بسم اهلل الرحمن الرحيم Alneelain University Graduate College
PHYTOCHEMICAL, ANTIOXIDANT AND ANTIMICROBIAL ACTIVITES OF BOSCIA SENEGALENSIS SEED
A thesis submitted for partial Fulfillment of the Requirement for M.SC. Degree in Chemistry
By Mnahil Suliman Omer (B. SC. Nyala University 2010)
Supervidor : Dr : Omer Abdalla Ahmed Hamdi
February 2019
I
Dedication
To spirit of my father,
To my lovely mother,
To my sisters and brothers,
To my friends,
&
To all those whom I love
Mnahil
II
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 deep gratitude to my supervisor Dr .Omer abdalla Ahmed Hamdi. Also deepest gratitude to Dr. Ragaa Mohammed satti the door of her office was always opened whenever I ran into a trouble spot or had a question about my research .she 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 .
I must express my very profound gratitude to my greet mother , my brothers , my sisters . And to my friends , special gratitude to my friend Mawda Ibrahim for help .
III
Abstract This study was conducted with the aim to extract and analyze the seeds powder of Boscia senegalensis , which collected from forest on the north of Alfasher in Darfour state, Sudan. Two methods of extractions from the seeds powder of B.seneglensis were performed ;method 1: extraction by maceration using three different solvents including methanol, water and ethyl acetate their yields were 29% ,9.6% and 17.3% , respectively. These extracts were subjected to phytochemical analysis which showed the presence of saponins, coumarins, carbohydrates, tannins, flavonoids and alkaloids. Method 2: successive extractions using soxhlet extractor from seeds powder using n- hexane, chloroform and methanol yielded 4.7%, 4.1% and 15.7 %, respectively.
GC-MS was used for quality and quantity evaluation of the oil extract of seeds revealed the presence of a total of 14 compounds were completely identified and the major compounds were classified as unsaturated fatty acid including Tetratiacontane (22.71%),. Hexaatriacontare (18.41%),Linolenic acid(16.75%) Hexadecanoic acid(11.85),Oleic acid (7.14% ) and Squalene(5.16%) .
Anti oxidant activity was evaluated for all extracts. The results showed that the methanol extract (mac) possessed the highest activity with 76.7% at the concentration of 0.1mg/ml using DPPH assay. The most active extract was subjected to TLC and visualized with vanillin –H₂SO₄ reagent, afforded nine pure compounds were analyzed by spectroscopic methods (Infra-red and Ultra-Violt ).
IV
V
List of Contents:
Main topic Page no.
I اآلية
Dedication Ii
Acknowledgments Iii
Abstract Iv
V مستخلص البحث
Table of Content Vi
List of Tables. Vii
List of Plates. Xi
List of Figures Xii
List of Abbreviations Xiii
List of Appendices. Xiiii
Chapter One
1. Introduction and Literature review 1
1.1 General Introduction 1
1.2 Literature Review 2
1.2.2. Taxonomy of B. senegalensis 2
1.2.3. common names 3
1.2.4.Discription of B. senegalensis 3
1.2. 5.Habitat and Distrbution 4
1.2.6. Constituents of Bos. Senegalensis 6
1.2.7 Nutritition value 7
VI
1.2.8 Uses of B. senegalensis 8
1.2.8.1 Nutrition uses 8
1.2. 8.2 Medicinal uses 9
1.2.8.2.1 Traditional medicine 9
1.2.8. 2.2 Recent medicine 11
1.2.8.2..2.1 Role against pathogen 11
1.2.8.2.2.2 The effect on blood cells 11
1.2.8.2.2.3 Anti flammatory effect 11
1.2.8.2.2.4 Water clarification 11
1.2.9 Toxicity 11
Chapter two
2.1 . Materials 13
2.1.1 . sample collection 13
2.1.2 . Instrumentations 13
2.1.3 . Chemicals and Reagents 13
2.1.3.1 . Chemicals 13
2.1.3.2 . Reagents 14
2.1.4 . Chromatographic Materials 15
2.1.4.1 . Solvents 15
2.1.4.2 . Stationary phase 15
2.1.4.3 . Mobile phase 16
2.1.4.4 Detection 16
2.2 Methods 16
2.2.1 Maceration methods 16
2.2.1.1 Preparation of ethyl acetate extract 16
VII
2.2.1.2 Preparation of water extract 16
2.2.1.3 Preparation of methanol extract 16
2.2.1.4 Successive extraction 17
2.3 Phytochemical screening of Extracts 17
2.4.1 Alkaloids content 17
2.4. 2 Flavonoid content 17
2.4.3 Saponin content 17
2.4 .4 Tannin content 18
2.4 .5 PH content 18
2 .4 .6 Ash content 21
2 .5 Antimicrobial activity materials 21
2.5 .1 Bacterial microorganism 21
2.5.2 Media 21
2.5. 3 Method 21
2. 6 Anti oxidant activity extract 21
2.7 Chromatographic methods 22
2.7 .1 Examination of extract 22
27.2 Preparative thin-layer chromatography 22
2 .8 Spectroscopic methods 23
2.8 . 1 preparation of sample for GC –MS 23
2.8. 2 Infra red spectra 23
2.8.3 Ultraviolet-spectra 23
Chapter three
VIII
3-1 Extraction and phytochemical screening 24
3-2 Quantitative determination of the chemical constituency 25
3-3 PH and ash content of B.senegalensis seeds 27
3-4 Antioxidant activity of extracts 28
3-5 Antimicrobial activity of B. senglensis seeds extract 29
3-6 The chemical composition of oil extract 31
3-7 Thin layer chromatography of methanol extract 38
3-8 Spectroscopic analysis of isolated fractions from methanolic extract 41
Chapter Four 4. Conclusion and Recommendation 44
4.1. Conclusion 44
4.2. Recommendations 45
References 46
IX
List of figures NO Name of Table Page
1.1 B.senegalensis (plant ) 4
1..2 B. senegalensis(map 5
1.4 B. Senegalensis(seeds) 6
3.2 phytochemical screening of B.senegalens 26
3.3 Figure (3.3)determination of the chemical 27 constituency of B.Seneglensis seed
3.4 fgure (3.4)Ash of B senegalensis (seeds ). 28
3.5 Figure (3-5) oil of seeds extract of B. Sengelansis 31
3.6 Figure ( 3-6 ) Gas chromatogram of the oil of 32 seeds extract of B.senegalensis
X
List of Plates NO. Name of plate Page
3-1 Plate inhibition zone of B. senegalensis seed 31
3.2 plate (3-2) :Thin layer chromatography of methanol extract 38
3.3 plate ( 3-3) Preparative thin layer chromatography of methanol 41 extract
XI
List Of Table NO. Name of Table Page 3-1 42 Percentage yields and properties of three solvent extracts using maceration method 3-2 42 Percentage yields and properties of three solvent extracts using soxhlet method Table 3-3 General phytochemical screening of B.seneglensis seed) 42 3-4 42 Quantitative determination of the chemical constituency 3-5 42 PH and ash content of B.sengalensis seed 3-6 Antioxidant activity of B. seneglensis 42 3-7 42 Antimicrobial activity of B.senglensis seeds extract
3-8 Chemical composition of oil extract of seeds of B. sengalensis 33 3-9 Maximum absorption of methanol isolated fractions 32
3-10 IR spectral data the nine isolated fractions 24 3-11 23 Maximum absorption of the nine isolated fractions of maceration methanol
XII
List 0f Abbreviations
Abbreviation Meaning
Maceration Mac Strong +++ Medium ++ Weak + Negative - Factor Retardation RF Thin layer chromatography TLC Gas chromatography – Mass spectroscopy GC-MS 2-2-Di phenyl 1- picryl hydrazyl DPPH Rt Retention time
Infra red IR Ultra violt UV Soxhlet Sox
XIII
Chapter one Introduction and Literature
Review
1- Introduction and Literature Review 1-1 General Introduction People have known the use of medicinal plants since ancient times. These plants have been used for the treatment of various diseases worldwide, because of ready availability, their low cost of production and less toxic action compared to those of synthetic compounds (Asima and Pkrashi, 1995).Africa is a continent endowed with an enormous wealth of plant resources over 5.000 distinct species are known to occur in the forest region most of them have been used for several countries in traditional medicine (Maurice , 1993). In Sudan, numerous plants or plant products are commonly used by rural and urban citizen for the treatment of a wide variety of condition, often without prior research to prove their claim (Bakhiet, 1995; Omer et al., 1992 and Ahmed,1988). B. senegalensis or Aizen, is a member of the family Capparaceae. The plant originated from West Africa. Still a traditional food plant in Africa, this little-known fruit has potential to improve nutrition, boost food security, foster rural development and support sustainable land care ( NRC , 2008). Synonyms are Porrdia Senegalensis (pers)-and Boscia octandra ( Hochste). The local name in Sudan ,Mukheit Umnkeit and krusan and widely spread in North kordofan and Darfour (ELgazalietal.,1987). Mukheit or Aizens are not fruit crops in normal sense. It is the combination of foods and useful qualities that makes them important. The species produces enough different products to sustain human life almost by itself. In at least a dozen countries, at times people virtually live off Aizen fruits, seeds, leaves and berries are commercially available in many parts northern Nigeria and Sudan as good condiment for soups in times of scarcity ( NRC , 2008). B.senegalensis has a lot of uses, the leaves are traditionally used in west Africa in cereal protection against pathogens, Pharmacologic application and food processing . In sudan whole plant use as an thelimentic were as the emulsion of leaves used as an eye wash ( ELgazalietal., 1997).
1
Objectives:
General objectives:
The aim of this study was to assess antioxidant activity and antimicrobial activity of methanol and essential oil extracts and determine the physicochemical properties of oil from the seeds of B. senegalensis
Specific objectives:
1- To evaluate antioxidant activity and antimicrobial activity of methanol extract and essential oil from the seeds of B. senegalensis 2- To investigate the best extract that can be give the active antioxidant and antimicrobial ingredients. 3- To fractionate the active antioxidant extract by using chromatographic methods. 4- To analyze the isolated pure fraction using UV and IR spectroscopic techniques. 5- To analyze the extracted oil using GC-MS spectroscopy.
1-2 Literature review
1.2 .1 Taxonomy of B. senegalensis: Kingdom : Planate – Plants
Subking : Tracheobionta - Vascular plants
Superdivision : Spermatophyta - Seed plants
Division : Magnoliophyta - Flowering plants
Class : Magnoliopsida Dicotyledons
Subclass : Dilleniidae
Order : Capparales
Family : Capparaceae – Caper family
Genus : Boscia Lam. – boscia Species : Boscia senegalensis (pers) lam. ex poiret
2
1.2.2 Common names Aizen (sometimes spelled ayzen) is a Berber word and is the one most commonly used in the literature dealing with West Africa. Other names include mandiarha (Berber),mokheit, mukheit, umkheit (all Arabic), bere (Bambara); ngigili (Fulani). Other common names for the fruit include dilo (Hausa); bokkhelli and kursan (hant, tadent, tadomet (Tamachek); harrenya (Sonrai); nabedega (More), and nkiandam and diendoum (NRC,2008) 1.2.3 Description of B.senegalensis Small trees, up to 3 m high with stiff grey branches. leaves are alternative, cariaceous,petiolate ; laminas oblong-ellaptic to obovate, 3.8-10x1.7-4.9cm, apex apiculate or mucronate, basecuneate, margin entire ; petioles 0.5-1cm long. In florescences dense corymbs,, peduncles up to 3cm long ;pedicels 2-3 cm long, gynophores 2-4 mm long .Flowers greenish. Fruits drupe, globose, warted up to 1-3 cm long , pale yellow when ripe, 1-2seeded (Elghazali et al.,1994).
3
Figure( 1.1 ) : B. senega lensis (plant ) 1.2 .4 Habitat and distribution B.senegalensis(Pers.) Lam. ex Poiret: is distributed south of 16 N°(Andrews,1950) . The aizen occurs across area that in recent decades has faced more hunger than any other in the world—the vast swath of Sahel and Sahara savannas stretching frome Mauritania, Senegal, and Mali all the way to southeastern Egypt, Sudan, Ethiopia,Somalia ,and Somalia, and Kenya (NRS,2008). B.senegalensis endurance is remarkable. It tolerates temperatures as high as 45°C, a level not rare in its habitat. It occupies most types of arid-land environment stony slopes, sand dunes, and cracking-clay plains, just for starters. It often occurs in desiccated, barren, hard, and even fire-scorched sites. As to soils, they are usually poor, sandy, rocky, worn-out laterite, orclay. Commonly it sprouts directly out of termite mounds. and it
4 survives in areas receiving as little as 100 mm annual rainfall, although it grows best where there is at least 250 mm. Contributing to the plant’s built-in drought to lerance is its remarkable leaf structure the cuticle is up to 20 microns thick, the stomata are sunk in deep cavities, and each stomata has thickened walls and a protective armoring of papillae ( Killian , 1937).
Figure(1.2) Boscia senegalensis(map)
5
Figure(1.4) B. Senegalensis(seeds)
1 .2 .5 Constituents of B. Senegalensis The genus has been shown to elaborate several flavonoids (walter and sequin , 1990 ) Terpenes and their glycosides (Pauli etal,1990), sulfur compound ( kjaeretal , 1973) , and a lipid( Grindley , 1948 ) . Leaves constitute choline and sterols (β-sisterol, campesterol and stigmastrol) as well as aliphatic alcohole (predominantly C30,C34,C38),carbohydrates and four glycosinolates of unknown structure could be detected ( Kerharo and Adam,1974). Three of glucosinolate from leaves as methyl and 2-propylisothiocyanate and in trace amount 2-butylisothiocynate (Kjaeretal., 1973). Proto alkaloid Lstachydrine and 3hydroxy from leave sample from Senegal (Delaveauetal., 1973). Root sample from Sudan give a positive reaction for sterol, triterpenes a negative reaction alkaloid ,cardilenpolides, tannin, saponin ,flavonoids and quinine (ELkheir and Salih , 1980). Saponin was detected in leave sample from Niger, alkaloid ,tannins, quinines and flavonoids were not present ( Baouaet al,1976). Methyl iso thiocyanate which is liberated from a glucosinolate precursor; glucocapparin found in the fruits and leaves (Seck, et al. 1993).
6
S
CH3 N N S
CH3 H3C CH3 S H3C N
2-butylisothiocynate 2-propylisothiocyante methyle iso thiocyanate
– O OH S O O – O O O HO S HO N + N OH O R H3C CH3 Aglucosinolate Lstachydrine
1 .2 .6 Nutritional value Detailed nutritional analyses seem unavailable, but the fruits are reportedly rich in calcium, phosphorus, iron, and B vitamins. They are also said to contain a little protein (NRC, 2008). Seeds of B .senegalensis ( dilo in Niger) Contained 70mg/100g from essential fatty acid, linolic acid.Ca 29mg/100 g dry Wt Mg 4.2/100g and traces copper selenium manganese, Zinc Iron. However were presented at relatively high level ,(6.0 and 5.3 mg /100 dry wt) respectively. The chemical composition of dilo harvested in Niger differs significantly from that of B senegalensis seeds grown in Sudan.(kim and pastuzyn,1997) . The nutritionl value of seeds of mukheit B.senegalensis (Pers) Lam ex Poir) and maikah (Doberaroxburghi Planch) widely used as food in times of Famine in western Sudan have been investigated. The chemical composition of each Food before and after the traditional debittering process is reported. Both foods Compared Favourably with the local staple cereals as regards the content of soluble Carbohydrate and crude protein
7 with values of 690 and 250 g kg-1 respectively for Mukheit debittered by soaking in water at ambient temperature and 8 ,150 g kg-1 Respectively for maikah debittered by boiling in water. The nutritional quality of Protine in mukheit debittered by soaking in water at ambient temperature appeared similar to that of sorghum but was markedly enhanced if debittering was performed By boiling, although the crude protein content of the food fell substantially during The latter procedure (Salih and Nour ,1991). 1 .2 .7 Uses of B. senegalensis
1.2.7 .1 Nutritional uses
The mixing a suspension of powder prepared from the leaves or roots with Cereal flour (or porridge) results in sweet products (Salih and Nour , 1991). The rationale behind the use of these leaves for the production of foods with improved taste (through probably release of reducing sugars) has never been elucidated (Dickoetal ,2001 ) . According to a Sudanese famine-food specialist , Aizen was the ―number one‖ famine food during the horrific 1984 famine in the western Sudan. ―It proved to be people’s lifesaver,‖ he reports, ―and it saved more lives than all the food aid that was given. (Abdelmuti ,1991) . In famine times, people in rural Sudan rely on aizen. Typically, they collect the Fruits ; sun-dry them, separate the pea-sized seeds, and remove the hard outer seed coat. Seeds are then subjected to ―sweetening‖ to remove bitter and possibly toxic components . The traditional procedure involves soaking for a week, changing the water every day. Sometimes ―kambo,‖ local potash prepared from plant ash, is added to aid the debitteing. Less commonly, sweetening is conducted by boiling for 3 hours, with the water changed hourly. After such treatment, the sun-dried sweet‖ seeds are stored until required, at which time they are boiled until soft, changing the water once during the process. The resulting food is usually eaten with oil and salt. Alternatively, seeds are ground to flour which is consumed in the form of kisra, flat thin bread popular in
8
Sudan or Asida, a local form of porridge. The taste of the final product can be improved by blending with millet or sorghum flour(NRC, 2008). 1 .2 .7 .2 Medicinal uses The leaves of B. senegalensis are traditionally used for human and animal nutrition, Protection of cereals against pathogens, and pharmacologic purposes(Salih and Nour , 1991). 1.2 .7.3 Traditional medicine In Senegal B. senegalensis used to treat colic without acting as a purgative, and for sever Jaundice and feverishy cholecyctitis. Also it used as antihelmentic (Schistosoma) . Schistosoma by decoction of leave ,for itching eye. Wolof treats cold with leave of B. Senegalensis also for catarrh in horse; pulverize the leave and rub into the nose or let animal inhale the vapor of the heated leave. A greul of leave or flour a medicine or heamorrhoid is made by adding Salvadora persica leave to that of B. senegalensis(eat every day ) . In Cameroon : plant is used for chronic ulcer, syphilis , colic and filariases as a fumigation the leave are said to relive cough in horses. In Niger hausa women take the powder of leave during the first 7 days of having birth, also use leaves powder or roots in the form of watery paste to ulcer and eczema. the root are considered to be a medication for male sexual weakness for exhaustion and fatique the decoction of the above ground leave plant part is drunk leave powder in food is cold medicine for horses and monkey. In Chad Bagirmi: wound and skin rashes are treated with bark extract, tooth ache and gingivitis with root infusion(mouth wash) . In Sudan Kordofan province leave infusion is widely used medication for ventral disease, inflamed eye are also bathed with it . Root prepared are used for Jaundice root, leave and bark are used as coagulating agent to clarify water .in west Of Sudan and blue Nile B. senegalensisis considered as one of the potent plant for treated ,turbid water .Water coagulation is believed to protect from disease like; Diarrhea ,gastro-intestile disorderr ,gastric fever (Neuwinger,1996).
9
1.2.8. Recent medicine \ 1.2.8 .1 Role against pathogen B. senegalensis possesses antimicrobial and anti fungal activities (Almagbouletal, 1988, It has been show to be effective uterine stimulation (Bulloughetal., 1982). The leave used in the preparation of malaria remedy and for the treatment of jaundice, Fungal infection and viral diseases ,it applied externally for wound. The fruits and roots are used as aphrodisiac, and the roots decoction is used for stomach ache and to facilitate labour(Maurice,1993). Activities of a-amylase, B-amylase, exo-(1-3, 1-4)-B-D glucanase, and endo- (1-3)-B- Dglucanase were detected in leaves of B. senegalensis (Dickoetal,2001) Synthesis of (1→3)-B-D-glucanases is enhanced in response to pathogen Infection or a biotic stress condition (Ryalsetal ., 1996). (1→3)-B-D-glucanse are associated with antitumor, antibacterial, anti coagulator , and wound healing properties the soluble ones are the most active (Bohn and BeMiller, 1995). 1.2.8.2 The effect on blood cells Leaves contain the alkaloids L-stachydrine and hydroxy-3 stachydrine. Stachydrine Affects aggregation of Thrombocytes and shortens the bleeding time (DFID et-al , 2009 ). 1.2.8.3 Anti inflammatory effect B. senegalensis is a bush of African savannah, used in folk Medicine for the treatment of articulatory pain. The aqueous and alcoholic extracts at dose of 400 mg/kg inhibited the development of oedema. The extract showed the presence of alkaloids saponosides, flavonoids and tannins on phytochemical screening. These findings indicate that the leaves extracts of B.senegalensis contain anti inflammatory active principles for the acute inflammation treatment. Moreover, these results justify the use of the leaves of B. senegalensis for the treatment of many inflammatory diseases (Nongoniermsierm ,2007 ) .
11
1.2.8.4 Water clarification Aizen contains natural coagulants. In Sudan, Niger, and Nigeria, for example ,bark,twigs leaves, and roots are used to scaveng suspended and colloidal compounds from unclean water (such as that from ponds churned up by storms or from baobab-tree cisterns contaminated with soil). Normally the plant parts are sliced up and placed on the water surface. Compounds leach out and catch the clay and other particulates like magnets, causing them to clump and settle to the bottom. It is reported that truly turbid water can be safely drunk after just a day of such treatment. For even faster results, aizen branches are swirled in the water. Indeed, certain Tuareg groups in the Sahara region fill sacks with aizen leaves and dunk them into the muddy pools that comprise their only source for drinking . Following a rare desert downpour they may also place these giant tea bags across ditches so that the run off clarifies itself as it oozes through (NRC,2008) . B.senegalensis is reduced turbidity of water by 94.26% ,many factors were Effect the efficiency of plant ,such as the methods of coagulant, the dose ,filtration and extraction time (long contact time led to deterioration on water quality ) (ELgaddal , 1993) . 1.2.9 Toxicity The nature glycosinolate proved to be a source of anti thyroid drug; the inhibit the organic binding of iodine in the thyroid gland, enlarge the thyroid and cause goiter .β sisterol is it show a significant anti inflammatory and antipyretic activity similar to acetyl salysilic acid by oral administration. Now it fails to induce gastric ulceration or it is analgesic activity (Gupta et- al., 1980). β sisterol posses anti bacterial effect (Chi and choi , 1985). In laboratory animal β sisterol in female may cause abortion (Burcketal.,1982; Pakarashi and Basak 1976) , it can exert oestorogenic effect, In male β sisterol produced an antifertility effect (it decreases the testicular weight and sperm concentration significantly with High doses ,but also after long term treatment with a low dose (Malani and Vanitha kumara , 1991) . Phytosterol fraction it is used for hyper cholestremia for avoid gallstones, it combination with chenodeoxy-cholic acid for cholelitholysis ; it cause increased excretion of
11 cholesterol (Neuwinger,1996). Leafless twigs contain glucosinalates, which can hydrolyze to mustard oils, which are highly toxic and irritant to mucous membranes (DFID et-al., 2009).
12
Chapter two
Materials and methods
2-Materials and Methods
2-1 Materials
2-1-1 Sample collection The B. senegalensis seed were collected from forest in the north of Alfasher on north Darfour state in Sudan. The mesocarp was removed with knife and dried at room temperature for two weeks.
2.1.2 Instrumentations and apparatus -Weighing balance
-Water bath
-Hot air oven
-UV lamp model 34015L(U.S.A)
-IR(Infrared Spectroscopy)
- UV\ Vis 1800 Double beam recording spectrophotometer Shimadzu - Japan
-GC-MS-Qp2010Ultra-Shimadzu- Japan.
2-1-3 Chemicals and Reagents
2-1-3-1 Chemicals -Ethanol
-Hexane
-Methanol
-Chloroform
-Toluene
13
-Petroleum ether (60-80) ℃
-Acetic acid(glacial)
-Acetic anhydride
-Ethyl acetate
-Ferric chloride
-Gelatin powder
-Magnesium turnings
-Sodium chloride
-Potassium iodide
-Iodine
-Picric acid
-Hydrogen peroxide
-Sodium hydroxide
-Ammonium hydroxide
-Hydro chloric acid
-Distilled water
2.1.3.2 Reagents
Baljects reagent Solution A; 1g 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.
14
Mayers reagent: Solution A: 13.36 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 g of iodine and 2 g of KI were dissolved in 5 ml of water and the solution was diluted to 100 ml with 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%AlCl3 w/v in water
FeCl solution ;5% w/ v of anhydrous ferric chloride in water.
Gelatin solution: 50 ml of distilled water were added to 1g of gelatin and allowed to stand for one hour, frequently shaking then the water was decanted and a fresh portion of 60 ml of water were added to the gelatin, and the last was dissolved with shaking and warming to 60C0. 10 g of NaCl were then added to the resulting solution, mixed, cooled, filtered and completed to 100 ml with water
2-1-4 Chromatographic materials
2-1-4-1 Solvents Methanol , ethyl acetate , aceton , petroleum ether
2-1-4-2 Stationary phases Silica gel for thin layer chromatography type 60 GF254 with fluorescent indicator (BDH);U.K.
15
2-1-4-3 Mobile phases -Petroleum ether ; Acetone ; Ethy acetate (3:1:1)
-Toluene :Ethyl acetate: Formic acid (5:4:1)
2-1-4-4 Detection Detection of compounds was achieved using short (254 nm) and long (365 nm).
UV radiation, using Handy lamp.Model R/34015L chromate VUE cabinet in U.S.A. then spraying with Vanillin –sulphuric acid- Methanol (1.5 ml :0.1g :8.5 ml ) followed by heating in an oven at 110℃ for 10 minutes.
2-2 Methods
2.2.1 maceration methods
2.2.1.1 Preparation 0f ethyl acetate extract 10 g of dried powder of sample were transferred into a beaker and 100 ml of ethyl acetate were added. The contents of the beaker were left at room temperature for three days with frequent shaking. The extract was filtered using funnel. The clear. yellow solution was evaporated, and the residual extract was dried and weighed. percentage yield calculated and subjected to preliminary phytochemical analysis.
2.2.1. 2 Preparation of water extract 10 g of dried powder of sample were transferred in to beaker and 100 ml of water was added. The contents of the beaker were left at room temperature for three days with frequent shaking The extract was filtered using 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 10 g of dried powder of sample were transferred in to beaker and 100 ml of (80%) methanol was added. The contents of the beaker were left at room temperature for
16 three days with frequent shaking . the extract was filtered using 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 Extraction of oil for B. Senegalensis seeds (200g) dry seeds powder were macerated with n-hexane at room temperature for 48h . The solvent was removed and oil was kept in the dried container at 40℃ .
2.2.1.5 Successive extraction A dried seed powder (30g) was successively extracted using a soxhlet extractor with hexane (300 ml) chloroform (300 ml) and methanol (300 ml). The solvent was carefully evaporated from each extract and the extractibility of each solvent was determined.
2.2.2 Phytochemical screening of B. senegalensis
The ethyl acetate, water and methanol extract of B. senegalensis were used for the following tests according to method of (Harbone 1984).
2.2.2.1 Screening for flavonoids 1ml of plant extract was taken in test tube 5 ml of ammonia solution were added, 5 ml of sulphuric acid (conc) was added car fully .formation of yellow color indicates to the presence of flavonoid.
2.2.2.2 Screening for alkaloids To 1 ml of plant extract in a test tube 10 ml of Hcl was added and heated I n steam bath for 20 minute ,then the mixture was cold ,
2.2.2.3 Screening for saponin 100 g of the powdered sample were placed in to a clean dry test tube ,10 ml 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 . -
17
2.2.2.4 Screening for tannins
A volume equivalent to 10 mg plant material was evaporated to dryness on awater –bath 25 ml 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 salte out any non –tannin compound .The solution was filtered and to 3 ml of filitrat, 4-5 drops 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,(3 drops) of FeCl3 test solution were added. The formation of blue black or green colour was taken as presumptive evidence for the presence of tannin.
2.2.2.5 Screening for coumarin
The extract (3ml) was evaporated to dryness. The residue was dissolved in hot water. After cooling the solution was divided in to two tubes : one tube contained the reference , and the aqueous solution of the second tube was alkaline with 0.5ml of ammonia solution 10%. The occurrence of an intense fluorescence under UV light indicates the presence of coumarins and derivatives.
2.2.2.6 Screening for carbohydrates
To 1ml of molish reagent, 2.5 ml crude extract and 3ml concentrated H₂SO₄ were added and mixture was heated at 45℃, to formation of a violet ring between two solution surface present which spread by shaking.
2.2.2.7 Screening for reducing compounds
The methanol extract (1ml) was diluted with distilled water (2ml) and fehling solution (1ml) was added and heated. A brick-red precipitate denotes the presence of reducing compounds.
10g of dried powder seed were transferred In to the a conical flask and 100 ml of 10% aqueous ethanol were added. The sample was heated over a hot water bath for 4 hours
18
about 55c. The mixture was filtered and the residue re-extracted with another 100 ml of 10% ethanol .The combined extracts were reduced to 20 ml over water –bath about 90c The concentrate was put in to a separatory funnel and 10ml of diethyl ether were added and shaken . The aqueous layer was recovered while the other layer was discarded. The purification process was repeated. 30 ml of n-butanol were added to combined butanol extracts were washed twice with 10 ml of 5% aqueous sodium chloride. The reaming solution was heated in water bath. After evaporation, the sample was dried in the oven to constant weight and the percentage yield of saponins was calculated .
2.2.2.8 PH content: 2g of the dried seeds powder were weighed and transferred to a beaker and extracted with 20 ml of distilled water and left for 15 minutes. The extract was filtered and the P H for filtrated was measured using PH– meter. (AOAC, 1990). 2.2.2. 9. Ash content: Accurately 2 g of the dried seeds powder were weighed in dry porcelain crucible and ignited at 550 ℃ over5h,in a muffle furnace until free from carbon, cooled in desiccators for 1h,sample and crucible was weighed to the nearest 0.0001 g. Then the total ash % was calculated as follows: (FAO, 1998) Ash (%) = W3 -W1 ×100/W2 - W1 Where: W1 = Weight of the empty crucible (g). W2 = Weight of the crucible + the sample (g). W3 = Weight of the crucible + ash (g) 2.2.4 Anti-microbial activity materials: 2.2.4.1. Bacterial microorganisms: Staphylococcus aureus ATCC 25923 (Gram +ve bacteria) Bacillus subtilis NTCC 8236 (gram +ve bacteria) Escherichia coli ATCC 25922 (Gram -ve bacteria) pseudomonas aeruginosea. *ATCC: American Type Culture Collection Rockville, Meryl and USA. *NCTC: National Collection of Type Culture, Colindale England. 2.2.4 .2 Media Mueller Hinton Agar
19
2.2.4.3 Method: Antimicrobial activity was determined by the well diffusion method Petri plates containing 20 ml of nutrient agar medium were inoculated by streaking the swab over the entire sterile agar surface. This streaking procedure was repeated and the plate approximately rotated 60 degrees each time so as to insure an even distribution of inoculums. Wells were cut into the agar and 100μl of the B. senegalensis seeds were in different concentrations (100, 50, 25. 12.5) mg/ml. The inoculums size was adjusted so as to deliver final inoculums of approximately 1.5×107 colony-forming units (CFU)/ml. Incubation was performed at 37°C for 24 hours. The assessment of antibacterial activity was based on measurement of the diameter of the inhibition zone formed around the well (Miles et al,1938).
2-2-5 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 – di phenyl 1picrylhydrazine (DPPH) (SOHN,2003). Briefly, three solutions were prepared the control (1m DPPH +0.9 mlTris-cl+0.1ml MeOH), the second blank (1ml MeOH +0.9 ml Tris-cl) the third was sample (1ml DPPH+0.9 ml Tris-C+0.1ml sample in MeOH 1mg\1ml). The mixture incubates in the dark for 30 min at room temperature. Afterward, the absorbance read using Spectrophotometer, at 517nm wave length. The antioxidant activity measure using the following formula (Lie.et.al,2005) Absorbance control- Absorbance sample-Absorbance control ×100 .
2.2.6 Chromatographic Methods
2.2.6.1 Examination of extracts using TLC About 2g of methanol (mac) extract were dissolved in 100 ml of methanol and spotted onto the preparative TLC plate by mean of capillary tube. Sample was spotted at about 2cm from the bottom of the plate. After evaporation of the plate by air ,the plate was placed vertically on glass tank which contained a suitable solvent to a depth of 1.5
21 cm and the chromatogram developed. The plate was removed out, 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 compound which appeared in day light or under UV lamp, or after sprayed and heated was calculated as follow .
RF = Distance traveled by spot / Distance traveled by solvent front
2.2.6.2 Preparative Thin layer chromatography A concentrated alcoholic extract solution was chromatography on preparative TLC plate . The plates were twice developed in a mixture of (3:1:1 ) (petroleum ether : ethyl acetate: acetone ) By examining plates (under uv light ), detection of the plates by 1% vanillin H₂SO₄ spray reagents different zones were located . Each was scrapped off in a separate container, and each zone was removed by washing the dry silica gel obtained with (chloroform : methanol )(80:20) several times ,decanting the solvents and removing the excess silica gel by filteration. The extracts were concentrated and the resulting solution of different zones were passed separately through a sintered glass (porosity no₃) to remove any trace of silica gel. The two filtered extracts were then concentrated (Stahl,1969). 2.2.7 Spectroscopic methods
2.2.7.1 Preparation of sample for GC-MS
2ml from the sample were taken in to the test tube, 7 ml from alcoholic NaOH were added. It was prepared by dissolving 2g NaOH in 100ml methanol Then 7ml of alcoholic H₂SO₄ 1% were added to the sample . The mixture was shaken by vortex for 3 minutes, left for overnight. After that 2ml from super saturated NaCl was added, then 2ml normal hexane was added and shaken for 3 minutes, the hexane layer was collected, 5µI from hexane was diluted with 5ml diethyl ether, then 1g from Na₂SO₄ was added as drying agent , filter through syringe filter 0.45µm . 1µl of the filtrate was injected directly to the GC-MS.
21
2.2.7.2 Infra red Spectra Few mg of the fraction was dissolved into chloroform made up to 100 cm, placed in the machine and the spectrums were recorded . The finished spectrum consist of chart showing down-ward peaks corresponding to transmittance potted against wave length . Fourier transform Infra-red spectrometer Model(FT-IR 8400 s-Shimadzu Japan) was used.
2.2.7.3 Ultra –Violet spectra The compound was dissolved in methanol and made up to 10 ml a portion of this was transferred to silica cell of 1 cm thickness. Matched cell containing pure methanol was also prepared and each cell was 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 through pure solvent.
22
Chapter Three
Results and discussion
Results and Discussion
3-1 Extraction and phytochemical screening of B. senegalensis
The B. senegalensis were extracted using different solvent and various methods(soxhelt and maceration). The yield% and properties of extracts are described in table (3-1) and table (3-2) respectively.
Table(3-1): Percentage yields and properties of three solvent extracts using maceration method NO Extracts Yield% Properties
1 Ethyl acetate 17.3 Yellow/Oily 2 Methanol 29.5 Deep brown Oily 3 Water 9.6 Yellow /Oily
Table (3-2): Percentage yields and properties of three solvent extracts using soxhlet method Table
NO Extracts Yield% Properties
1 Hexane 4.7 Yellow Oily 2 Chloroform 4.1 Yellow Oily 3 Methanol 15.7 Brown Oily
Tables (3.1) and (3.2) show that methanol is by far is a good solvent for soxhlet extraction of B.sengalensis seeds gave (4.7%) ,choloroform (4.1%) and finally methanol (15.7%) have lowest ratio of extraction . hexan is less polar solvent removed all fatty acids and terpenoids, choloroform is mordent polar solvent removed the pigments and methanol is more polar detected the all polar compounds and non polar compounds.so the maceration extraction methods is considered better comparative with soxhlet method .
23
3-2 General phytochemical screening of B.senegalensis seed
Phytochemical screening of B.Senegalensis seed extracts (water, ethyl acetate and methanol were carefully carried out using methods described in section (2-2) The results presented in Table (3-3.) summarize the classes of natural compounds present
Table(3-3 ): General phytochemical screening of B.senegalensis seed
Class of compounds Water Ethylactate Methanol Alkaloid + _ + ++
Steroide + + _ + + +
Glycoside + + + _ + + +
Reducing sugar + + _ + + +
Flavonoid + + _ ++ +
Saponin + + + ++ + + +
Cardenolides(Balajact) + _ +
Tannins + _ + + +
Coumarins + + _ + + +
Phenol + + + _ + + +
Key: (+++)=high(++)= Medium (+)= weak (-) Negative
The results of the phytochemical test summarized in table (3.3) revealed that flavonoids ,alkaloids , tannins, coumarins, glycosides, steroids , and saponin present highly in
24 methanol extract , and the phenol, reducing sugar and saponins present highly in aqueous extract, but in ethyl acetate extract, tannins , coumarins, flavonoid,alkaloid and phenol were not detected flavonoids, coumarins, reducing sugars and steroids are present moderately in aqueous extract, finally the cardenolides is not found in all extracts.
Fig(3.2) : Phytochemical screening of B.senegalensi
3-2 Quantitative determination of the chemical constituency
The determination of the crude compound, alkaloid, flavonoid, saponin and tannin showed in table(3-4) .
Table (3-4) Quantitative determination of the chemical constituency Compound Yield% Alkaloid 8.6% Flavonoid 19 Saponin 35 Tannin 12
The results in table(3.4) showed that B.senegalensis is rich source of flavonoids(19%), tannins(12%), alkaloid (8.6%) and contain a high amount of saponin (35% ).
25
40 35 30 25 20 35 15 yield
10 19 5 12 8.6 0 flavoniods tannins saponins alkaloids
Figure (3.3) : determination of the chemical constituency of B.Seneglensis seed
3-3 PH and ash content of B.senegalensis seeds PH and ash content of B.senegalensis seed are presented in table (3-5)
Table (3-5) PH and ash content of B.senegalensis seed
Test Percent/ppm
PH 6.2 Ash 7.67
The present study showed that the pH of B.senglenes (6.2%).The ash content represents the inorganic materials found in the plant, for (7.6%). Table (3-5)
26
Figure (3.4)Ash of B. senegalensis (seeds )
3-4 Antioxidant Activity of extracts: The methanol (sox) and methanol (mac) extracts of B. senegalensis seeds were carefully assessed for their antioxidant activity using DPPH method , the results were explained in table (3-6).
Table (3-6) : Antioxidant activity of B. senegalensis :
No Sample Activity 1 Methanol maceration 76,6 %
2 Methanol (sox) 63.2%
The methanol (sox) and methanol (mac) extracts were evaluated for antioxidant activity using DPPH method . percentage of inhibition of DPPH free radical scavenging activity of B. senegalensis seeds extracts were explained in Table (3-6.). An antioxidants test was conducted at 0.1 mg/ ml for all extracts ; we found that the methanol maceration extract showed the highest antioxidant activity (76.6%) .
27
3-5 Antimicrobial activity of B. sengalensis seeds extract :
The methanol (sox) and methanol (mac) extracts of B. senegalensis seeds were tested for their anti- microbial activity against Bacillus subtilisand Staphylococcus aureusas (Gram + ve bacteria). Escherichia coli and Pseudomonas aeruginosaas (Gram –v bacteria) and (Candida albican) fungi the results presented in (table 3-7). and plates(3.)
Table (3-7) Antimicrobial activity of B.sengalensis seeds extract . Extracts Organism
Inhibition zones (mm) Concμg/ml C.a S.a P.S B.S E.C
Methanol 100 9 10 - 12 13 (mac) Methanol 100 10 - - - - (sox )
Key :Sa =Staphylococcus aureus , Ps = Pseudomonas aeruginosea ,Bs = Bacillus subtilis , Ec = Escherichia coli .
The antibacterial activity results were expressed in term of the dimeter of zone of inhibition ≤ 9mm zone inactive;9-12 mm spartially active ; 13-18mm as active .
Ec gave highest anti-bacterial (12mm ),
Bs= moderate susceptibility for extract (12mm ) .
The gram- negative bacteria (Pseudomonas aeruginosea ), this may be due to the structural differences in cell wall of these bacteria .
28
(a ) (b)
(c ) ( d)
( e) ( f )
( g) Plate (3-1)inhibitition zone of B. senegalensis seed of methanol extract against (a) staphylococcus aureus, (b) pseudomonas aeruginosa, (c) Bacillus subtilis, (d) Escherichia coli . and inhibition zones of methanol (sox) extract of B. Senegalensis against (e) Escherichia coli (f) pseudomonas aeruginosa (h) Bacillus subtilis (k) candida albicas
29
3-6 The chemical composition of oil extract of seeds of B. Senegalensis The essential oil of B. senegalensis fig (3-5) was analyzed by Gas chromatography–mass Spectroscopy. The major compounds were fatty acids showed that in table (3-8)
\
31
Figure (3-5) oil of seeds extract of B. Sengelansis
Figure ( 3-6 ) Gas chromatogram of the oil of seeds extract of B.
Senegalensis
31
Table(3-8) Chemical composition of oil extract of seeds of B. sengalensis
Common NO R .Time Area % IUPAC name M.Wt name M. Formula 16.403 11.85 Hexadecanoic acid 270 c₁₇H₃₄O2
1
18.152 3.43 9,12 octadecadinoic acid Methyl C₁₉H₃₄O2 294 lionleate
2 18.198 7.14 9-octatecenoic acid Oleic acid 296 3 C₁₉H₃₆O2
4 18.233 16.75 9,12,15-octadecatrienoic Linolenic acid C₁₉H₃₂O2 292 acid
5 18.339 3.70 Phytol C₂ₒ H₄ₒ O 296 2-Hexadecen-1-01- 3,7,11,15 – tetra methyl
6 18.420 5.16 Octadecanoic acid Squalene C₁₉H₃₈O2 298
7 20.274 0.91 Eicosanoic acid Methyl C₂₁H₄₂O2 326 arachisate
8 20.503 2.17 Alpha- 472 C₃₁H₅₂O3 2H-1-Benzopyran- Tocopheryl 6o1,3,4dihydro- acetate 2,5,7,8tetramethyl-acetate
32
9 20.973 0.69 Docosanoic acid Methyl C₂₃H₄₆O2 354 behenate
10 22.778 0.40 Tricosantane acid Methyl C₂₄H₄₈O2 368 tricosanate
11 23.269 1.13 n- Tetracontane Tetracontant C₄ₒ H₈₂ 562
12 24.345 5.56 2,6,10,14,18,22- Squalene C₃ₒ H₅ₒ 410 tetracosahexaene,2,6,10,15 ,19,23- hexamethyl 13 24,794 22.71 n- Tetratriacontane Tetratiaconta C₃₄H₇ₒ 478 ne
14 26,628 18.41 n- hexatriacontare hexaatriacont C₃₆H₇₄ 506 are
GC–MS), which showed the presence of 14 compounds such as Tetracontane(22.71%), hexatriacontare(18.41%),Linolenicacid(16.75%) Hexadecanoic acid(11.85), Oleic acid (7.14%), Squalene(5.56%).
33
O H 3 C
CH3 H3C
O
Hexadecanoic acid
H 3 C
O
H 3 C O
9-12 Octadecadienoic acid
C H 3
O H 3 C O
9- Octadecenoic acid
CH3 H3C O
9-12-15 Octadecatrienoic acid
34
HO CH3
CH3 CH3 CH3 H3C
Phytol
O
CH3 H3C O
Octadecanoic acid
O
CH3 H3C O
Eicosanoic acid
O H 3 C C H 3 O C H H 3 C 3 C H 3
H C O 3 C H 3 C H C H 3 3
Alpha –Tocopheryl acetate
O
O CH3 H3C Docosanoic acid
35
O
O
H3C
CH3 Tricosanoic acid
CH3 H3C
n- Tetracontane
HC CH CH 3 3 3
H3C C H C H 3
3 CH3 CH3 2,6,10,14,,18,22-Tetracosahexaene,2,6,10,15,19 hexamethyl
CH3 H3C
Tetratriacontane
CH3 H3C Hexatriacontane
36
3-7 Thin layer chromatography of methanol extract The results in tables (3-9) revealed that the methanolic extract has high antioxidant activity (76,6%) also antimicrobial results in table (3-6) quite obviously indicate that methanol extract has inte mediate anti-bacterial activity . These promising results encourage so much to investigate The methanol extract was fractionated in a TLC using different mobile phase ;finally, nine fractions of methanol extract were separated by preparative thin layer chromatography (PTLC) . The characteristic colors and RF values of these fractions are tabulated in table (3-9) and plate (3-2) and (3-3).
plate (3-2) :Thin layer chromatography of methanol extract
37
Table : maximum absorption of methanol isolated fractions
Spraying Fraction Rf value Vis Uv n 254 reagent
Dark blue
1 0.98 - -
Blue
2 0.94 - -
3 0.89 - - Dark violet
Blue
4 0.83 - -
Blue
5 0.75 - -
Greey
6 0.41 - -
Violet
7 0.15 - -
8 0.08 - - Greey
9 0.03 - - Violet
38
Plate ( 3-3) Preparative thin layer chromatography of methanol extract Mobile phase: petroleum ether : ethyl acetate :Acetone (3:1:1)
3-8 Spectroscopic analysis of isolated fractions from methanolic extract: Preparative Thin Layer Chromatography ( PTLC), ended up with the isolation of nine pure fractions were subjected to further analysis using different spectroscopic techniques such as UV, IR, and spectroscopy. Tables (3-10) & (3-11) below .
39
Table (3-10 ) IR spectral data the nine isolated fractions
No Fraction ((cm)) Group fractional Rf Appendix
1 F1 3018.39 (c-H) :2925.81 (c-H) 0.987 1 : 2399.28(c-H) :1523.66 and 1425.30 (C=C) :1215.07 (C- OH):1016.42 (C-OH)
2 F2 3618.21 (O-H):3018.39 (C- 0.945 2 H):2974.03(C-H) :2399.28 (C-H):1215.07 ,1081,99 and 1047.27 (C-OH)
3 F3 3627.85 (O-H) :3018.39 (C- 0.896 3 H): 2975.96, 2434.00 and 2343.35 (C- H) 1215.07 and 1045.35 (C- OH)
4 F4 3679.93and 3620.14 (OH) 0.836 4 :3018.39 (C-H) :2975.96 ,2894.95,and 2399.28 (C-C) :1521.73 and 1423.37(C=C) 1217.00 and 1045.35(C-OH) .
41
5 F5 3018.39(C-H):2399.28(C-H): 0.757 5 1523.66 and 1423.37 (C=C) 1215.07 and 1045.35 (C-OH)
6 F6 3681.86 and 3627.85 (OH) 0.412 6 :3018.39 (C-H ) 2399.28 (C- H ): 1521.73 and 1490.87 (C=C): 1215.07 and 1045.35 (C-OH )
7 F7 3620.14 ((OH) : 3421.48 0.157 7 (OH) : 3018.39 (C-H ) 2975.96 and 2894.95 (C-H ) ;1215.07 (C- OH )
8 F8 3629.78 (O-H) : 3018.39 (C- 0.084 8 H ) : 2399.28 (C-H ) 1215.07 and 1045.35 (C-OH )
9 F9 3018.39 (C-H ):2974.03 (C-H 0.03 9 ): 2399.28 (C-H ) :1215.07 (C- OH )
The Result of identification of functional group of methanol maceration extracts fraction , many fractions have the same functional group it may be isomers.The
41 functional group detected or identified confirmed the presence of flavonoids in the B.senegalensis seeds which is equal to the results of phytochemical screening .
Table (3-11): Maximum absorption of the nine isolated fractions of methanol maceration :
NO Fraction Max (nm) RF 368.00 1 F1 0.978 364.00 2 F2 0.945 368.00 3 F3 0.896 368.00 4 F4 0.836 368.00 5 F5 0.757 358.00 6 F6 0.412 312.00 7 F7 0.157 310.00 8 F8 0.084 9 F9 306.00 0.03
42
Chapter four
Conclusion and Recommendations
References
4. Conclusion and Recommendations
4.1 Conclusion
In conclusion , the results obtained in the present study are in agreement to certain degree with traditional of the plant estimated .
- The phyto chemical screening on B. Senegalensis seeds extracts demonstrated the presence of different type of compound like carbohydrate , flavonoid , tannin , alkaloids , coumarins and saponins
-GC-MS spectrum for oil extract confirmed the presence of 14 compounds un saturated fatty acids. Which means that the high value of antioxidants due to the presence of these acids .
- Preparative thin layer chromatography of methanol extract gave nine compounds isolated and analysed by infra- red confirmed the presence of ( C-O), (C-H) , (C=O), (C-OH ) and (OH) .
4.2 Recommendations
It is recommended to :
- Use modern spectroscopic techniques such as Nmr and MS for structure elucidation of the pure compounds . - Do further pharmacological studies for macerated methanol extract
43
References
Abdelmuti,O.M. (1991). Biochemical and Nutritional Evaluation of Famine Foods in Sudan. Ph.D. Thesis, University of Khartoum. Ahmed , O.M.M. (1988). Toxicological studies on fruit of Balanites aegyptiaca (Hijlij tree). Ph.D .Thesis , University of Khartoum. Sudan. Anderews, F.W. ( 1950).The flowering plant of the Anglo Egyptian Sudan, Pup.T.BUNCLE and CO.LTD ARBR0ATA.Scotland. Asima , C . and Pakrashi, S.C. (1995) .The treatise on Indian medicinal plants .Vol.4. P: 325. Akanji, M.A., Olagoke, O.A. and Oloyede, O.B. ( 1993). Effect of chronic con sumption of metabi-sulphite on the integrity of the kidney cellular system. Toxicology, 81:173-179. Almagboul,A .Z, Bashir.AK .,Karim.A, Salih.M,Farouk.A, Khalid,S.A. (1988) Antimicrobial activity of certain Sudanese plants used in folkloric medcine. Screening antifungal activity. Fitoterapia.(59) : 393-396 . Bakhiet, A.O. (1995). Comparative effects on chicks of some indigenous plants .Ph.D. Thesis, University of Khartoum, Sudan. Baoua,M.,Fayn,J. and Bessiere. J.M.(1976). Essais Phytochemique preliminatary on some medicinal plant in Niger .Plant Med.10 :251-266. Bohn JA, Be Miller JN. (1995). (1,3)-ß-D-glucans as biological response modifiers: A review of structure-functional activity relationships. Carbohydr. Polym. 28: 3-14. Bullough.C.W,Leary.W.P,1982) Trop.Geograph.Med.34:810 . Burck.P.J.,Thakkar,A.L.,Zimmermann.R.E. (1982). Antifertility action of a sterol sulphateinrabit .Jou Reproduction and fertility .66.109:112 . Cheesbrough, M., (1991). Medical Laboratory Manual for Tropical Countries.University Press, Cambridge.P: 508-511.
44
Chi,H.J. , Choi,J.R.Yu.S.C. (1985) . Pharmacological studies on (Ho_jang). Phytochemical study of the rhizome of poly gonum elliprum Miso korean. Jou.Pharmmacognsy. 13. P: 145_152. Chubb, L.G.(1982). In Recent Advances in Animal Nutrition. W. Harvesign Butterworths, London, p: 21-37 Coles,E.H .(1974) .Veterinary Clinical Pathology .2nd edition. Pub.W.B SaunderCompany USA. Connig, D.M.( 1993). The Basic Issues. Anderson D. and D.M. Conning (Eds.),2nd Edn. P: 1-3. Delaveau ,P.,Koudogbo,B.and Pousset.(1973). Alkaloid from the Capparidaceae. Jou Phytochemistry .12. P: 2893-2895. DFID: The British Department for International Development ,The European Union and the World Agroforestry Centre. (2009) Agroforestree database . Dicko,MH. ,Marjo S., leeuwen,V., Alfred S., Hilhorst, T. R and Beldman, G.(2001). Polysaccharide hydrolases from leaves of Boscia senegalensis Properties of endo-(1→3)-β-d-glucanase . Jour Applied Biochemistry and Biotechnology, Publisher Humana Press Inc. 94: 225-241 Drury, R.A. and Wallington, E.A. (1980). Carleton’s Histological Techniques. 5th ed., Oxford, New York, Toronto. http://www.worldagroforestry.org/af/treedb/index.php?keyword=Poison Duke,H.H,D.V.M.,MS. (1955).The physiology of domestic animals.7th edn .vol 1020 . P: 26_28. ELGaddal, J.A. (1993).Thesis. The use of plant coagulants for water treatment in rural Sudan, University of Surrey . Elgazali. G. E.B, Eltohami .M.S.,, Elegami.A.B.,Abdalla .W.S., Mohammed .M.G.(1997).Medicinal plant of Northern Kordofan .National Center for Researrch( MAPRI) Khartoum. Sudan .Part5 Elgazali, G.E.B., Eltohami .M.S., Elegami.A.B.,Abdalla .W.S., Mohammed .M.G.,(1987).Medicinal plant of Sudan part II. National Center for Researrch( MAPRI) Khartoum.
45
Elgazali, G.E.B., Eltohami .M.S., Elegami,A.B.(1994). Medicinal plant of Sudan part III (Medicinal plant of The White Nile province). National Center for Researrch( MAPRI) Khartoum . ELkhier , Y.M.,Salih,MH. (1980)Investigation of certain plant used in Sudanese folk Medicine. Fitoterapia.143-147. Gupta ,M.B.,North,R.,Srivastava,N.,Shanker,K.,Kishorbhargava,KP.(1980). Antiinflamotory and Antipyretic activity of B sisterol, Plant.Medica., 39:157-163. Grindely,D.N. (1948) .Jou soc,chem. Ind 67-230 Harborne, J.B. (I972). Phytochemical Ecology. 8th Edn., Academic Press Inc., London,NewYork . P :182-195.. Harborne , J. B (1984). Phytochemical methods . 2nd edition. Kerharo, J. ,Adams, J.R. (1974). La pharmacopée sénégalaise traditionnelle, plantes médicinales et toxiques (Vigot Frères eds.),Paris, France Kim, T. R., Pastuszyn, A. (1997). The nutritional composition of seeds from Boscia senegalensis (Dilo) from the Republic of Niger. Jour of Food Composition and Analysis. 10(1): 73-81. {A} Killian , C. (1937) . Contribution à l’étude écologique des végétaux du Sahara et du Soudan tropical. Bull. Soc. Hist. Nat. Afrique du Nord 28:12-18. Kjaer, A .,Schuster,A.,Delaveau, P., koudogbo ,B. (1973).Glucosinolate in Boscia senegalensis Phytochemis12:725-726. Source was an original research paper Laurens ,A .(1985).Pharmazie 40(7):482 Longoay,G ., Marlier,M .,Seck,D., Haubruge ,E.,Wathelet,J.P.,Coulibally A.D., Gaspar ,C.and Severin,M. (1994).Bull.Res.agro.Gembloux,Belgium.:117-124. Malani,T.,Vanithakumari,G.(1991) .The anti fertility effect of in male albino rats. Jour Ethno pharmacology. (35) :14 7_153. Maurice ,I.W.U.(1993).Hand book of tropical plant of Africa. vol 435. p :132-133. CRC press , printed in USA. Mendehall, W. S. (1971). Introduction of Probability Statistics. 3rd ed., Wadswarch Publishing Co., Belmont, California, USA. Monica, C. (1992). Medical Laboratory Manual for Tropical Countries. Butterworth-Heinemann Ltd, UK p: 465-472 .
46
Bos 1
Abs Wavelength(nm) 38133 368833 38261 336833
38196 274833
38125 358833
38159 286833
38237- 256833
Bos 2
Abs Wavelength(nm) 38397 348833 38152 336833
38354 278833
38333 224833
38371 364833
38359 356833
38321 286833
38255- 262833
38426- 256833
Bos 3
Abs Wavelength(nm) 38225 368833 38329 336833
38241 278833
38237 358833
38238 286833
38324- 262833
38172- 256833
Bos 4
Abs Wavelength(nm) 38387 368833 38536 336833
38473 274833
38371 358833
38424 286833
38221 262833
38382 256833 38318- 244833 Bos 5
Abs Wavelength(nm) 38251 368833 38334 336833
38226 274833
38237 358833
38191 286833
38195- 256833
Bos 6
Abs Wavelength(nm) 38376 336833 38329 274833
38381 358833
38234 288833
38466- 248833
Bos 7
Abs Wavelength(nm) 38957 312833 38766 274833
38739 288833
38634 256833
38646 253833
Bos 8
Abs Wavelength(nm)
38721 313833
38563 274833
38486 286833
38514 262833
Bos 9
Abs Wavelength(nm)
18389 336833
18323 274833
38917 286833
38793 256833
38775 253833