatogram of the ethanol extract of Pumpkin
Leaves:
Page 88
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.
Page 89
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
Page 90
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)-
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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
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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
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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:
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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
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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
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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
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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
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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]-
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Chapter four:
Discussion
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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:
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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:
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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
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(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
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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
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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).
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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- :
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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:
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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)- :
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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).
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(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:
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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
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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:
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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).
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(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).
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(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.....
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Chapter Five:
CONCLUSION & RECOMMENDATIONS
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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]-).
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- 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.
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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.
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