Chemical Composition and Antioxidant Activity of Hyphaene Thebaica

Chemical Composition and Antioxidant Activity of Hyphaene thebaica

A thesis submitted in fulfilment of the requirements for the degree of M.Sc. in Chemistry

By Makarim Abdelmalik Abdalla (B.Sc Chemistry)

Supervisor Dr. Omer Abdalla Ahmed Hamdi

Sudan April 2018

اآليـــــــــــــــــــــــــــــــــــــــة

ﭧ ﭨ

ﭽ ﭑ ﭒ ﭓ ﭔ ﭕ ﭼ

صدق هللا العظيم سورة الرحمن )68(

Dedication

To the soul of my father, to my mother To my husband and children To my brothers and sister

Acknowledgments

First and foremost, I am offering my thanks, obedience and gratitude to Allah who helped me and gave me the strength to achieve this thesis. I would like to express my deepest gratitude to my supervisor Dr. Omer Abdalla Ahmed Hamdi. The door of his office was always open whenever I ran into a trouble spot or had a question . He steered me in the right direction whenever he thought I needed it. Also gratitude and thanks are extended to all staff and technicians at Department of Chemistry, Alneelain University. Special thanks are due to The National Center for Agricultural Research, Alawia Laboratory at University of Medical Sciences and Technology, Ministry of Minerals Chemical Laboratory and The National Center for Industrial Research and Consultancy. Finally, I must express my very profound gratitude to my husband, my brothers, my sister, my sons and my daughters for continuous encouragement throughout my years of study and through the process of researching and writing this thesis.

iii

Abstract

This study was conducted with the aim to extract and analyze the fruits powder of Hyphaene thebaica, which brought from forest on the north of Atbara River, in the Nile River State, Sudan. Two methods of extractions from the fruits powder of H. thebaica were performed; extraction by maceration using three different solvents including methanol , ethyl acetate and n-hexane, their yields were 21.67%, 0.66% and 0.64%, respectively. These extracts were subjected to phytochemical analysis which showed the presence of saturated and unsaturated sterols, saponins, coumarins, carbohydrates, tannins, flavonoids and alkaloids .Yield of successive extractions were methanol (26.77%) , n-hexane (0.621%) and ethyl acetate (0.388%). GC-MS was used for quality and quantity evaluation of the hexane extract revealed the presence of twenty compounds saturated fatty acids (38.66%) including palmitic acid (31.48%), Hydrofol acid (2.57%) , unsaturated fatty acids (27.22%) including oleic acid (7.74%%) Linoleic acid (2.97%), and Palmitoleic acid (0.91%). The essential oil obtained from n-hexane extract of seeds of H. thebaica revealed a total of twenty five compounds. The major compounds were saturated fatty acids (69.32%) including lauric acid (28.68%), myristic acid (15.53%) and unsaturated fatty acids (30.00%) including oleic acid (24.67%) and linoleic acid (4.75%). Antioxidant activity was evaluated for all extracts. The results showed that the methanol extract (sox) possessed the highest activity with (70.5%) at the concentration of 0.1mg/ml using DPPH assay. The most active extract was subjected to TLC and visualized with vanillin-H2SO4 reagent. Seven compounds were isolated by TLC, analyzed by Infra-red and UV. Visible spectroscopy.

The element composition of H. thebaica fruits showed the presence of K (1.95 %) Cl (1.67%), Si (0.425%), P (0.33%) , Mg (0.27%), Na (0.158%), Ca (0.05%) and Fe (0.009%).

المستخلص

أجريت هذه الدراسة بهدف استخالص و تحليل مسحوق من ثمار الدوم Hyphaene thebaica ، والتي أحضرت من غابات شمال نهر عطبرة، في والية نهر النيل السودان. تم تنفيذ طريقتين لإلستخالص من ثمارالدوم Hyphaene thebaica ؛ شملت اإلستخالص بالنقع بواسطة ثالثة مذيبات مختلفة هي الميثانول، خالت اإليثيل والهكسان، وكانت نسبة اإلستخالص%21.67 ، 0.66% ، 0.64% على التوالي. أخضعت هذه المستخلصات لتحليل كيميائي نباتي أكد وجود ستيرول مشبع ، ستيرول غير مشبع ، صابونين ، كومارين ، كربوهيدرات ، تانين ، فالفونويد وقلويدات.ثم باإلستخالص باستخدام جهاز السوكسليت من مسحوق ثمار الدوم بواسطة الميثانول( 26.77%)، الهكسان)%0.62 ( وخالت اإليثيل ) 0.388%(. تم استخدام جهاز كروماتوغرفيا الغاز الملحق بمطياف الكتلة لتحديد انواع وكميات المركبات الموجودة في مستخلص الهكسان من مسحوق ثمار الدوم التي كشفت عن وجود عشرين مركب تم تحديدها بالكامل والمركبات الرئيسية من االحماض الدهنية المشبعة بنسبة 38.66٪ مثل حمض البالمتيك 31.48٪ ،حمض االستياريك 2.57٪،ومن االحماض الدهنية غير المشبعة بنسبة 27.22 ٪ مثل حمض اللينوليك 14.98٪, حمض االولييك 7.74 ٪،حمض البالميتوليك0.91 ٪. تم الكشف ايضا عن الزيت الذي تم الحصول عليه من مستخلص الهكسان من مسحوق بذور ثمرة الدوم بوجود خمسة وعشرين مركبًا وتم التعرف عليها تما ًما. كانت المركبات الرئيسية األحماض الدهنية المشبعة بنسبة 69.32٪، مثل حمض اللوريك 28.68 ٪ ، وحمض المريستيك 15.53 ٪ واألحماض الدهنية غير المشبعة كانت بنسبة 30.00٪ مثل حمض األوليك 24.67٪، حمض لينوليك ٪4.7. تم تقييم فعالية مضادات األكسدة لجميع المستخلصات. وأظهرت النتائج أن مستخلص الميثانول جهاز السوكسليت يمتلك أعلى فعالية بنسبة 70.5٪ عند تركيز 0.1 ملجرام / مل باستخدام اختبار DPPH. عليه تم اختبار مكونات المستخلص بواسطة كروماتوغرافيا الطبقة الرقيقة باستخدام الكاشف ) فانيلين –حمض كبريت (. تم عزل سبعة مركبات بواسطة كروماتوغرافيا الطبقة الرقيقة التحضيري ، تم تحليلها بواسطة أجهزة التحليل الطيفي باألشعة تحت الحمراء و االشعة فوق البنفسجية. أظهرمسحوق ثمرة الدوم وجود عناصر مثال البوتاسيوم )1.95%(، الكلور )1.67%( ، السيلكون )0.425%( ، الفسفور)0.33%( ، المغنسيوم)0.27%( ، الصوديوم )0.158%( ، الكالسيوم )0.05%( ، الحديد )%0.009( .

List of Abbreviations

Abbreviations Meaning and concept

Mac Maceration

Sox Soxhlet

+++ Strong

++ Medium

+ Weak

_ Negative

IR Infra red

UV Ultra violet

Rf Retardation Factor

Rt Retention time

DPPH 2.2-Di phenyl 1-picryl hydrazyl

GC-MS Gas chromatography-mass spectroscopy

XRF X-Ray Flouresencee

DW Dry weight

vii

Table of contents

Subject Page no.

I اآلية

Dedication II

Acknowledgments III

English Abstract IV

V المستخلص

List of abbreviations VII

Table of contents VIII

List of tables XII

List of figures XIII

List of appendices XIV

Chapter One Introduction and Literature review 1 1 1.1 Introduction 1

1.1.1 Objectives 3 Chapter one 1.1.1.1 General objective 3

1.1.1.2 Specific objectives 3

1.2 Literature review 4 1.2.1 Family of of Arecaceae 4

1.2.2 Taxonomy of H. thebaica 6

1.2.3 Botanical Description of H. thebaica 6

1.2.4 Geographical distribution of H. thebaica 7

1.3 Chemistry of the H. thebaica 8

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1.3.1 Elements composition of fruit H. thebaica 8

1.3.2 Phytochemical Composition of H. thebaica 9

1.3.2.1 Antioxidant 9

1.3.2.2 Flavonoids 11

1.3.2.3 Glycoside 12

1.4 Uses of H. thebaica 14

1.4.1 General uses of H. thebaica 14

1.4.2 Medicinal uses of H. thebaica 14

Chapter two

2 Materials and methods 17

2.1 Materials 17

2.1.1 Sample collection 17

2.1.2 Equipments 17

2.1.3 Chemicals and reagents 18

2.1.3.1 Chemicals 18

2.1.3.2 Reagents 19

2.1.4 Chromatographic materials 20

2.1.4.1 Stationary phases 20

2. 1.4.2 Mobile phases 20

2.1.4.3 Detection 20

2.1.4.4 Visualization 20

2.2 Methods 20

2.2.1 Extraction methods 20

2.2.1.1 Preparation of hexane extract 20

2.2.1.2 Preparation of ethyl acetate extract 21

2.2.1.3 Preparation of methanol extract 21

2.2.1.4 Successive extractions 21

2.2.2 Phytochemical screenings of H. thebaica 22

2.2.2.1 Screening for triterpenes and sterols 22

2.2.2.2 Screening for saponins 23

2.2.2.3 Screening for tannins 23

2.2.2.4 Screening for flavonoids and Leucoanthocyanins 24

2.2.2.5 Screening for Alkaloids 24

2.2.2.6 Screening for coumarins 25

2.2.2.7 Screening for 2-Deoxy sugars 25

2.2.2.8 Screening for carbohydrates 25

2.2.2.9 Screening for reducing compounds 26

2.2.3 Quantitative determination of the chemical constituency 26

2.2.3.1 Alkaloids contents 26

2.2.3.2 Flavonoid contents 26

2.2.4 Chromatogaphic methods 26

2.2.4.1 Examination of extracts using TLC 26

2.2.4.2 Preparative Thin Layer Chromatography 27

2.2.5 Spectroscopic methods 27

2.2.5.1 GC-MS Parameters 27

2.2.5.2 Infra red Spectra 27

2.2.5.3 Ultra-Violet spectra 28

2.2.6 Antioxidant activity of extracts 28

2.2.7 Determination of cationic composition of H.thebaica 28

Chapter three 3 Results and discussion 29

3.1 Extraction and phytochemical screening of H.thebaica 29

3.2 Antioxidant activity of H.thebaica extracts 32

3.3 Minerals content in H. thebaica 34

3.4 GC-MS analysis of hexane extract 36

3.5 Thin layer chromatography of methanol (sox) extract 45 3.5.1 Identification of compounds from fractions 1-7 47

3.6 Conclusions and Recommendations 49

3.6.1 Conclusions 49

3.6.2 Recommendations 50

References 51

List of Tables

No Name of Tables Page

1.1 The classification of H.yphaene 6

3.1 Percentage yields and properties of different solvents 29

3.2 Percentageextract using yields (mac) and method properties of different solvents 29

3.3 Geneextractral using phytochemical (sox) method screenings of H. thebaica fruit 31

3.4 Evaluationextract of Antioxidant activity of H.thebaica fruit 32

3.5 Evaluationextract of antioxidant activity of different 32

3.6 Mineralsconcentrations content of ofH. H. thebica thebaic methanola fruits extract 34

3.7 GC-MS analysis of hexane extract of fruit of H .thebaica 37

3.8 composition of essential oil in seeds of H.thebaica 38

3.9 Compoundscompounds of the methanol (sox) extract 46

3.10 Characteristic Infra –red of the seven isolated compounds 47

3.11 Maximum absorptions of the seven isolated compounds 48

xii

List of Figures

No Subjects Page

1.1 Description of H.thebaica 5

1.2 Geographic Distribution of H.thebaica 7

1.3 Structural formula of vitamin A 10

1.4 Structural formula of vitamin E 10

1.5 Structural formula of vitamin C 10

1.6 Basic Structural formula of flavonoids 12

1.7 Structural formula of glycoside 13

2.1 Hexane extract (Sox) 22

3.4 Chromatogram of antiocxident activity of H.thebica 33

3.5 Gas chromatogram of the hexane extract of H.thebaica 36

3.6 Gas chromatogram of oil of seeds of H.thebaica 36

3.7 Thin layer chromatography of methanol extract 45

(sox)

xiii

List of Appendices

No Name of Appendix

1 IR Spectrum of compound 1

2 UV Spectrum of compound 1

3 IR Spectrum of compound 2

4 UV Spectrum of compound 2

5 IR Spectrum of compound 3

6 UV Spectrum of compound 3

7 IR Spectrum of compound 4

8 UV Spectrum of compound 4

9 IR Spectrum of compound 5

10 UV Spectrum of compound 5

11 IR Spectrum of compound 6

12 UV Spectrum of compound 6

13 IR Spectrum of compound 7

14 UV Spectrum of compound 7

xiv

Chapter One

Introduction and Literature Review

Chapter (1): Introduction and literature review

1. Introduction and Literature Review 1.1 General Introduction Medicinal plants are part and parcel of human society to combat diseases, from the dawn of civilization. Information and benefits of herbal drugs in our ancient literature of Ayurvedic (Traditional Indian Medicine), Siddha, Unani and Chinese medicine. According to the World Health Organization, (2003) about 80% of the population of developing countries being unable to afford pharmaceutical drugs rely on traditional medicines (Stray and Hans 1998). The use of plant extracts and phytochemical, both with known antimicrobial properties can be of great significance in therapeutic treatments. In the last few years, a number of studies have been conducted in different countries to prove such efficiency. Many plants have been used because of their antimicrobial traits, which are, chiefly, due to synthesized during secondary metabolism of the plant (Prust et al., 2008). In modern days, antioxidants and antimicrobial activities of plant extracts formed the basis of many applications in pharmaceuticals, alternative medicines and natural therapy. Recently extracts of plant have provoked interest as sources for their potential uses as alternative medicines for the treatment of many infectious diseases (Acharya et al.,. 2010). H. thebaica is an Africa palm tree, common in Upper Egypt, originally native to the Nile valley, bearing an edible fruit which is glubose- quandrangular, about 6x5cm with a shinny orange-brown to deep chestnut skin (epicarp). The rind (mesocarp) in some palm is unedible but of other it is very palatable, highly aromatic and sweet with ginger bread taste hence the English name. When eaten it serves as verifies and parasite expellant (Burkill, 1997). (Aboshora et al., 2014; Admassu et al., 2013) showed that in sudan H. Thebaica has remained in use to prepare activated carbon from its by-products via chemical activation method .Nutritionally, doum fruit is

Chapter (1): Introduction and literature review an excellent source of carbohydrate and fibre. Additionally, micronutririents such as vitamins (especially B vitamins ) and minerals including K ,Na,,Ca ,Mg and P also help to regulate the biological process in body and impart health benefits. Various studies have revealed the fact that doum ftuit extracts contain high levels of phenols and flavonoids, which possess significant antioxidant and antibacterial activities(Aboshora et al., 2015; Hsu et al., 2016). Roots of H.thebaica were used in treatment of Bilharziasis, while the resin of the tree has demonstrated, diuretic, diaphoretic properties and also recommended for tap worm as well as against animal bites (L.Boulos, 1983). ( Sharaf et al., 1972) reported that the aqueous extract of H.thebaica stimulated the contractions of frog’s heart and rat intestine but inhibited uterine contractions in rats. On the arterial blood pressure, the extract proved to be capable of lowering the blood pressure both in normotensive and hypertensive anaesthetised dogs. Meanwhile, (Hetta and Yassin 2006), reported that constituents of the Hyphaene exhibited a significant decrease in serum total cholesterol and Non- HDL cholesterol in rats, this can reduce the risk of atherosclerosis and, subsequent, cardiovascular diseases. Hypertension is a major health problem throughout the world because of its high prevalence and its association with increased risk of cardiovascular disease. The higher the blood pressure the greater the chance of myocardial infarction, heart failure, and stroke. For individuals aged 40 -70 years, each increment of 20mmHg in systolic blood pressure (SBP) or 10 mmHg in diastolic blood pressure (DBP) doubles the risk of cardiovascular disease (Chobanian et al., 2003). The use of some plants as medicines is due to the presence of flavonoids and saponins (Waterhouse 2003). Hyphaene, which is rich in flavonoids and saponins, in folk medicine is not surprising.

Chapter (1): Introduction and literature review

1.1.1 Objectives 1.1.1.1 General objective The aim of this study was to assess antioxidant activity of n-hexane, ethyl acetate and methanol extracts. 1.1.1.2 Specific objectives The objectives of this work can be summarized as follows  To evaluate antioxidant activity of different extracts from H. thebaica fruits .  To investigate the best solvent that can be give the active antioxidan ingredients.

 To Analyze n-hexane extracts of the fruits and seeds using GC- MS.  To Fractionate active extract with a view to isolate pure compounds by TLC.  To identify the pure fractions by IR and UV spectroscopy.  To evaluate element composition of Hyphaene thebaica fruits by XRF.

Chapter (1): Introduction and literature review

1.2 literature review 1.2.1 Family of Arecaceae The family Arecaceae is a large group comprising approximately 2,500 arboreal species found throughout equatorial, tropical and subtropical areas of the world where they feature as very peculiar element of the landscape. The main geographical areas having played the role of differentiation centres are African equatorial coasts, Brazilian coasts ,Amazonian and the Antiles. It was during the cretaceous period that this group had its largest diffusion and differentiation,leaving behind several fossil remain of trunks and leaves .Habits of palms are quite typical ,in that they are characterized by a tall unbranched stem or rarely, by a dichotomous branching stem of H.thebaica and of the same diameter all along from base to top at apex . It bears a rosette consisting of coriaceous either palmate or pinnate, leaves, up to some metres long.The Arecaceae include plants of enormous economic importance for humans beings. Particularly, a large number of foodstuffs are made from the coconut palm to be found along equatorial sea coasts in the Old World. (Christenhusz and Byng 2016). The sap of Borassus yields a sugar, which on fermentation gives alcoholic drink “Toddy” .Fruits of Phoenix dactylifera. Are very delicious and eaten throughout the Arab world.The nuts of Arecaceae serve as a astringent and used with betel leaves. The milk of cocos nucifera makes a refreshing drink,endosperm is eaten raw and stored when dry. Medicinal Tender leaves of Calamus travancoricus are given in biliousness, worms and dyspepsia. Fibres Mesocarps of drupes are used for stuffing pillows and sofa sets are used for making mats, baskets and other furniture. The leaves are used in the manufacture of hand fans, umbrellas, baskets and mats. The wax is used in

Chapter (1): Introduction and literature review making gramophone records, candles and models. Coconut oil is obtained from cocos nucifera and is used as hair oil, in soap industry and also for cooking .

Figure (1.1): Description of H.thebaica

Chapter (1): Introduction and literature review

1.2.2Taxonomy of H. thebaica Table (1.1): The classification of Hyphaene palm (Factsheet 2014)

Kingdom Plantae– Plants

SuK Kingdom Tracheobionta – Vascular plants

Superdivision Spermatophyta–seedplant

Division Magnoliophyta – flowering plants

Class Liliopsida- Monocotyledons

Subclass Arecidae

Order Arecales

Family Areaceae – Palm family

Genus Hyphaene

Species Hyphaene thebaica

1.2.3 Botanical Description of H. thebaica H. thebaica is a deciduous palm 10-17 m high, with a girth of 90 cm. Trunk is Y-shape and the tree is easily recognizable by the dichotomy of its stem forming up to 16 crowns. Bole fairly smooth but clearly showing the scars of the fallen leaves. Bark dark grey. Leaves 120 180 cm fan shaped, in tufts at the end of branches with the blade divided into segments about 60 cm long, margins entire, leaf stalk about 60cm long armed with curved thorns petiole more than 1m long sheathing at the base with numerous upwardly curving hooks.(Baker W.J et al., 2008) Fig.1.1. Male and female flowers on separate trees. The inflorescence is similar in both sexes up to 1.2 m long with short branches at irregular intervals and 2-3 spikes arising from each branch. Male flowers shortly stalked in pits of the spadix, spathe-bracts encircling the

Chapter (1): Introduction and literature review spadix pointed .Branches of female spadices stouter, in the fruiting stage marked by densely tomentose cushion after the fall of the fruit. Hyphaene is derived from the Greek word hyphaino (web), referring to the fibres from the leaves which are used for weaving (Orwa et al,.2009). 1.2.4 Geographic Distribution of H. thebaica H. thebaica is one of the 11 species of the genus found in Africa. Widespread in the Sahel, it grows from Mauritania to Egypt from Senegal to central Africa and east to Tazania. The tree tends to grow close to ground water but can also grow further away. It is also found in oases and is widely distributed near rivers and stream, sometimes on rocky slopes. It does not do well in water logged areas; it is very resistant to bush fires. Trees occur on silty soils on river and stream banks and on rocky hilly slopes throughout the Sudan (Kiran and Das 2010).

Figure (1.2): Geographic Distribution of H.thebaica .

Chapter (1): Introduction and literature review

1.3 Chemistry of the H. thebaica The essential oil isolated from doum fruits H. thebaica revealed the presence of fifty-seven compounds. Fifty of them could be identified with monoterpenes represent 15.97 % including compounds such as sabinene (0.82 %), β-pinene (1.98 %), limonene (2.42 %), terpinen 4-ol (1.77 %) %), α-terpineol (0.95 %). While diterpenes represent 40.49 %, of which incensole (17.52 %) and incensole acetate (19.81 %) were found to be the main components. Oxygenated compounds constituted 66.78 % of the total compounds identified which indicated the economical value of this oil. The scent of doum fruits oil may be attributed to the presence of volatile diterpenes as cembrene A, cembrene C, incensole and incensole acetate; these compounds are reported here for the first time in family Palmae. (Nwosu. et al.,2008). The fruit of H.thebaica was found to contain 12.65%ash, 89.25% carbohydrate,0.95%oil .316mg/g glucose, very low protein content of 0.01%. It also contains Ca 245.1mg/100g, Mg 236.45mg/100g, Fe 47.9mg/100g, Cu 0.38mg/100g and 0.62. (Nwosu. et al.,2008) (Hinar 2015) reported that the chemical composition of doum fruit powder was protein 7.05 %, total fat 2.57 %,crude fibre 20.88 % , ash 6.60 % ,total carbohydrate 62.72 % . (Salih and Yahia 2015) showed that the chemicals composition fruit powder was moisture 7.50 % , fat 0.09 % ,protein 2.62 % , ash 7.04 % , carbohydrates 64.46 % , fibre 17.48 % . 1.3.1 Elements composition of the fruit H.thebaica (Salih and Yahia 2015) reported that the elements analysis indicated that the fruit contained high levels of Ca (0.48 mg /kg DW), K (8.02 mg /kg DW), Mg (0.54 mg / kg DW), Na (133.58 mg / kg DW), Fe (13.4 mg / kg DW)

Chapter (1): Introduction and literature review

,Cu( 1.5 mg / kg DW), Zn (2.1 mg /kg DW),Mn ( 1.5 mg /kg DW), Al ( 20.8 mg /kg DW). 1.3.2Phytochemical Composition of H. thebaica 1.3.2.1Antioxidant An antioxidant is a molecule that inhibits oxidation of other molecules. Oxidation is a chemical reaction that can produce free radicals, leading to chain reactions that may damage cells. Antioxidants such as thiols or ascorbic acid (vitamin C) terminate these chain reactions. The term "antioxidant" is mainly used for two different groups of substances: industrial chemicals which are added to products to prevent oxidation, and natural chemicals found in foods and body tissue which are said to have beneficial health effects. To balance the oxidative state, plants and animals maintain complex systems of overlapping antioxidants, such as glutathione and enzymes (e.g., catalase and superoxide dismutase) produced internally or the dietary antioxidants: vitamin A figure1,3, vitamin C figure1,4, and vitamin E figure1,5. Antioxidant dietary supplements do not improve health nor are they effective in preventing diseases as shown by randomized clinical trials including supplements of beta- carotene, vitamin A, and vitamin E single or in different combinations having no effect on mortality rate or cancer risk. Supplementation with selenium or vitamin E does not reduce the risk of cardiovascular disease. Oxidative stress can be considered as either a cause or consequence of some diseases, an area of research stimulating drug development for antioxidant compounds for use as potential therapies( Eunok C.and David B.,2009).

Chapter (1): Introduction and literature review

Figure (1.3): Structural formula of vitamin A

Figure (1.4): Structural formula of vitamin E

Figure (1.5): Structural formula of vitamin C

Chapter (1): Introduction and literature review

1.3.2.2 Flavonoids Flavonoids in broad sense of term are virtually, plant pigments. Almost always water-soluble. They are responsible for color of flower, fruit, and leave When they are not, directly, visible they contribute to the color by acting as copigments. Flavonoids are also universally present in the leaf cuticle and epidermal where they ensure tissue protection against the damaging effect of UV radation. All flavonoids have a common biosynthetic origin and therefore possess the same basic structural element. They are phenolic and hence change in colour when treated with base or with amonia, thus they are easily detected on chromatograms or in solution. Flavonoids contain conjugated aromatic system and thus show intense absorption band in the UV and visible regions of the spectrum. Flavonoids are present in all vascular plants but some classes are more widely distributed than others; while flavones and flavonols are universal, Isoflavones and bioflavonyls are found in only a few plant families. Flavoniod are present in plant as mixture and it is very rare to find only single flavoniode component in plant tissue there are often mixture of different flavonoids classes. Falvonoids can be separated by chromatographic procedure and individual components identitied by chromatographic and spectral comparison with known markers ( McNaught; et.al 1997). .

Chapter (1): Introduction and literature review

Figure (1.6): Basic Structural formula of flavonoids

1.3.2.3 Glycoside In chemistry, a glycoside is a molecule in which a sugar is glycosidicully lenked to another functional group. Glycosides play numerous important roles in living organisms. Many plants store chemicals in the form of inactive glycosides. These can be activated by enzyme hydrolysis, which causes the sugar part to be broken off, making the chemical available for use. Many such plant glycosides are used as medications. In animals and humans, poisons are often bound to sugar molecules as part of their elimination from the body. In formal terms, a glycoside is any molecule in which a sugar group is bonded through its anomeric carbon to another group via a glycosidic bond. Glycosides can be linked by an O- (an O-glycoside), N- (a

Chapter (1): Introduction and literature review glycosylamine), S-(a thioglycoside), or C- (a C-glycoside) glycosidic bond. According to the IUPAC, the name " C -glycoside" is a misnomer; the preferred term is “C -glycosyl compound. The given definition is the one used by IUPAC, which recommends the Haworth projection to correctly assign stereochemical configurations. Many authors require in addition that the sugar be bonded to a non- sugar for the molecule to qualify as a glycoside figure1,7, thus excluding polysaccharides. The sugar group is then known as the glycone and the non-sugar group as the aglycone or genin part of the glycoside. The glycone can consist of a single sugar group (monosaccharide) or several sugar groups (oligosaccharide). The first glycoside ever identified was amygdalin, by the French chemists Pierre Robiquet and Antoine Boutron-Charlard, in 1830.

Figure (1.7): Structural formula of glycoside

Chapter (1): Introduction and literature review

1.4 Uses 1.4.1 General uses Fuel palms are occasionally used for fire wood and charcoal. Leaves may also be used as fuel. Fiber leaves are providing sketching the raw material used in basket making brooms course textiles ropes thatching and string fruit fibers obtained after 2-3 days of soaking and beating of the roots are used for making fishing nets. Timber: Wood can be cut using an axe but is difficult to saw due to the many fibers that constitute the wood. Timber from the male palm is said to be better than that from the female, as it is borer and termite proof decorative and durable. It is often used for construction, providing supports and rafters for house sweater ducts and raft construction, Tannin or dye stuff. Dried bark is used to produce a black dye for leather wear.; . The hard seed inside the fruit known as vegetable ivory is used to make buttons and small carvings and as artificial pearls, Ashes from the stipes of trees can b e used as a substitute for salt. The trunk is used for house construction, fences, railway sleepers and canoes. Cut into planks it is made into canoes and water wheels. Hollowed trunks are used as water troughs and irrigation pipes. In Mali the wood is used for poles, shafts and harpoons. The wood is also used as fuel and for making charcoal. The leaves, rachis and fruits are used as fuel as well. Ash from the stem is used as a vegetable salt. A member of the family Arecaceae (palmae), it is native to Nile region and was considered a sacred tree in ancient Egypt. It s seeds are often eaten as vegetables(Elgendy 2008). 1.4.2 Medicinal uses Roots are used in the treatment of bilharzias .The fruit of the H. thebaica palm has been used in folk medicine to treat hypertension .in a trial a group

Chapter (1): Introduction and literature review of patients with raised blood pressure was all given an antihypertensive drug but in half the individual this was supplemental with fruit H. thebaica extract. It was found that those receiving the supplement had lower systolic and diastolic pressures and lower total cholesterol, and the blood lipids and lipoproteins were changed in such a way as to decrease the risk of cardiovascular diseases (Elgendy 2008). In vitro antimicrobial properties of solvents extracts of H.thebaica (L.) Mart fruits showed that ethyl acetate extract was active against all five pathogenic bacteria, Staphylococcus aureus, Eschericia coli, Bacillus subtilis, Pseudomonas aeruginosa and Klebsiella pneumoniae , while methanol extract was active against Pseudomonas aeruginosa and Klebsiella Pneumoniae. Hexane extract showed little inhibition on the growth of Bacillus subtilis and out of the three fungi, Aspegillus niger, Candida albicans and Penicillium sp . used in this study, only Penicillium sp. Growth was slightly affected by high concentration of methanol extract. The phytochemical screenings showed that tannins and flavonoids are present in the ethyl acetate fraction while saponin was found in methanol fraction (Dosumu, et al,. 2006). The antioxidant capacity and the total phenol content were analyzed in the fruit extract of H. thebaica L. The antioxidant capacity was estimated by DPPH and iron chelating assays. Quercetin, ascorbic, BHT and tannic were used as positive controls. Phenolic compounds of doum fruit extract were identified by GC-Mass. Also the effect of doum extract on the acute myeloid leukemia cells (AML) was studied. Nine major phenolic compounds were identified by GC-Mass as, Gallic acid, P- coumaric, Catechol, Apiginin, ferulic acid, carvacrol, resorcinol, pyrogallol and cinnamic acid. In iron chelating assay the results showed that 8.0 µg/ml doum extract gave the best antioxidant activity (21% inhibition).

Chapter (1): Introduction and literature review

In DPPH assay 10. µg/ml extract exhibited 50% antioxidant activity (IC50) but 15. µg/ml extract exhibited 80 % antioxidant activity. In the viability test of AML cells, the results showed that the half maximal inhibitory concentration (IC50) of doum extract was 3. µg/ml. The results indicated that the water doum extract could be an important dietary source of phenolic compounds with high antioxidant and anticancer activities (Dosumu, et al,. 2006).

Chapter Two

Materials and Methods

Chapter (2): Materials and Methods

2. Materials and methods 2.1 Materials 2.1.1 Sample collection Ripe H. thebaica fruits were collected from forest in the north of Atbara river on the River Nile State in Sudan. The mesocarp was removed with knife and air dried at room temperature for two weeks. 2.1.2 Equipments - Weighing balance. - Water bath. - Hot air oven - UV lamp model 34015L (U.S.A.). - UV spectrometer . UV-240 (PC)S,Double beam recording spectrophotometer Model :/UV/1800 Shimadzu ,Japan. - FT- IR . Fourier transform Infra–red spectrometer, Model( FT-IR 8400 s- Shimadzu Japan) was used. - GC-MS-QP2010 Ultra-Shimadzu-Japan. Detector: Mass Spectrometer Model Number: Shimadzu.GC-MS-QP2010 Ultra. Company:Shimadu. Country:Japan. Capillary column:RTX- 5MS..Length(30m).Diameter(0.25mm).Thickness(0.25) Column oven temperature :60 C . Injection temperature: 300C . Injection Mode :Split.

Chapter (2): Materials and Methods

Total Flow: 50ml/ min. Column Flow :1.61ml/min. Purge Flow :3.0ml/min. - XRF (X-ray fluorescence) Model of machine:axios max 4.0 kw.Country manufacturers: Netherlands. 2.1.3 Chemicals and reagents 2.1.3.1 Chemicals - Ethanol (Analytical grade) - Hexane - Methanol - Chloroform - Toluene - Petroleum ether - Ethyl acetate - Acetic acid (glacial) - Acetic anhydride - Ferric chloride - Gelatin powder - Magnesium turnings - Sodium chloride - Potassium iodide - Iodine -Picric acid - Aluminium trichloride - Hydrogen peroxide - Sodium hydroxide - Ammonium hydroxide

Chapter (2): Materials and Methods

- Hydrochloric acid - Distilled water 2.1.3.2 Reagents Baljects reagent: Solution A; 1 g picric acid was dissolved in 100 ml 95% alcohol. Solution B: 10 g of NaOH in ml water .Solution A and B were mixed before use. Mayer‘s reagent: Solution A: 1.36 g of mercuric chloride was dissolved in distilled water

Solution B: 5 g of potassium iodide were dissolved in 10 ml water. Solution A and B were mixed and diluted to 100 ml with water. Wagner‘reagent: 1.27 gm of iodine and 2 g of KI were dissolved in 5 ml of water and the solution was diluted to 100 ml water.

Molish‘reagent: To 10 g of 2- naphthol, 50 ml of ethanol were added and the volume was Completed to 100 ml with water.

AlCl3 solution: 1% AlCl3w/v in water.

FeCl3 solution: 5% w/v of anhydrous ferric chloride in water. Gelatin solution: 50 ml of water were added to 1g of gelatin and allowed to stand for one hour, shaking frequently then the water were decanted and a fresh portion of 60 ml of water were added to the gelatin, and the later were dissolved with shaking and warming to 60oC. 10 g of NaCl were added to solution, mixed, cooled, filtered and completed to 100ml with water.

Chapter (2): Materials and Methods

2.1.4 Chromatographic materials 2.1.4.1 Stationary Phases Silica gel for thin layer chromatography type. 60 GF254 with fluorescent indicator (BDH); U.K. 2. 1.4.2 Mobile phases -Toluene: ethyl Acetate: formic acid (5:4:1). -Toluene: chloroform: formic acid (5:9:1). -Toluene: ethyl acetate: (93:7). -Petroleum ether: Acetone: chloroform (3:1:1). -Chloroform: methanol (8:2). -Chloroform: Ethyl acetate (4:1), chloroform: Ethyl acetate (6:4). 2.1.4.3 Detection Detection of compounds was achieved using short (254nm) and long (365nm) UV radiation, using Handy UV lamp. Model R /34015L chromato- VUE cabinet in U.S.A. then spraying with vanillin -sulphuric acid. 2.1.4.4 Visualization Vanillin-sulphuric acid solution was prepared by dissolving 0.1gm of vanillin crystals in 1.5ml conc. H2SO4 and 8.5 ml of absolute methanol followed by heating in an oven at 110oC for 10 minutes. 2.2 Methods 2.2.1 Extraction methods 2.2.1.1 Preparation of hexane extract 50 g of dried powder of sample were transferred into a beaker and 300 ml of hexane were added. The contents of the beaker were left at room temperature for three days with frequent shaking. The extract was filtered using a funnel. The clear yellow solution was evaporated, and the residual extract was dried

Chapter (2): Materials and Methods and weighed. Percentage yield calculated and subjected to preliminary phytochemical analysis. 2.2.1.2 Preparation of ethyl acetate extract 50 g of dried powder of a sample were transferred into a beaker and 300 ml of ethyl acetate was added. The contents of the beaker were left at room temperature for three days with frequent shaking. The extract was filtered using a funnel. The clear solution was evaporated, and the residual extract was dried and weighed. Percentage yield calculated and subjected to preliminary phytochemical analysis. 2.2.1.3 Preparation of methanol extract 50 g of dried powder of a sample were transferred into a beaker and 300 ml of {80%} methanol was added. The contents of the beaker were left at room temperature for three days with frequent shaking. The extract was filtered using a funnel. The clear brown solution was evaporated, and the residual extract was dried and weighed. Percentage yield calculated and subjected to preliminary phytochemical analysis. 2.2.1.4 Successive extraction A dried fruits powder (40g) was successively extracted using a Soxhlet extractor with hexane (250ml), ethyl acetate (250ml) methanol (250 ml). The solvent was carefully evaporated from each extract and the extractability of each solvent was determined.

Chapter (2): Materials and Methods

Figure (2.1) Hexane extract (Sox)

2.2.2 Phytochemical screening of Hyphaene thebaica The hexane, ethyl acetate and methanol extracts of H.thebaica were used for the following tests according to method of (Harbone 1984) 2.2.2.1 Screening for triterpenes and sterols The prepared extract the equivalent of 10gm of plant material were evaporated to dryness on a water bath, 10ml of pet-ether were added and stirred for a few minutes and allowed to settle. The supernatant liquid was decanted and discarded. The above treatment was repeated several times to remove most of the pigments. 10ml of hexane were added to the residue and stirred thoroughly for 5minutes, and were then decanted into a clean dry test tube. 100gm of (anhydrous Na2SO4) were added to the extract, shaken gently, filtered and divided equally into 2 clean dry test tubes. (a)The Liebermann Barchard test for saturated sterols To the first portion of the extract, 3drops of acetic anhydride were added and mixed gently by swirling the tube. One drop of conc. H2SO4 was added and mixed gently. The gradual appearance of a green to blue colour was taken as an indication for possible presence of sterols, while a pink to purple colour indicates the possible presence of triterpenes.

Chapter (2): Materials and Methods

(b) The Salkouaski test for unsaturated sterols To the second portion of the extract 1-2 ml of concentrated sulphuric acid were added by allowing it to run gently down the side of the test tube. Any immediate colour change at junction of the extract and the sulphuric acid is an indication of possible presence of unsaturated sterols. The sulphuric acid and the extract were mixed and observed for an immediate and a gradual colour change over a period of one hour. A cherry colour is taken also as a presumptive evidence for the presence of unsaturated sterols. 2.2.2.2 Screening for saponins 100mg of the powdered sample were placed into a clean dry test tube. 10ml of distilled water were added, stoppered and vigorously shaken for about 30 seconds. The tube was allowed to stand in a vertical position and observed for 3 minutes. If honey comb froth persisted after 30 minutes, the sample is presumed to contain saponins. 2.2.2.3 Screening for tannins A volume equivalent to 10mg plant material was evaporated to dryness on a water-bath 25ml of hot distilled water were added to the residue and stirred well and allowed to cool. 3-4 drops of 10% NaCl solution were added to salt out any non-tannin compounds. The solution was filtered and to 3ml of filtrate, 4-5drops of 1% gelatin solution were added. Formation of an immediate precipitate was taken as presumptive evidence for the presence of tanning in the plant sample. To another portion of the above prepared solution, (3drops) of FeCL3 test solution were added. The formation of blue- black or green colour was taken as presumptive evidence for the presence of tannins.

Chapter (2): Materials and Methods

2.2.2.4 Screening for flavonoids and leucoanthocyanins An equivalent of 3% of extract was evaporated to dryness on a steam bath. The residue was cooled and triturated with 15ml of pet-ether and filtered. Trituration of the residue was prepared with additional volumes of pet-ether until the last volume of pet-ether was essentially colourless. The defatted residue was dissolved in 30ml of 80% ethanol and filtered: a- To 3ml of the filtrate in a test tube 1 ml of 1% ALCl3 solution in methanol was added. Formation of a yellow colour indicates the possible presence of flavonols, flavones or/and chalcones. b- To 3ml of the filtrate in a test tube, 1 ml of KOH solution was added, a dark yellow colour indicates the possible presence of flavonoid compounds (flavones, flavonones, chalcones and/or flavonols). c- To 2ml of the filtrate 0.5ml of concentrated HCl and few magnesium turnings were added. Production of a definite colour change to pink or red was taken as presumptive evidence that flavonols or were present in the plant sample. (d) To 5ml of the filtrate in a test tube 0.5ml of concentrated HCL was added and warmed on a water-bath for 5 minutes. Red violet colour development is an indication of the possible presence of leucoanthocyanins. 2.2.2.5. Screening for alkaloids A volume of the prepared extract equivalent to 50 gm of plant material was taken and evaporated to a syrup on a water-bath. 10ml of 2N HCl were added to the extract in an evaporating dish and stirred while heating on a water-bath for 5 minutes. The dish was removed from the water-bath and cooled to room temperature.0.5gm of NaCl were added, stirred and then filtered. The residue was washed with sufficient volume of HCl to bring to a final volume of 10ml and was divided into two equal portions in clean dry test tubes:

Chapter (2): Materials and Methods

1-To one of the test tubes a few drops of Mayer s reagent were added. 2-To other test tube afew drop of Wagner s reagent were added. 2.2.2.6 Screening for coumarins The extract (3ml) was evaporated to dryness. The residue was dissolved in hot water. After cooling the solution was divided into two tubes: one tube contained the reference, and the aqueous solution of the second tube was alkaline with o.5ml of (10%) ammonia solution .The occurrence of an intense fluorescence under UV light indicates the presence of coumarins and derivatives. 2.2.2.7 Screening for 2-Deoxy sugars 10ml of alcoholic (80%) extract were placed in an evaporating dish and evaporated to dryness on a water-bath. The dried extract was defatted triturated with pet-ether to remove as much pigments as possible. The pet- ether was decanted and the deffatting process was repeated twice and the residue was dried by evaporating the residual pet- ether 3ml of FeCL3reagent were added, the mixture was well stirred and then transferred to small test tube, and 2ml of concentrated H2SO4were added by allowing then to run down side wall of the test tube.Apurple ring at the interference indicates the possible presence of 2-deoxy sugar. 2.2.2.8 Screening for carbohydrates To 1ml of Molish reagent, 2.5 ml crude extract and 3ml concentrated

o H2SO4were added and mixture was heated at 45 C, to formation of a violet ring between two solution surfaces present which spread by shaking indicates the presence of carbohydrate.

Chapter (2): Materials and Methods

2.2.2.9 Screening for reducing compoundsThe methanol extract 1ml was diluted with distilled water 2ml and Fehling s solution 1ml was added and heated. A brick-red precipitate denotes the presence of reducing sugar. 2.2.3 Quantitative determination of the chemical constituents 2.2.3.1Alkaloids content 5g of plant sample were weighed into 250ml beaker and 100ml of 10% acetic acid in ethanol were added and the beaker was covered and allowed to stand for 4hours. This was filtered and the exract was concentrated on a water bath to one quarter of the original volume, concentrated ammonium hydroxide was added dropwise to the extract until the precipitation was complete. The whole solution was allowed to settle and the precipitate was collected and washed with dilute ammonium hydroxide and then filtered. The black oily residue was dried and weighed. 2.2.3.2 Flavonoid content 10 g of the plant sample was extracted, repeatedly, with 100 cm3 of 80% aqueous methanol at room temperature. The whole solution was filtered through whatman filter paper No 42 (125mm).The filtrate was later transferred into a crucible and evaporated to dryness over water –bath and weighed to a constant weight. Flavonoids content was calculated as percentage yield. 2.2.4 Chromatographic Methods 2.2.4.1 Examination of extracts using TLC About 2g of methanol (sox) extract were dissolved in ml of methanol and spotted onto the preparative TLC plate by means of a capillary tube. Sample was spotted at about 2cm from the bottom of the plate. After air evaporation of the plate, the plate was vertically placed on a glass tank which contained a suitable solvent to a depth of 1.5 cm and the chromatogram developed. The

Chapter (2): Materials and Methods plate was removed, allowed to evaporate, the plate was inspected in day light, then examined under UV-lamp, and finally sprayed by vanillin reagent. Retardation Factor (Rf) values of separated compounds which appeared in day light or under UV0lamp, or after sprayed and heated were calculated as follows. Distance traveled by spot/ distance traveled by solvent. 2.2.4.2 Preparative Thin Layer Chromatography Followed the TLC method in (2.2.4.4), and using Tollouene: Ethylacetate: formic acid.(5:4:1) as mobile phase (Stalh,E. 1969). 2.2.5 Spectroscopic methods 2.2.5.1 Preparation of samples for GC-MS 2ml from the sample were taken in to the test tube, 7ml from alcoholic NaOH prepared by dissolving 2g NaOH in 100ml methano were added.

Then 7ml of 1% alcoholic H2SO4 were added to the sample. The mixture was shaken by vortex for 3 minutrs, left overnight. After that 2ml from supersaturated NaCl were added , then 2ml normal hexane were added and shaken for 3minutes, the hexane layer was collected. 5µl from hexane was diluted with 5ml diethyl ether ,then 1g from Na2SO4 was added as drying agent, filtered through syring filter 0.45µm .1µl of the filtrate was injected in the GC-MS. 2.2.5.2 Infra red Spectra Few mg of the compounds were dissolved in chloroform, placed in the machine, and the spectrums were recorded. The finished spectrum consist of chart ( showing down-ward )peaks corresponding to transmitance plotted against wave length. Fourier transform Infra–red spectrometer, Model( FT- IR 8400 s- Shimadzu Japan) was used. 2.2.5.3 Ultra-Violet Spectra

Chapter (2): Materials and Methods

UV spectrometer was used for scanning of compounds. The compounds were dissolved in methanol and made up to 10 ml .Apportion of this was transfered to silica cell of 1cm thickness. Matched cell containing pure methanol was also prepared and each cellwas placed in appropriate placed in the spectrophotometer arranged that two equal beam of UV or visible light are passed,one through the solution of the sample and one thro ugh the pure solvent . 2.2.6 Antioxidant activity of extracts The method is based on the reduction of alcoholic DPPH solution in the presence of a hydrogen-donating antioxidant due to the formation of the non- radical 2,2- diphenyl 1-picrylhydrazine (DPPH) (SOHN, 2003) .Briefly, three solutions were prepared the control (1ml DPPH+0.9 ml Tris-Cl+0.1ml MeOH), the sample( 1ml DPPH+ 0.9mlTris-Cl+0.1ml sample in MeOH 1mg/1ml). The mixture was incubated in the dark for 30 min at room temperature. The absorbance was read using Spectrophotometer, at517nm wave length. Percent antioxidant activity was estimated using the following formula. Absorbance control- Absorbance sample / Absorbance control X 100(Lie.et .al., 2005) . 2.2.7 Determination of cationic composition of H.thebaica 12g were weighed (3:1) 9g from the sample and 3g of wax. The two weights were added to mix . The sample was added in aluminum cub, then was pressed in pressing machine . Then the sample was placed in the instrument.

Chapter Three

Results and Discussion

Chapter (3): Results and Discussion

3. Results and Discussion 3.1 Results of extraction and Phytochemical Screening of H. thebaica Table (3.1) and (3.2) show percent yields and colors of the extracts using maceration and soxhlet methods respectively. Table (3.1): Percentage yields and colors of three solvent extracts using maceration method

No. Extracts Weight (g) Yield % Colors

1 Hexane 0.7 0.64 Yellow

2 Ethyl acetate 0.132 0.66 Yellow

3 Methanol 2.305 21.67 Deep brown

Table (3.2): Percent yields and colours of three solvent extracts using soxhlet method

No. Extracts Weight (g) %Yield Colours

1 Hexane 0.248 0.621 Yellow

2 Ethyl acetate 0.25 0.388 Yellow

3 Methanol 10.7085 26.77 Brown

Extraction of fruit using maceration showed that the methanol extract was high yield (21.67%) followed by ethyl acetate extract (0.66%) and finally hexane extract (0.64%) these yields are presented in Table (3.1). In case of successive extractions, the yields obtained from n-hexane, ethyl acetate and methanol were 0.62175%, 0.388% and 26.77%, respectively. These yields are presented in Table (3.2). Phytochemical screening of H.thebaica fruit extracts (hexane, ethyl acetate and methanol were carefully carried out using methods described in

Chapter (3): Results and Discussion

(Harbone 1984). The results presented in Table (3.3) summarize the classes of natural compounds present. The results of phytochemical tests showed that the extract has few alkaloids but it is very rich in reducing sugar showing , high concentration of glycoside in the methanol extract and medium concentration in the ethyl acetate extract. The methanol extract has high concentration of saponin while high fiavonoid concentration was recorded in the ethyl acetate and methanol extracts. The use of some plants as medicinal plant for traditional treatment of diseases is due to the presence of flavonoids and saponin ( Zwady.,1992), hence the use of H. thebaica which is very rich in saponin and flavonoids in folk medicine is not surprising . Medium concentration of tannin is found in ethyl acetate extract, it is not out of place then to tag the activity of this fraction to tannin. The presence of tannin in some medicinal plants have been found to responsible for the antiviral and antibacterial activities exhibited by the plant, hence the local use of H. thebaica for the treatment of virus and bacteria induced diseases is supported (Elegami., 2002).

Chapter (3): Results and Discussion

Table (3.3): General phytochemical screening of (H. thebaica fruit)

Class of compounds Hexane Ethylacetate Methanol

Sterols and Triterpenes a-Un saturated sterols +++ +++ +++ b-Saturated sterols ++ +++ ++ Saponins +++ - +++ Deoxy sugars - +++ +++ Cardenolides (Balajact) ++ - +++

a- ALCl3 - +++ +++ Flavonoids b- KOH - ++ ++ c- HCl + Mg - +++ +++ d- HCl - ++ ++

For a- Mayer’s reagent - - + alkaloids b-Wagner’s reagent - - +

Coumarins ++ + +++

Tannins + ++ +++ Glycoside - - ++ Carbohydrate +++ ++ + Reducing compounds - + ++ ReducingKey: (+++) strongcompound (++) =Medium (+) =Weak (-) = Negative Quantitative determination of the alkaloids and flavonoids constituent Weight (g) %

Alkaloids 0.5863 11.72

Flavonoids 4.284 42.84

Chapter (3): Results and Discussion

3.2 Antioxidant Activity of extracts The extracts of H. thebaica fruits were tested for antioxidant activity and the results were shown in Table (3.4) and (3.5) and figure (3.4). Table (3.4) Evaluation of antioxidant activity of H. thebaica fruit extract

Sample Activity %S ± /D IC50S±D Methanol (sox) 70.5 ± 0.007 0.029 ± 0.08

Ethyl acetate(mac) 57.67 ± 0.001 0.053 ± 0.01

ــ Hexane (sox) 1.3 ± 0.038

Ethylacetate (sox) 29.49 ± 0.024 _

Table (3.5) Evaluation of antioxidant activity of different concentrations of H. thebica methanol extract

Conc. mg/ml Activity%

0.1 70.5

0.05 68.24

0.025 41.94

0.0125 34.67

Chapter (3): Results and Discussion

Antioxidant activity of H.thebaica 80 70 60 50 40 30 antioxidant activity of 20 H.thebaica 10 0

Figure (3.4): Chromatogram of antiocxident activity of H.thebica Different concentration of the various H. thebaica extract were carefully assessed for their antioxidant activity. Powder of active H. thebaica fruits were extracted successively using continuous Soxhlet technique for fractionating the different groups and made it possible to determine which fraction was responsible for activity as antioxidant. For the total weight of the extraction of the H. thebaica fruit powder with different solvents, methanol (sox) gave the highest value (70.5%) followed by ethyl acetate (sox) (51.67%) . These results suggested that the methanol extract of H. thebaica fruit demonstrated high antioxidant activity with inhibition percentage of (70.5%), ethylacetate (mac) extract demonstrated medium antioxidant activity with inhibition percent of (57.67%), ethylacetate (sox) extract demonstrated antioxidant activity with inhibition percent of (29.49%), and hexane (sox) extract demonstrated as weak antioxidant activity with inhibition percent of (1.3%) . These results agree with (Hinar 2015) which indicated that doum fruit is a rich source of antioxidants using the DPPH radical scavenging activity of methanolic extracts.

Chapter (3): Results and Discussion

3.3 Elements content in H. thebaica Table (3.6) show the element composition of H. thebaica

Elements %

Potacium (k) 1.9568

Chlorine (Cl) 1.679

Sulpher (S) 0.536

Silicon(Si) 0.4247

Phosphorus (P) 0.3279 Magnesium(Mg) 0.2735 Sodium(Na) 0.158 Calcium (Ca) 0.0528 Alaminium (Al) 0.0185 Iron(Fe) 0.008

This study indicated that Elements content were K (1.9568%), Cl (1.679%) , S(0.536%) , Si(0.4247%) , P(0.3279%) , Mg(0.2735%) ,Na (0.158%) ,Ca(0.0528%) , Al(0.0158%) , Fe(0.008%). Carbohydrates, protein, lipids and vitamins (organic substances). Previous studies on doum focused on the fruit because, besides its nutritional value, the fruit drink brewed from water infusion of the dried fruits are widely consumed as a health tonic and has been used for its many anecdotal medicinal properties .Research on the fruit of H.thebaica showed that it contains nutritional minerals ,proteins and fatty acids, in particular the nutritionally essential linoleic acid .The doum palm is the more important plant families that supplied human with dietary fibers ,carbohydrates and antihypertension substances. Essential Elements sometimes divided up into major Elements and trace Elements.These two groups are equally important, but trace Elements are

Chapter (3): Results and Discussion needed in smaller amounts than major Elements. The amounts needed in the body aren’t an indication of their importance. Elements composition in the samples was determined from the ash which was prepared dissolve in 10ml of concentrated nitric acid made up to 25 ml. Results of Na and K agree with (Salih and Yahia., 2015) and disagree in presence of Fe and Al. Potassium helps regulate mineral and balance throughout the body. It has been associated with a decreased risk of cardiovascular disease. Although sodium is often maligned as acause of pressure, it also plays several essential roles in the body .It helps control blood pressure and regulates the function of muscles and nerves.

Chapter (3): Results and Discussion

3.4 GC-MS analysis of n-hexane extract

Figure (3.5): Gas chromatogram of the hexane extract of H.thebaica

Figure (3.6) Gas chromatogram of the oil of seeds of H.thebaica

Chapter (3): Results and Discussion

Table (3.7): GC/MS Composition of hexane extract of fruits of H.thebaica compounds

No R.Time Area% IUPAC Name Common name Formula M. Wt

1 11.735 0.23 Dodecanoic acid Lauric acid C121H24O2 200

2 13.226 0.61 Heptadecane Normal heptadecane C17H36 240

3 13.287 0.48 Pentadecane,2,6,10,14-tetramethyl- Norphytane C19H40 268

4 13.982 1.41 Tetradecanoic acid Myristic acid C14H28O2 228

5 14.30 1.13 Heneicosane Henicosane C21H44 296

6 15.169 13.12 1,2Benzenedicarboxylic acid, bis(2-methyl) Phthalic acid C16H22O4 278

7 15.341 1.19 Hexacosane Hexacosane C26H54 366

8 15.633 2.37 Hexadecanoic acid, methyl ester Palmitic acid C17H34O2 270

9 15.869 0.91 Palmitoleic acid Palmitolinoleic C16H30O2 254

10 16.110 31.48 n-Hexadecanoic acid Palmitic acid C16H32O2 256

11 16.238 1.10 Octadecane n-Octadecane C18H38 254

12 16.991 0.60 Pentadecanoic acid Pentadecylic acid C15H30O2 242

13 17.285 2.97 9,12-Octadecadienoic acid(z,z)-methyl ester Lenoleic acid C19H34O2 294

18 14 17.331 0.62 9-Octadecanoic acid(z)-, methyl ester Oleic acid C H32O2 280

15 17.740 14.98 9,12-Octadecadieoic acid (z,z) Grapeseed oil oil C18H34O2 282

16 17.768 7.74 9,-Octadecadieoic acid (z,z)- Cis-oleic acid C18H34O2 282

17 17.933 2.57 Octadecanoic acid Hydrofol acid C18H36O2 284

18 21.168 14.02 Bis(2ethylhexyl) phthalate Phthalic acid C24H38O2 390

19 24.599 1.70 Hexacontane _ C60H122 842

20 25.606 0.77 Cholesterol Cholesterin C27H46O 386

100.00

Chapter (3): Results and Discussion

Table (3.8) GC/MS of essential oil in seeds of H.thebaica compounds

No R.Time Area% IUPAC name Common name M.Formula M. Wt

1 3.332 0.21 Hexanoic acid, Caproic acid C6H12O2 118

2 5.906 4.04 Octanoic acid, Caprylic acid C8H16O2 144

3 8.663 2.45 Decanoic acid Capric acid C10H20O2 172

4 11.270 28.68 Dodecanoic acid Lauric acid C12H24O2 200

5 12.406 0.17 Tridecanoic acid n-Tridecanoic acid C13H26O2 214

6 13.557 15.53 Methyl tetradecanoate Tetradecanoic acid C14H28O2 228

7 14.343 0.02 5-Octadecanoic acid Stearic acid C18H34O2 282

8 14.610 0.04 Pentadecanoi cacid n-Pentadecanoic acid C15H30O2 242

9 15.406 0.06 7-Hexadecanoic acid Palmitic acid C16H30O2 254

10 15.441 0.03 9-Hexadecanoic acid Palmitoleic acid C16H30O2 254

11 15.649 11.09 Hexadecanoic acid Palmitic acid C16H32O2 256

12 16.404 0.06 Cis-10-Heptadecenoic acid, Margaric acid C17H32O2 268

13 16.613 0.14 Heptadecenoic acid Margaric acid C17H34O2 270

14 16.791 0.05 Methyl octyl phthalate -

15 17.295 4.75 9,12 Octadecadienoic Linoleic acid C18H342O2 280 acid(z,z) 16 17.373 24.67 9-Octadecenoic acid(z)- Oleic acid C19H36O2 296 methyl ester 17 17.555 5.73 Methyl stearate - C19H38O2 298

18 19.107 0.41 Cis-11-Eicosenoic acid, - C20H38O2 310

19 19.305 0.66 Eicosanoic acid, Methyl arachisate C20H40O2 312

20 20.925 0.24 Docosanoic acid, methyl Behenic acid C23H46O2 340 ester 21 21.156 0.14 Bis(2-ethylhexyl)phthalate Phthalic acid C23H36O2 376

22 21.691 0.06 Tricosanoic acid, Tricosanoic acid C23H46O2 354

23 22.429 0.26 Tetracosanoic acid, methyl Lignoceric acid C24H48O2 368 ester 24 23.164 0.26 Squalene Trans Spinacen C30H50 410

25 23.829 0.05 Hexacosanoic acid, Cerotic acid C26H52O2 396

100.00

The experimental results obtained by GC-MS analysis of hexane extract figure(3.5) and (3.6) performed highlighted the major (38.66%) saturated and (27.22%) unsaturated. The main fatty acids contained in hexane extract

Chapter (3): Results and Discussion of doum fruit were n-Hexadecanoic acid (31.48%) ,9,12-Octadecadienoic acid (14.98%) ,Oleic acid (7.74 %) Octadecanoic acid (2.57%)Heptadecane (0.61 %) ,Tetradecanoic acid (1.41%) ,Hexadecanoic acid (2.37%),Octadecane(1.1%) ,9-Octadecenoic acid ( 0.62%),Hexacontane(1.7%),Palmitoleic acid (0.91%) , but the analysis of essential oil in the seed extract of H. thebaica indicated that the major saturated and unsaturated the main fatty acids contained in oil were Dodecanoic acid (28.68%),Methyl tetradecanoate (15.53%), hexadecanoic acid (11.9%),9-Octadecenoic acid (Z) (24.67%) , 9,12- Octadecadienoic acid (Z,Z) (4.75%) .Fatty acids were extracted from doum fruit by hexane solvent by the soxhlet method. The fatty acid profile was determined by GC/MS after converion of samples into fatty acid methyl esters. From the GC-MS analysis, some similar compounds were observed in fruits and seeds of H. thebaica specially, unsaturated fatty acids such as oleic acid and linoleic acid. In seeds, the total percentages of both acids (29.42%) are greater than of the same acids in fruits (22.72%). However some compounds were observed in fruits and were not detected in seeds. From the current research, it is observed that the percent yields of saturated and unsaturated fatty acids in seeds were greater than the percent yields of fruits. This study showed that method two is the best choice for extraction than method one because the yields obtained by soxhlet were greater than extraction by maceration.

Chapter (3): Results and Discussion

The following forms of some common saturated compounds:

1- dodecanoic

2- Heptadecane

3- Tetradecanoic acid

Chapter (3): Results and Discussion

4- Pentadecane

5- Heneicosane

Chapter (3): Results and Discussion

6- Hexadecanoic acid

7- Hexacosane

8- palmitic acid

The following forms of some unsaturated compounds :

1- 9- Octadecenoic acid

Chapter (3): Results and Discussion

2- 9,12- Octadecadienoic acid

3- Cholesterol

Chapter (3): Results and Discussion

4- Palmitoleic acid

Chapter (3): Results and Discussion

3.5 Thin layer chromatography of methanol (sox) extract Figures (3.1) show then layer chromatography of methanol (sox) extract. table(3.9) shows colours and Rf of seven compounds.

Figure (3.1): Thin layer chromatography of methanol extract (sox)

Chapter (3): Results and Discussion

Table (3.9): Compounds isolated from the methanol (sox) extract

Color

Compounds Rf Vis UV(365nm) UV(254nm) Spray value reagent

1 0.06 - - - Light blue

2 0.14 - - - Light blue

3 0.36 Light - - Yellow yellow

4 0.59 - - Light blue Light violet

5 0.73 - - - Light violet

6 0.87 - - Light blue Violet

7 0.94 - - - Gray

The active methanol (sox) extract was fractionated in TLC using different mobile phases, seven compounds were successively isolated by preparative thin layer chromatography (PTLC). The characteristic colors and Rf values of these compounds are tabulated in Table (3.9).Mobile phase Toluene: Ethyl acetate: Formic acid (5:4:1).

Chapter (3): Results and Discussion

3.5.1 Identification of compounds of fractions Table (3.10) show the IR spectral data for the identified fractions and table (3.11) show the maximum absorption of the isolated fractions Table (3.10): IR Spectral data the seven isolated fractions

-1 Appendix Fractions Functional Group (cm) Rf

1 Fraction 1 2918.4(C-H)Alkane,1100.5(C O)Stretching,1480(C=O) 0.08 3624 (O-H)

3 Fraction 2 758.05(C-H) Bending,3100(=C-H)Stretching 0.20

5 Fraction 3 3024.48(C-H)Stretching,795.63(C- H)Bending,3016.77(C-H)Aromatic 0.32 ring,1228.7(C-N),1203.62 (C-O ), 1524 (C=O)

7 Fraction 4 2400(N-H), 1215(C-O), 3018 (C-H) Aromatic ring ,760(C-H)Bending, 0.62 2926 (C-H) Stretching

9 Fraction 5 758.05(C-H) Bending,1215 (C-O), 1508.38 0.77 (C=O), 2926 (C-H) Stretching

11 Fraction 6 750(C-H) Bending, 1230 (C-O),2400 (N- H),3020.63 (C-H)Aromatic ring, 1521 (C=C) 0.83 Stretching

13 Fraction 7 1215.07 (C-O), 750 (C-H) Bending, 2399.53(N- H), 3010.63(C-H)Aromatic ring, 1732 (C=O), 0.88 3686 (O-H)

Chapter (3): Results and Discussion

Table (3.11): Maximum absorption of the seven isolated fractions

Appendix Fractions (ʎ Max) nm Rf

2 Fraction 1 306 0.08

4 Fraction 2 306 0.20

6 Fraction 3 306 0.32

8 Fraction 4 306 0.62

10 Fraction 5 306 0.77

12 Fraction 6 306 0.83

14 Fraction 7 306 0.88

The identification of fractions were based on different spectroscopic techniques such as IR and UV spectroscopy. Spectroscopical (IR and UV) data are recorded for the isolated major fractions which were found to be isomeric flavonoids in nature and in agreement with those spectral data reported by (Pedro, .F.P and Goncalo, C., J., 2012).

Chapter (3): Results and Discussion

3.6 Conclusion and Recommendations 3.6.1 Conclusion  The phytochemical screening on H. thebaica fruit extracts demonstrated the presence of sterols, triterpenes, carotenoids, tannins, coumarins , carbohydrate , flavonoids , 2-deoxysugar and saponins.  The methanol (sox) and ethyl acetate (mac) extracts were effective against the tested antioxidant activity, the highest activity percent (70.67%) was demonstrated by the methanol extract (sox).  GC-MS spectrum for hexane extract of fruits show the presence of twenty compounds saturated and unsaturated fatty acids. Also the hexane extract of seeds show twenty five compounds saturated and unsaturated fatty acids which means that the high value of antioxidants due to the presence of these acids .  Extraction of H. thebaica fruits using methanol (sox) represent the highest yield ( 26.77%).  XRF indicated that H. thebaica has elements like k(1.9568%) ,Cl(1.679%) , S(0.536%) , Si(0.4247%) , P(0.3279%) , Mg(0.2735%) ,Na (0.158%) ,Ca(0.0528%) , Al(0.0158%) , Fe(0.008%)  Thin layer chromatography of methanol(sox) extract gave seven compounds isolated and analysed by infra-red showed the presence of (C-O), (C-H) Alkane, (C=O), (=CH)Stretching, (C-H)Aromatic ring ,(C-H) Bending, (N-H), (CO) ,(O-H).  The richness of these fruits in organic, minerals and antioxidant compounds makes them considerable sources of nutrition and of potential impact on human health.

Chapter (3): Results and Discussion

3.6.2 Recommendations  The need for modern equipments for extraction, purification, isolation and identification of plant active constituents.  Further research needs of most compounds act as antioxidant.  Further research on oil extracted from seeds of H. thebaica.

References

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Appendices

Appendix(2) UV Spectrum of Compound 1

Absorbance Wavelength(nm)

0.004 572.00

0.005 396.00

0.137 306.00

00.00 216.00

10.00 210.00

04-0.0 340.00

0.012- 234.00

Appendix (4) UV Spectrum of Compound 2

Abs Wavelength(nm)

0.002 560.00

0.003 402.00

0.140 306.00

0.001- 216.00

0.007- 340.00

0.012- 234.00

Appendix(6) UV Spectrum of Compound 3

Abs Wavelength(nm)

0.219 306.00

0.001 206.00

0.012- 234.00

Appendix(8) UV Spectrum of Compound 4

Absorbance Wavelength(nm)

0.003 580.00

0.218 306.00

0.000 212.00

-0.014 234.00

Appendix(10) UV Spectrum of Compound 5

Absorbance Wavelength(nm)

0.006 366.00

0.181 306.00

0.001 210.00

0.014- 234.00

Appendix(12) UV Spectrum of Compound 6

Absorbance Wavelength(nm)

0.008 366.00

0.191 306.00

0.000 212.00

0.000 206.00

0.015- 234.00

Appendix(14) UV Spectrum of Compound 7

Absorbance Wavelength(nm)

0.007 384.00

0.178 306.00

0.001- 210.00

0.017- 234.00