i

TITLE PAGE USMANU DANFODIYO UNIVERSITY, SOKOTO

(POSTGRADUATE SCHOOL)

PHYTOCHEMICAL SCREENING AND ANTIBACTERIAL STUDIES OF Euphorbia balsamifera LEAVES (Aguwa)

A DISSERTATION

SUBMITTED TO THE POSTGRADUATE SCHOOL,

USMANU DANFODIYO UNIVERSITY, SOKOTO, NIGERIA

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF

THE DEGREE OF MASTER OF SCIENCE IN APPLIED CHEMISTRY

BY

BELLO LYDIA BASHIR 09211312008

APRIL, 2015.

ii

DEDICATION

This dissertation is dedicated to my late husband Femi Bello Bashir. May your soul

rest in peace, amen.

iii

CERTIFICATION

This dissertation by Bello Lydia Bashir has met the requirements for the award of

the degree of masters of Science in Applied Chemistry of the Usmanu Danfodiyo

University, Sokoto and is approved for its contribution to knowledge.

________________ Prof.S.M.Dangoggo (Major Supervisor) Date ______________ Dr.K.J.Umar (Co-Supervisor I) Date _______________ Prof.M.J.Ladan (Co-Supervisor II) Date

External Examiner Date _______________ Date H.O.D

iv

ACKNOWLEDGEMENTS

I thank God for the grace and opportunity given to me to pursue this work

and accomplish it successfully. I wish to express my sincere gratitude and

appreciation to my Supervisors Prof. S. M. Dangoggo, Dr K. J. Umar and Prof. M.

J. Ladan, for the academic and moral advice I received from them.

My special regards goes to the Head of Department Dr M. U. Dabai who has

been a true shepherd in the course of my studies. To the entire academic Staff of

the Department of Pure and Applied Chemistry, more especially; Prof. A. A. Zuru,

Prof. L. G. Hassan, Prof. U. A. Birnin Yauri, Dr. P. O. Ikeh Dr. A. I. Tsafe, Dr. A. B.

Muhammad, for the academic training given to me may God bless you all

I acknowledge my parents Deacon and Mrs S.A. Onifade for their supports

and advice; to my Siblings Funmi, Nike, Seyi, Femi and Wumi, I’m proud of you all

for your supports and encouragement that have given me serious inspiration all

along and my daughter Triumph you make me whole.

The following personalites cannot be left out for their immense contribution

in my academic pursuit Rev and Mrs J.A. Oyedepo and members of First Baptist

Church Birnin Kebbi for their prayer and contribution. God bless you all.

I cannot end this acknowledgement without mentioning my friends,

Colleagues, Course mates, well wishers such as Faith, Joshua, Musa, Mary, Bola,

Charity, Folake, Bunmi, Mulikat, Sola, my Colleagues at Old Prison Birnin-Kebbi,

Bridget, Aisha, Zainab, Hauwa, Teni, in- charge DCP Aliyu M. Bukar and others

whom their friendship, supports and encouragements have given me serious

inspiration. May God bless you all.

v

TABLE OF CONTENTS

TITLE PAGE ............................................................................................................. i

DEDICATION ........................................................................................................... ii

CERTIFICATION .................................................................................................... iii

ACKNOWLEDGEMENTS ....................................................................................... iv

TABLE OF CONTENTS ........................................................................................... v

LIST OF FIGURES................................................................................................ viii

LIST OF TABLES .................................................................................................... ix

LIST OF PLATE ....................................................................................................... x

ABSTRACT ............................................................................................................. xi

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW ....................................................... 1

1.1 Introduction ................................................................................................... 1

1.2 LITERATURE REVIEW ................................................................................. 2

1.2.1 Description of Plants ..................................................................................... 2

1.2.2 Medicinal Uses of Euphorbia balsamifera ..................................................... 4

1.4 Phytochemicals ............................................................................................. 6

1.4.1 Tannins ......................................................................................................... 7

1.4.2 Saponins ....................................................................................................... 9

1.4.3 Steroids ....................................................................................................... 10

1.4.4 Flavonoids ................................................................................................... 12

1.4.5 Cardiac glycosides ...................................................................................... 14

1.4.6 Anthraquinones ........................................................................................... 16

1.4.7 Alkaloids ...................................................................................................... 17

1.4.8 Terpenoids .................................................................................................. 20

1.5 Soxhlet Extraction ....................................................................................... 24

1.6 Thin Layer Chromatography ........................................................................ 25

1.7 Column Chromatography ............................................................................ 25

1.8 Ultraviolet Spectroscopy .............................................................................. 26

1.9 Infrared (IR) Spectroscopy .......................................................................... 26

vi

1.10 GC – MS (Gas Chromatography – Mass Spectroscopy) ............................. 27

1.11 Justification ................................................................................................. 28

1.12 Aim and Objectives of the work ................................................................... 28

CHAPTER TWO

MATERIALS AND METHODS ............................................................................... 30

2.1 Plant Materials Collection and Identification ................................................ 30

2.1.1 Instruments.................................................................................................. 30

2.1.2 Chemicals ................................................................................................... 31

2.1.3 Preparation of Reagents ............................................................................. 31

2.2 Processing of Plant Samples ...................................................................... 33

2.3 Extraction of Plant Materials ........................................................................ 34

2.3.1 Ethanol Extracts .......................................................................................... 34

2.4 Melting Point................................................................................................ 34

2.5 Phytochemical Screening ............................................................................ 35

2.5.1 Test for Alkaloids ......................................................................................... 35

2.5.2 Test for Saponins ........................................................................................ 35

2.5.3 Test for Steroids .......................................................................................... 35

2.5.4 Test for Anthraquinones .............................................................................. 36

2 .5.5 Test for Flavonoids ...................................................................................... 36

2.5.6 Test for Tannins .......................................................................................... 37

2.5.7 Test for Cardiac Glycosides ........................................................................ 37

2.5.8 Test for Volatile Oils .................................................................................... 37

2.6 Quantitative Phytochemical Analysis ........................................................... 37

2.6.1 Determination of Total Alkaloids .................................................................. 37

2.6.2 Determination of Total Tannins ................................................................... 38

2.6.3 Determination of Total Phenolic Compounds .............................................. 39

2.6.4 Determination of Total Flavonoids ............................................................... 39

2.6.5 Determination of Total Saponins ................................................................. 40

2.7 Fractionation of Ethanol Extract .................................................................. 40

2.8 Tests for Antibacterial Activity ..................................................................... 41

2.8.1 Antibacterial Assay ...................................................................................... 41

vii

2.8.2 Minimum Inhibition Concentration (MIC) ..................................................... 42

2.8.3 Minimum Bactericidal Concentration (MBC) ................................................ 42

2.9 Thin Layer Chromatography ........................................................................ 42

2.10 Column Chromatography ............................................................................ 43

2.11 Isolation of Bioactive Compounds Using Preparative TLC .......................... 43

2.12 Ultra-violet (UV) ........................................................................................... 44

2.13 Infrared Spectroscopy IR ............................................................................. 45

2.14 GC-MS (Gas Chromatography-Mass Spectroscopy) .................................. 45

CHAPTER THREE

RESULTS AND DISCUSSION ............................................................................... 46

3.1 Results ........................................................................................................ 46

3.1.1 Percentage Yields of the Plant .................................................................... 46

3.1.2 Phytochemical Screening ............................................................................ 47

3.1.3 Melting point ................................................................................................ 47

3.1.4 Quantitative Estimation of Phytochemical Contents 48

3.1.5 The Yields of Fractionated Components 49

3.1.6 The Antibacterial Activities .......................................................................... 50

3.1.6.2 Minimum Bactericidal Concentration (MBC) .............................................. 51

3.1.7 Thin Layer Chromatography of Chloroform Fraction of Leaves ................... 52

3.1.8 Results of Ultra Violet/Visible Analysis 53

3.1.9 Infra-red Spectroscopy Analysis .................................................................. 54

3.1.10 GC-MS Analysis .......................................................................................... 55

3.2 Discussion ................................................................................................... 56

CHAPTER FOUR

CONCLUSIONS AND RECOMMENDATIONS ...................................................... 62

4.1 Conclusions ................................................................................................. 62

4.2 Recommendations ...................................................................................... 63

REFERENCES ............................................................................................ 64

APPENDICES ............................................................................................. 70

viii

LIST OF FIGURES

Figure 1.1 Structure of penta-galloyl glucose ...........................................................7

Figure 1.2- Catechin–(4-alpha-8) catechin ...............................................................8

Figure 1.3: Tetracyclic triterpenoids skeleton of saponin (R = sugar component). ...9

Figure 1.4: Pentacyclic triterpenoids skeleton of saponins ..................................... 10

Figure 1.5 Stigmasterol (Plant).............................................................................. 11

Figure 1.6 Cholesterol (Animals) .......................................................................... 11

Figure 1.7 Ergosterol (Fungus) .............................................................................. 12

Figure1.8: Skeleton structure of flavonoids ............................................................ 13

Figure1.9: Skeleton structure of isoflavonoids ....................................................... 13

Figure: 1.10: Skeleton structures of neoflavonoids ................................................ 14

Figure 1.11 Cardiac glycoside (R=Glycone) ........................................................... 15

Figure 1.12 (an S-Glycoside) ................................................................................. 15

Figure1.13 Anthracenedione .................................................................................. 16

Figure 1.14: Structure of Ephedrine ....................................................................... 18

Figure1.15 Structure of hygrine .............................................................................. 18

Figure 1.16 Structure of coniine ............................................................................. 19

Figure 1.17 Structure of nicotine ............................................................................ 19

Figure 1.18: Structure of Quinine ........................................................................... 19

Figure 1.19 Structure of papaverine ....................................................................... 20

Figure 1.20 Structure of gramine............................................................................ 20

Figure 1.21 Structure of limonene .......................................................................... 21

Figure 1.22 Structure of curcumenenene ............................................................... 22

Figure 1.24 Ceroplastol .......................................................................................... 23

Figure1.25 Structure of Gonane ............................................................................ 23

Figure 1.26 Structure of lycopene . ........................................................................ 24

Figure 3.1 6H-Indolo [3, 2, 1-de][1,5]naphthyridin-6-one ..................................... 61

ix

LIST OF TABLES

Table 2.1 List of instruments .............................................................................. 30

Table 3.1: Percentage Yields of the Plant ........................................................... 46

Table 3.2: Preliminary Phytochemical Screening of Ethanolic Extracts of

Euphorbia balsamifera leaves, stem and root .................................. 47

Table 3.3: Quantitative Estimation of` Phytochemical Contents of the

Chloroform Extract of the Leaves of Euphorbia balsamifera .............. 48

Table 3.4: Results of the Yields of Fractionated Components using various

Solvents ............................................................................................. 49

Table 3.5: Zone of Inhibition Results (mm) .......................................................... 50

Table 3.6 Minimum Inhibition Concentration of Tested Organism ...................... 51

Table 3.7 Minimum Bactericidal Concentration of Tested Organism .................. 51

Table 3.8 TLC of Chloroform Fraction ................................................................ 52

Table 3.9: Ultra Violet /Visible Spectroscopy Analysis Results ........................... 53

Table 3.10: Infra-red Spectroscopy Analysis Results ............................................ 54

Table 3.11: GC-MS Analysis Results .................................................................... 55

x

LIST OF PLATE

Plate1.1 a photograph showing section of Euphorbia balsamifera plant ..................3

xi

ABSTRACT

Euphorbia balsamifera plant belongs to the family of Euphorbiaceae. Leaves, stems and roots of Euphorbia balsamifera were extracted using ethanol and the extracts were screened for phytochemical components. The phytochemical results indicated that the plant contained tannins, flavonoids, saponins, steroids, glycosides, terpenoids, alkaloids, and anthraquinones. Ethanol extract of leaves and its fractions were tested for antibacterial activity using agar well diffusion method and were found to show inhibitory activity against Escherichia coli, Staphylococcus aureus, Micrococcus species and Pseudomonas aeruginosa. The sensitivity test results showed highest activity of 25mm zone of inhibition at 120mg/ml of chloroform fraction against Micrococcus species. The MIC and MBC of the chloroform fraction were 5mg/ml and 7mg/ml respectively. The TLC of chloroform fraction of leaves showed six components of different Rf value using benzene: ethyl acetate (3:1) solvent system. The chloroform fraction was further subjected to column chromatography, after which preparative TLC was carried out on the fraction obtained from column. The isolated compound was subjected to IR, UV and GC-MS Spectroscopy which revealed the presence of (6H-Indolo[3,2,1-de][1,5]naphthyridin-6-one) and fatty acid in the isolated compound. Therefore the bioactive of Euphorbia balsamifera leaves can be served as a lead for the development of new pharmaceutical therapeutic needs.

1

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

1.1 Introduction

Plants have long served mankind as sources of food, shelter and medical

agents. The medicinal plants have been used for many years in daily life to treat

diseases all over the world (Ates and Erzdogrul, 2003). According to reports of the

World Health Organisation, 80% of the world’s population relies mainly on

traditional therapies for their primary health care (WHO, 1993) because of better

cultural acceptability, fewer side effects and a steady rise in antibiotic resistance of

bacteria due to indiscriminate use of commercial drugs (Ahmed et al., 1998) and

these urgently calls for the discovery of alternative therapeutic agents by using

medicinal plants, which involve the use of plant extracts or their active substance.

Secondary plant metabolites (phytochemicals) have been extensively investigated

as sources of medicinal agents (Krishnaraju et al., 2005). The medicinal value of

plants lies in some chemical substances that produce definite physiological action

on the human body and the most important of these bioactive compounds of plants

are alkaloids, flavonoids, tannins and phenolic compounds, they have defence

mechanism and protection from various diseases (Chidambara et al., 2003).

Knowledge of the chemical constituents of plants is very important not only for the

discovery of drugs and other therapeutics agents but also in disclosing new

sources of such economic materials as tannins, oils, gums and precursors for the

synthesis of complex chemical substances (Mojab et al., 2003).

2

1.2 LITERATURE REVIEW

1.2.1 Description of Plants

Euphorbia balsamifera belongs to the family Euphorbiaceae commonly

known as balsam spurge in English and espurge in French; it is native to all

Canary Islands and is also present in West Africa including Senegal and Nigeria

popularly known as aguwa among Hausa in Nigeria (Burkill, 1985). The botanical

name Euphorbia derives from Euphorbous, the Greek Physician of king Juda II of

Numidia Botanist and Taxonomist Carl Linnaeus assigned the name Euphorbia to

the entire genus in honour of the physician (Nancy Dele, 1986). Euphorbia is

having almost 2000 species among which are Euphorbia albomarginata, Euphorbia

amygdaloides, Euphorbia decidue, Euphorbia elastica etc. (Bruyns et al., 2006)

These plants are found in dense community in rocky places and on less

mobile sand dune (Bruyns et al., 2006). The plants are annual or perennial herbs,

woody shrubs with caustic, poisonous milky sap latex. Euphorbia balsamifera plant

is thick, short and hardly rising above the ground to small trees up to five meter tall.

The plant is commonly grown as a hedge and field boundary marker in the

northern part of Nigeria. It is easily raised by cutting and is said to be one of the

best hedge plants for low rainfall area. The plant’s stem is thick, fleshy, semi-

succulent, spineless with bases and becoming very thick at minimum injure. It

gives out pungent smelling white latex. The leaves are long oval shaped green to

glaucous cluster at branches tip; the dioecious leave may be opposite or alternate.

The root is fine or thick and fleshy or tuberous. Euphorbia flower are tiny and the

3

collection of many flowers may be shaped and arranged to appear collectively as a

single individual flower. Majority of species are monoecious (bearing male and

female flower on the same plant) although some are dioecious with male and

female flower occurring on different plants. The fruits are three compartment

capsules, sometime fleshly but almost ripening to a woody container that then

splits open, the seed are oval or spherical in shape (Carter and Smith 1987).

Plate1.1 a photograph showing section of Euphorbia balsamifera plant

4

1.2.2 Medicinal Uses of Euphorbia balsamifera

Euphorbia balsamifera is used as a traditional antalgic in the treatment of

acute dental pulpits and as anti diabetic (Rau et al., 2006).

The latex has an anti eczema property and it is also used as antidotes for

venomous (snake) bites (Burkill, 1985). It promotes fertility and milk production in

cattle (Lin et al., 1983). The leaves and latex are used for insect bites and

treatment of erysipelas, cough, bronchial paroxysmal asthma, hay fever and

catarrh (Gill, 1992).The leaves are used in treating skin irritation. It promotes

healing in cases of fever by facilitating the production of platelets. It’s also known

for its antihelmintic properties and can be used to get rid of worms and other

parasitic organism (Burkill, 1985). Leaf also boost breast milk production in

lactating mothers and is used in treatment of venereal disease like gonorrhea,

impotence, premature ejaculation and other sexual disorders (Burkill, 1985).

The root of Euphorbia balsamifera can be made in paste and used for

healing stomach pain; it possesses antiviral properties and used in the treatment of

dysentery and diarrhea. In Chinese medicine, the herb is used as a diuretic and

laxative, it’s believed to expel water from the body and thus reduce edema and

inflammation on lymph nodes (Burkill, 1985). Euphorbia was reported to have anti-

bacterial activities against pathogenic strain of gram-positive and gram-negative

bacteria (Natarajan et al., 2005). Its ethanolic extract has anti-inflammatory

properties (Kamba and Hassan 2010).

5

1.3 Antimicrobial Agents

Plant medicines are the most widely used medicines in the world today.

The pharmaceutical industries have come to consider traditional medicine as a

source for identification of bioactive agents that can be used in the preparation of

synthetic medicine (Robbers et al., 1996). Many commercially proven drugs used

in modern medicine, were initially used in crude form in traditional healing

practices, or for other purposes that suggested potentially useful biological

activities. The primary benefits of using plant derived medicines are that, they are

relatively safer than synthetic alternatives, offering profound therapeutic benefits

and more affordable treatment (Donal, 2000).

Antimicrobial agents include naturally occurring antibiotic, synthetic

derivatives of naturally occurring antibiotic and chemical antimicrobial compounds.

Generally however the term antibiotic are used to described antimicrobial agents

(usually antibacteria) that can be used to combat infection (chemotherapy) and the

fundamental principle of chemotherapy, its collective toxicity (Cheesbrough, 2002).

Antibacteria is a drug that kills or slows down the growth of bacteria, it was labelled

magic bullet drugs which targeted disease without harming the host. Conventional

antibiotic are not effective in viral, fungal and other non-bacterial infection and

individual antibiotics can be categorized based on their target specific narrow-

spectrum antibiotics target particular types of bacteria such as gram-negative or

gram-positive bacteria, while wide-spectrum antibiotic affect a larger range of

bacteria. (www.en.wikipedia.org/wiki/antibiotic retrieved on 7/4/2006).

6

The effectiveness of individual antibiotics varies with the location of the

infection, the ability of the antibiotic to reach the side of infection and the ability of

bacteria to resist or inactivate the antibiotic. Some antibiotics usually kill the

bacteria (bactericidal) whereas others merely prevent the bacteria from multiplying

(bacteriostactics) so that the host immune system can overcome them

(www.en.wikipedia.org/wiki/antibiotic retrieved on 7/4/2006

1.4 Phytochemicals

Phytochemicals are chemical compounds produced by plants that have

pharmacological importance to human health’s. It is the studies of plant physiology

by determine the chemical structure of different plants constituents. It is concerned

with the enormous variety of organic substance that is elaborated accumulated by

plants. They are divided into two groups, namely primary and secondary

metabolites (Donal, 2000).

(a) Primary metabolites: Primary metabolites are organic compounds that are

directly involved in the normal growth, development or reproduction of organism,

their absences may result in immediate death of the organism e.g. proteins, fats,

etc

(b) Secondary metabolites: Secondary metabolites are synthesized during

secondary metabolism of plants, they work as biocatalysts they are the basic

source for the establishment of several pharmaceutical industries since they have

enormous medicinal properties many of these are known to provide protection

7

against insect attacks and plant diseases Examples are: tannins, saponins,

flavonoids, alkaloids and phenolic compounds (Harborne, 1999).

1.4.1 Tannins

Tannins are defined as substances of plant origin which because of their

ability to cross-link with protein are capable of transforming raw animal skin into

leather (Zaki, 2000). They are non-crystallizable complex compounds, which

normally form colloidal solution in water. They are usually of high molecular weight

(1000 - 5000) with the ability to react with protein forming stable water insoluble

copolymers. They are located in the vacuoles or surface wall of the plants. Tannins

are usually divided into hydrolysable and condensed tannins (Pro anthocyanidins)

(Calvi et al., 1995).

(a) Hydrolysable tannins: Hydrolysable tannins are esters of gallic acid.

Glycosides of these esters are hydrolyzed upon heating with dilute acid, giving

glucose and gallic acid, gallitannins or ellagitannins (Zaki, 2000). A structural unit

of hydrolysable tannin is shown in figure 1.1 (Zaki, 2000).

CH2OR

H

OR

H

H

RO

OR

OR

H

O

Figure 1.1 Structure of penta-galloyl glucose

8

(b) Condensed tannins: Condensed tannins are polymers derived from various

flavonoids. They do not hydrolyze on heating with dilute acids but oxidize and

polymerize giving insoluble red amorphous precipitate (El-Olemy, et al., 1994).

They are polymers of 2 to 50 (or more) flavonoid units that are joined by carbon

bonds. A structural unit of condensed tannin is shown in figure 1.2 (Zaki, 2000).

O

O

OH

OH

OH

OHOH

OH

OH

OH

OH

Figure 1.2- Catechin–(4-alpha-8) catechin

Uses

Tannins play an active anti nutritional role in the body where they affect the

gastrointestinal tract, interfere with the absorption of iron and with a possible

carcinogenic effect (Ademoroti, 1996). It also shows potential antiviral, antibacterial

and anti parasitic effects (Calvi et al., 1995). It is very important ingredient in the

process of tannin leather e.g. oak bark is a source of tannery tannins.

9

1.4.2 Saponins

Saponins are secondary plant metabolites that occur in a wide range of

plant species. They are group of steroidal glycosides that has the properties of

forming foam in water like soap (Finar, 1975). As glycosides they are hydrolyzed

by acid to give an aglycone (sapogenin) and various sugar related uranic acid

(Figen, 2005).

Classification of Saponins

Saponins are classified into two major groups; steroidal glycoside,

commonly called tetracylic triterpenoids and triterpenoid saponins called

pentacyclic triterpenoid. Saponins have bitter acid taste and drugs containing them

are usually sternutatory and irritating to the mucous membrane of eye and nose.

All saponins have ability to cause haemolysis of red blood corpuscles or cells

(RBC) and destroy them (Trease & Evan, 1989).

O

R

O

Figure 1.3: Tetracyclic triterpenoids skeleton of saponin (R = sugar component).

10

O

R

Figure 1.4: Pentacyclic triterpenoids skeleton of saponins

Uses of saponins

Saponins were reported to have many health benefits including effects on

blood cholesterol levels, cancer, bone and stimulation of the immune system

(Nassiri and Hosseinzedeh, 2008).

1.4.3 Steroids

The term steroid applies to compounds containing a hydrogenated

cyclopentanophenanthrene carbon skeleton. Most steroids are alcohols, and

accordingly are named as sterols. Important examples include cholesterol,

ergosterol, estradiol, and stigmasterol. `As you can see from their structures, most

possess the same ring skeleton but vary considerably in their peripheral structural

features, stereochemistry, and in the degree of ring unsaturation. Sterols are

widely distributed in plants, fungus and animals. Many are of vital importance to

animal physiology such as cholesterol, the bile acids, vitamin D, sex hormones,

and corticoid hormones. Many are medicine, such as cardiac glycosides,

hormones, and steroidal antibiotics. Examples of steroids include the dietary fat

11

cholesterol, the sex hormones; estradiol, testosterone, and the anti-inflammatory

drug, dexamethasone (Dussourd, 2000).

Classification of Steroids

Steroids are classified in to three based on their source as follows:-

(a) Animal steroids, example insect steroids: such as ecdysteroid, vertebrate

steroids: such as steroidal hormones (e.g. sex steroids like progesterone,

testosterone, estrone, cortisone and androsterone) and cholesterone.

(b) Plant steroids, example stigmasterols.

(c) Fungus steroids or mycosteroids, example ergosteroids (Dusssourd,

2000).

C2H

5

HO

Figure 1.5 Stigmasterol (Plant)

H

OH

H

C8H

17

Figure 1.6 Cholesterol (Animals)

12

CH3

OH

CH3

CH3CH

3 CH3CH

3

Figure 1.7 Ergosterol (Fungus)

Uses of steroids

Plants and fungus steroids are used in pharmaceuticals as medicine especially

Vitamin D2 formed by ultraviolet radiation of ergosterol from yeast is used to

regulate heart action. Some steroids are naturally occurring in plants and animals,

as hormones to regulate and develop sex and reproductions (Dussourd, 2000). β-

sitosterol reduces carcinogen-induced cancer of the colon. It shows anti-

inflammatory, anti-pyretic, antiarthritic, anti-ulcer, insulin releasing and oestrogenic

effects and inhibition of spermatogenesis. β-sitosterol is mainly known and used for

its cholesterol lowering property (Arjun et al., 2010). But studies have shown that

the phytochemicals may have other health benefits: such as easing symptoms of

prostatic enlargement, reducing risk of cancer and prevention of oxidative damage

through its antioxidant activity (Dussourd, 2000).

1.4.4 Flavonoids

Flavonoids are the largest group of naturally occurring phenols and occur in

the plant both in the free state and as glycoside. They consist of 15 carbon atoms

13

arranged in C6 – C3 – C6 orders. They are phenolic compounds consisting of two

benzene rings linked together through a heterocyclic pyrol-ring (Okeke, 2005).

Flavonoids are more abundant in leaves and flowers; they are responsible for

colour appearance in the petal of flower and leaves of plants (Herbone, 1984).

Types of flavonoids

They are categorized into three major classes depending on the position of

the linkage of the aromatic ring to the benzopyrans (chromano) moiety; 1.the

flavonoids (2-phenylbenzopyrans) 2.isoflavonoids (3-benzopyrans) and

3.neoflavonoids (4-benzopyrans) 3. These groups usually share a common

chalcone precursor, and therefore are biogenetically and structurally related.

(Erich, 2006)

O

Figure1.8: Skeleton structure of flavonoids

O

Figure1.9: Skeleton structure of isoflavonoids

14

O

Figure: 1.10: Skeleton structures of neoflavonoids

Uses of flavonoids

Flavonoids prevent cancers and cardiovascular diseases. Although

physiological evidence is not yet establishedthe beneficial effects of fruits,

vegetables and tea or even red wine have been attributed to flavonoid compounds.

Flavonoids are known for their antioxidant activity in vitro and they also protect

plant from attacks by microbes, fungi and insects (Njoku and Akumefula, 2007).

1.4.5 Cardiac glycosides

Cardiac glycosides are plant steroids which occur as glycosides, they are

colourless non-volatile crystalline and mostly bitter tasting solid compounds.

Chemically, glycosides are group of organic compound which can be resolved by

hydrolysis into sugar component (glycone) and other organic substances known as

aglycones. They have the properties of stimulating heart muscles and are

sometimes referred to as the cardiac active or cardiotonic glycosides (Zaki, 2000).

Cardiac glycosides are used as diuretic and heart tonics. Cardiac glycosides are

found in seeds, leaves, stem, roots or barks of plant of wide geographical

distribution in which tobacco plant is one of them (Conn, 1981).

15

Types of cardiac glycosides

There are two types of cardiac glycosides namely:

(a) Cardenolides: This is the most common type having an unsaturated lactone

ring; the structure is shown in figure 1.11

O

OR

O

Figure 1.11 Cardiac glycoside (R=Glycone)

(b) Scilladienolides: This is the less common one, with a ring which has a

conjugated system (Finar, 1975) (an S– glycoside) the structure is shown in figure

1.12 below.

O

CH2OH

H

H

OH

H

OH

OH

HH

S CH

CH2 - CH = CH

2

N - O - SO2 - OK

Figure 1.12 (an S-Glycoside)

Uses of cardiac glycoside

Cardiac glycoside are used therapeutically mainly in the treatment of cardiac

failure due to their anti-arrhythmic effects. These are caused by the ability to

16

increase cardiac output by increasing force of contraction by prolonging the plateau

phase of cardiac depolarization thus slowing ventricular filling (Harbone, 1984).

1.4.6 Anthraquinones

Anthraquinones are tricyclic in structure, having weak reducing properties

which may account for the use of anthranols and anthrones as antiseptic in certain

skin diseases (Balbaa, 1976). They are one of the four major groups of quinones

that have phenolic properties. They may occur either in combined form with sugar

as glycoside or in a colourless dimeric quinol form (Harbone, 1984).

Anthraquinones occurred most frequently in plants. Emodin is an example of

anthraquinones that is widely distributed in many plant families the structure is

shown in figure 1.13 below (Harbone, 1984).

O

O

Figure1.13 Anthracenedione

Toxicity of Anthraquinones

Anthraquinones are toxic as other groups of glycosides like saponins and

cardiac glycosides. It damages the red blood cells, destroys the heart muscles, and

increases the blood pressure and also leads to sudden death (Harbone, 1984).

17

Uses of Anthraquinones

They are used in the production of dye such as alizarin. They are used as

catalysts in the production of chemical pulp, repellant on seeds and also used as a

laxative (Witte 1993)

1.4.7 Alkaloids

Alkaloids are organic nitrogenous compounds that have complex molecular

structure and show significant pharmacological activity. They have bitter taste and

most of them are basic in nature and contain nitrogen in heterocyclic ring (Bahl and

Bahl, 1980).

Alkaloids occur naturally in seeds, root, leaves and barks of plants, they

generally occur as salts of various plant acids like acetic oxalic acid etc. (Finar,

1975). They are colourless, crystalline, non-volatile solid which are insoluble in

water but soluble in ethanol, ether and chloroform. Some are liquid which are

soluble in water e.g. conine and nicotine, a few are coloured e.g. berberine is

yellow (Finar, 1975).

Classification of Alkaloids

Alkaloids have been classified based on source or origin, nucleus present,

structures, pharmacological activities and chemical nature. The most chemically

satisfactory way to classify alkaloids is according to the nature of the nucleus

present in the molecules. Some of the types are:

18

(a) Phenyl ethyl amine group e.g. Ephedrine, many compounds of this group are

naturally occurred, their outstanding physiological action is to increase the blood

pressure hence they are often referred to as the pressure drug. Ephedra is one of

the most important drugs in action similar to that of adrenaline and can be taken

orally. It is used in treatment of hay fever and asthma. The molecular formular is

C10H15N (Finar, 1975) the structure is shown in Figure 1.14.

CH3

N

CH3

CH3

Figure 1.14: Structure of Ephedrine

(b) Pyrrolidine group e.g. Hygrine is one of the coca alkaloids, its reaction shows

the presence of keto group and tertiary nitrogen atom, the structure is shown in

Figure1.15

N CH2 COCH

3

CH3

Figure1.15 Structure of hygrine

(c) Pyridine and piperidine group e.g. Coniine, it is the first alkaloid to be

synthesized; the structure is shown in Figure 1.16

19

N

H

Figure 1.16 Structure of coniine

(d) Pyrrolidine – pyridine group: Many alkaloids have been isolated from

the tobacco leaf e.g. nicotine (C10H14N2). It is best known and most widely

distributed of the tobacco alkaloids, it occurs naturally. The structure is shown in

Figure 1.17.

N

CH3

Figure 1.17 Structure of nicotine

(e) Quinoline group e.g. Quinine: Quinine (C2OH24N2O2) is used as anti malaria,

the structure is shown in Figure 1.18 below

OHN

CH3O

Figure 1.18: Structure of Quinine

20

(f) Isoquinoline e.g. papaverine. Isoquinoline is a type of alkaloids that have been

isolated from opium e.g. papaverine, the structure is shown in Figure 1.19 below.

N

CH2

CH3O

CH3O

CH3O

CH3O

Figure 1.19 Structure of papaverine

(g) Indole group e.g. Gramine (C11H14N2) has been found in barley mutant. It

raises the blood pressure in dogs when administered in small doses.The structure

is shown in Figure 1.20 below.

N

CH2N(CH

3)2

H

Figure 1.20 Structure of gramine

1.4.8 Terpenoids

Terpenoids are groups of organic natural product compounds which occur

widely in both plants and animals. They can be found in nature either in their free

forms, or conjugated with one or more sugar units through the hydroxyl group at

21

the C-3 (Morrison and Boyd, 2007). They are produced by the cyclization of

squalene epoxide in different arrangements. It comprises framework of five-carbon

isoprene units. The isoprene unit is considered as one of nature’s favourite building

block in terpene’s biosynthesis. The isoprene unit is 2-methylbuta-1, 3-diene.

Isoprene units can easily be recognized by ‘’ head- to- tail’’ bonds, as well as

supplementary bonds. Isoprenes are compounds which appear to be formed by the

polymerisation of multiple unit of isoprene (Morrison and Boyd, 2007).

Classification of Terpenes

Terpenoids can be classified based on the number of isoprene units (methybuta -1,

3 – diene) incorporated in the basic molecular skelton (Morrison and Boyd, 2007).

(a) Monoterpenes

They are 10 carbon compounds derived by assembly of 2 isoprene units. Terpenes

have been known for several centuries as components of fragrant oil obtain from

leaves, flowers and fruits. The structure is shown in Figure 1.21 below.

Figure 1.21 Structure of limonene

22

(b) Sesquiterpenes

They are 15 carbon compounds derived by assembly of 3 isoprene units

and they are found mainly in higher plants e.g. curcumene the structure is shown in

Figure 1.22

Figure 1.22 Structure of curcumene

(c) Diterpenes: They are 20 carbon compounds derived by assembly of 4 isoprene

units. They are found mainly in fungal or plant e.g. abietadiene. The structure is

shown in Figure 1.23

H

Figure 1.23: Abietadiene

23

(d) Sesterpenes

They are derived from geranyl farnesol pyrophosphate by assembly of 5

isoprene units and have 25 carbon atoms. They are isolated from insect protective

waxes and from fungal sources (Zaki, 2000)

CH2OH

Figure 1.24 Ceroplastol

(e) Triterpenes: They form a large group of natural substances which includes

steroids and consequently sterols (Zaki, 2000). They have 30 carbon atoms and 6

isoprene units

R

CH3

H

CH3

H

Figure1.25 Structure of Gonane (Zaki, 2000)

24

(f) Carotenes: Carotenoids are derivatives of lycopene found in tomatoes, fruits

and flowers. Its long chain is highly unsaturated.

Figure 1.26 Structure of lycopene (Balbaa, 1976).

1.5 Soxhlet Extraction

When a compound of low solubility needs to be extracted from a solid

mixture a soxhlet extraction can be carried out. A soxhlet extractor is a piece of

laboratory apparatus that was originally designed for the extraction of a lipid from a

solid material. However it is not limited to the extraction of lipid but is required

when the desired compound has a limited solubility in a solvent and the impurity is

insoluble in that solvent. Normally a solid material is placed in a porous bag or

“thimble” made of strong filter paper, which is placed in chamber of the soxhlet

apparatus. The extracting solvent in round bottom flask is heated, and its vapors

condense in condenser. The condensed extractant drips into the thimble containing

the solid, and extracts it by contact. When the level of liquid in chamber rises to the

top of siphon tube, the liquid contents of chamber siphon into the round bottom

flask. This process is continuous and is carried out until a drop of solvent from the

siphon tube does not leave residue when evaporated (Taylor, 1996). The

advantage of this method, compared to other methods, is that large amounts of

25

solid can be extracted with a much smaller quantity of solvent (Abraham and

Sunol, 1997).

1.6 Thin Layer Chromatography

Thin layer chromatography is a chromatographic technique used to separate

mixture (Reich and Schiba, 2007). It is performed on a sheet of glass, plastic or

aluminium foil which is coated with a thin layer of adsorbent material, usually silica

gel, aluminium oxide or cellulose. This layer of adsorbent is known as the

stationary phase. After the sample has been applied on the plate, a solvent mixture

(mobile phase) is drawn up the plate via capillary action because different

compound ascend the thin layer chromatography plate at different rates,

separation is achieved. The more strongly a component of a mixture is absorbed

onto the stationary phase, the less time it will spend in the mobile phase and the

more slowly it will migrate up the plate (Skoog et al., 2004).

1.7 Column Chromatography

Column chromatography in chemistry is a method used to purify individual

chemical compounds from a mixture of compounds. There is dry and wet method,

for the wet method slurry is prepared of the eluent with the stationary phase

powder and then carefully poured into the column. Care must be taken to avoid air

bubbles. A solution of organic materials is pipette on top of stationary phase.

Eluent is slowly passed through the column to advance the organic materials. The

individual components are retained by the stationary phase differently and

separate from each other while they are running at different speeds through the

26

column with eluent and collected in a series of fractions. The composition of the

eluent flow can be monitored and each fraction is analyzed (Harrison, 2003).

1.8 Ultraviolet Spectroscopy

Ultraviolet Spectroscopy is one of the most important quantitative

spectroscopic techniques. The wavelength ranges from about 190nm to 750nm

which corresponds to electronic transitions of different origins. The ultraviolet (UV)

region scanned is normally from 200 to 400nm and the visible portion is from 400

to 800nm (Skoog, et al., 2004).

1.9 Infrared (IR) Spectroscopy

Infrared (IR) Spectroscopy is a chemical analytical technique, which

measure the infrared intensity versus wavelength. Infrared used for IR

spectroscopy falls between 400 and 4000cm-1 When an infrared light interacts with

matter chemical bonds will stretch, contract and bend, as a result a chemical

functional group tends to absorb infrared radiation in a specific wave number

range, regardless of the structure of the test molecular. The wave number positions

where functional groups absorb are consistent despite the effect of temperature,

pressure, sampling or change in the molecular structure in other parts of the

molecule (Gary, 2004).

In infrared spectroscopy, an infrared radiation is passed through a sample,

some of the infrared radiation is absorbed by the sample and some of it is passed

through (transmitted). The difference between the incident and transmitted

27

radiation is recorded by the machine and displayed as absorption bonus (peaks)

which result in a positive identification (qualitative analysis) of every different kind

of compounds and the size of the peaks in the spectrum is a direct indication of the

amount of compounds present (Gary, 2004).

1.10 GC – MS (Gas Chromatography – Mass Spectroscopy)

Gas chromatography mass spectroscopy: GC-MS is one of the so called

hyphenated analytical techniques. It is two techniques that are combined to form a

single method of analyzing mixture of chemicals. Gas chromatography separates

the component of mixture and mass spectroscopy characterized each of the

components individually by combining the two techniques. An analytical chemist

can both qualitatively and quantitatively evaluate a solution containing chemicals

(Skoog, et al., 2004). When GC is combined with MS, a powerful analytical tool is

created. Organic solution injected into the instrument will be separated into

individual components and identified. As individual compounds elute from GC

column they enter the mass spectrometer where they are bombarded with a

stream of electrons causing them to break apart into fragments. The mass of each

fragment divided by the charge is called the mass to charge ratio (M/Z). The

computer on GC – MS has a library of spectra that can be used to identify an

unknown chemical in the sample mixture based on its fragmentation characteristics

(Khopkar, 2008).

28

1.11 Justification

It is well known that infectious diseases account for high proportion of health

problems especially in the developing countries. Microorganisms have developed

resistance to the treatment of infectious diseases (Donal, 2000). This resistance

has increased due to indiscriminate use of commercial antimicrobial drugs

commonly used in the treatment of infectious diseases. This situation forced

various Scientists to search for new antimicrobial substance from various sources,

such as medicinal plant (Ahmed et al.,1998 ) and make entire world to shift its

interest to the development of alternative or new drug from medicinal plant (Abdul,

1990). Therefore this research work deemed it important to investigate the active

bioactive component of Euphorbia balsamifera with the view to verifying the

traditional claims of its medicinal activities for treating insect bites, venereal

diseases, cough, hay fever and boosts breast milk production (Burkill, 1985) and

identification of the active compounds for further development into full drug for the

benefit of all.

1.12 Aim and Objectives of the work

The main aim of this work is to extract, identify, assess the invitro antibacterial

activity, isolate and characterize the bioactive compounds in Euphorbia

balsamifera plant.

29

The objectives are:-

1. To extract the roots, stem and leaves of Euphorbia balsamifera with ethanol.

2. To conduct preliminary phytochemical screening of the ethanol extracts

3. To test for the antibacterial activity of the crude extract and fractions

4. To isolate bioactives compound(s)

5. Characterize the compound(s) isolated from Euphorbia balsamifera plant

UV, IR and GC-MS techniques

30

CHAPTER TWO

MATERIALS AND METHODS

2.1 Plant Materials Collection and Identification

The plant sample of leaves stems and root of Euphorbia balsamifera was

obtained from Birnin-Kebbi in Kebbi state, Nigeria during the month of August

2013. The leave of plant was identified at the Taxonomy unit of the Department of

Biological Sciences Usmanu Danfodiyo University, Sokoto, Nigeria by A.M. Umar.

A Voucher Specimen No (UDUH/ANS/0039) was deposited at the Herbarium of

Botany Unit Department of Biological Science.

2.1.1 Instruments

The instruments used in this research work are listed in Table 2.1 blow in

addition, the routine laboratory glass wares, common apparatus were also used

Table 2.1 List of instruments

Name of Instruments Model Manufacturers Source

Infrared

Spectrophotometer

FTIR-8400S Shimadzu, Japan Central Lab. Udus

Gas-

Chromatography/Mass-

Spectrometry

G890N Agilent

Technologies

Central Lab. Udus

Ultraviolet Visible UV 20550 Shimadzu, Japan Central Lab. Udus

Oven BS OV-160 Gallen Kamp,

England

Central Lab. Udus

Weighing balance PM 16-K Mettler Central Lab. Udus

Soxhlet extractor 24/29 Joint Quick fit brant

Germany

Central Lab. Udus

31

2.1.2 Chemicals

The chemicals used for this research work were of laboratory and analytical grades

as shown in Appendix 1

2.1.3 Preparation of Reagents

(a) Mayer’s Reagents

HgCl2 (1.3g) and KI (5g) were added into 6cm3 and 10cm3 of distilled water

respectively. The two solutions were then mixed and diluted to 100cm3 with distilled

water (El-Olemay et al., 1994).

(b) Dragendoff’s Reagents

Bismuth-nitrate (0.5g) was dissolved in 2cm3 of conc. HCl and diluted with 10cm3

of distilled water. 5.0g of KI was dissolved in 10cm3 of distilled water. The two

solutions were mixed together with 7cm3 conc. HCl then 15cm3 of water were

added and stirred. The solution was then made up to 100cm3 in a volumetric flask

(El-Olemay et al., 1994).

(c) Wagner’s Reagent

Iodine (1.3g) and 2.0g of KI were dissolved in 5cm3 of distilled water and diluted to

100cm3 in a volumetric flask (El-Olemay et al., 1994).

32

(d) Hydrochloric Acid (1%v/v) solution.

Conc. HCl (2.3cm3) of 36% purity and 1.19 density was diluted to 100cm3 in a

volumetric flask with distilled water (El-Olemay et al., 1994).

(e) Sodium Hydroxide (20%w/v) Solution Sodium hydroxide pellet (20%) was

dissolved in 50cm3 of distilled water in a beaker, allowed to cool, transferred to

100cm3 volumetric flask and made up to the mark with distilled water.

(f) Lead acetate (10%w/v) solution

The solution was prepared by dissolving 10g of lead acetate in 20cm3 of distilled

water and the content transferred into 100cm3 volumetric flasks and made up to the

mark.

(g) Ferric Chloride 5% (w/v) Solution

Ferric chloride (1.41g) was dissolved in 20cm3 of distilled water and the solution

was made up to the mark of volumetric flask.

(h) Acetic acid 5% v/v solution

The solution was prepared by adding 5cm3 of glacial acetic acid into 95cm3 of

distilled water in volumetric flask and mix thoroughly.

33

(i) Ammonia Solution 10% v/v

It was prepared by diluting 10cm3 of ammonia stock reagent in 30cm3 distilled

water, and transferred into 100cm3 volumetric flasks then made up to the 100cm3

mark of volumetric flask.

(j) Sodium Carbonate (10% w/v)

sodium carbonate (10g) was dissolved in 50cm3 of distilled water in a beaker,

transferred to a 100cm3 volumetric flask and then made up to the mark of

volumetric flask.

(h) Folin-Denis Reagent

Phosphomolybdic acid (10g), 50g sodium tungstate and 25cm3 orthophosphoric

acid were dissolved in 375cm3 of distilled water and it was refluxed for 2 hours, the

solution was allowed to cool and made up to the mark of 500cm3 volumetric flask

with distilled water (Gabriel et al.,2014)

2.2 Processing of Plant Samples

The fresh plant samples were properly washed with tap water and air dried

under shade. The dried samples were then pulverized in a wooden mortar and

stored in polythene bag for analyses (African pharmacopoeia, 1985).

34

2.3 Extraction of Plant Materials

2.3.1 Ethanol Extracts

The leaves (300g), stem (300g) and root (300g) of Euphorbia balsamifera

plant were extracted with 1200cm3 ethanol using soxhlet extractor for 6 hours at

temperature of 850C. The extracts were concentrated by using hot air sterilizing

cabinet at 600C and yields 35.28g of leaves, 25.51g of stem and 29.82g of root.

The crude ethanol extracts were used for preliminary qualitative test for

phytochemicals, antibacterial evaluation and further fractionation into different

fractions.

2.4 Melting Point

Melting Point Apparatus (Intech make) is comprises of 3600C thermometer,

capillary tube and apparatus, the apparatus has socket that connect to power

supply, four holes and viewing point. The three holes for three capillary tubes while

the other one is for thermometer. The crude ethanol extract was injected into the

capillary tube and the tube was placed in the hole at the same time with the

thermometer, then it was set at 500C. The thermometer reading was recorded

when the sample start to melt. The initial temperature was subtracted from the final

temperature of the thermometer.

35

2.5 Phytochemical Screening

2.5.1 Test for Alkaloids

(a) Mayer’s Test: five (5) drops of Mayer’s reagent were added into 2cm3 of each

extract in a test tube. Appearance of a yellowish precipitate was taken as indication

for presence of alkaloids (El-Olemay et al., 1994).

(b) Dragendorff’s Test: Five (5) drops of Dragendorff’s reagent were added to

2cm3 of each extract in a test tube. Formation of a brown precipitate was taken as

indication for the presence of alkaloids (El-Olemay et al., 1994).

(c) Wagner’s Test: Five (5) drops of Wagner’s reagent were added to 2cm3 of

each extract in test tube. Formation of dark brown precipitate was taken as

indication for the presence of alkaloids (El-Olemay et al., 1994).

2.5.2 Test for Saponins

2cm3 of each filtrate was diluted with 5cm3 of distilled water and vigorously shaken,

then allowed to stand for 30 minutes. Persistent frothing indicated the presence of

saponins. To the foam, 3 drops of olives oil were added and shaken vigorously.

Formation of an emulsion was considered as a confirmation for saponins

(Sofowora, 1984).

2.5.3 Test for Steroids

(a) Liebermann’s Test: Anhydrous acetic acid (5cm3) and 5cm3 of chloroform

were combined together with the extract and cooled in ice, then few drops of

36

concentrated sulphuric acid was added down the side of the test tube. A violet or

pink colour which changed gradually to blue and to bluish green was taken as

positive test (Abubaker 2009).

(b) Salkowski Test: A little quantity of each of the extracts was dissolved in 2cm3

of chloroform and few drops of concentrated H2SO4 was added down the test tube

to form two layers. A red or yellow colour was taken as indication for the presence

of sterol (Abubakar 2009).

2.5.4 Test for Anthraquinones

The extract (0.5g) was boiled with 10cm3 of sulphuric acid (H2SO4) and the filtered

using Whatman filter paper. No 1. The filtrate was shaken with 5cm3 of chloroform.

The chloroform layer was pipetted into another test tube and 1cm3 of 10% of dilute

ammonia was added. The resulting solution was observed for colour changes

(Trease and Evans, 1989).

2 .5.5 Test for Flavonoids

(a) Sodium Hydroxide Test: Each filtrate (2cm3) was acidified with 1% HCl

followed by drops of 20% of NaOH canary yellow colour indicates presence of

flavonoids (Gariapathi et al., 2011)

(b) Lead Acetate Test: Each filtrate (2cm3) was treated with 3 drops of 10% lead

acetate solution. A coloured precipitate indicates the presence of flavonoids

(Harbone, 1984).

37

2.5.6 Test for Tannins

Each extract (0.5g) was boiled in 100cm3 of water in a test tube and then filtered. A

few drops of 0.1% ferric chloride were added and observed for brownish –green or

a blue-black colouration (Sofowora, 1984).

2.5.7 Test for Cardiac Glycosides

Each extract (0.5g) was dissolved in 2cm3 of glacial acetic acid containing one drop

of ferric chloride solution and shaken vigorously. Then, 1cm3 of concentrated

sulphuric acid was added and carefully shaken. A positive test was indicated by a

blue layer at the interface (Trease and Evans, 1989).

2.5.8 Test for Volatile Oils

Each extract (1cm3) was mixed with dilute hydrochloric acid. Formation of white

precipitate confirmed the presence of volatile oil (Trease and Evans, 1989).

2.6 Quantitative Phytochemical Analysis

Quantitative phytochemical estimation was carried on the leaves of Euphorbia

balsamifera to know the percentage of secondary metabolites present, (Burkill,

1985) and (Natarajan et al., 2005) reported that the leaves of the plant is the most

useful part, having antiviral, antibacterial and anti parasitic effect.

2.6.1 Determination of Total Alkaloids

Powdered leaves of Euphorbia balsamifera (5g) was extracted with 100cm3

of methanol: water (1:1 v/v) mixture and the solvent was evaporated. The residue

38

was then mixed with 20cm3 of H2SO4 (0.0025M) and partitioned with ether to

remove unwanted plants material. The aqueous fraction was basified with NH4OH

solution and then extracted with excess chloroform to obtain the alkaloid fraction

and separated. The process was repeated several times, the extract was

concentrated to dryness. The remaining alkaloids were weighed and the

percentage was calculated with reference to the initial weight of the powder

(Trease and Evans, 1989).

% Alkaloid = Weight of Alkaloid Residue x 100………………………………… (2.1) Weight taken

2.6.2 Determination of Total Tannins

The powdered sample (0.1g) was transferred into a 100cm3 conical flask

containing 50cm3 of distilled water; it was heated for one hour and filtered. The

filtrate was kept, while the residue was washed several times with distilled water

and the combined solution made to the volume with distilled water in a volumetric

flask. To 0, 1, 2, 3, 4 and 5cm3 of the standard tannic acid in a 50cm3 volumetric

flask; 2.5cm3 Folin-Denis reagent and 10cm3 Na2CO3 solutions were added and

made to volume with distilled water. Similarly, 10cm3 of the sample solution was

treated in the same way. The flasks were allowed to stand for 20 minutes after

which optical density was measured at 760nm using a spectrophotometer. A

calibration curve was plotted from which the concentration of tannic acid (x) in the

sample was extrapolated and tannins content in the sample was calculated using

equation (2.2) below (Edeoga, et al., 2005).

39

Tannin acid (mg/100g) = x(ppm) Extracted volume …………………………(2.2) Aliquot (10cm3) x Sample weight (0.1g)

2.6.3 Determination of Total Phenolic Compounds

Total phenolic content was determined with the Folin-Denis reagent, extract

was mixed with 0.4 cm3 Folin-Denis, and after 5minutes 4 cm3 of Na2CO3 was

added. The final volume of the tubes were made up to 100cm3 with distilled water

and allowed to stand for 90 minutes at room temperature. Absorbance of sample

was measured against the blank at 750nm using a spectrophotometer. Total

phenolic content was expressed as gallic acid equivalent based on calibration

curve (Katasani and Damodar 2011).

2.6.4 Determination of Total Flavonoids

Total flavonoids content was determined by taking 1cm3 of leave sample and add

4cm3 of distilled water and 0.3cm3 of 5% sodium nitrite and 0.3cm3 of 10% AlCl3

were also added. After 6 minutes incubation at room temperature, 2cm3 of 1M

NaOH was added to the reaction mixture. Immediately the final volume was made

up to 100cm3 with distilled water. The absorbance of the reaction mixture was

measured at 420nm. Yellow colour indicates the presence flavonoids while total

flavonoids were expressed as gallic acid equivalent based on calibration curve

(Katasani and Damodar 2011).

40

2.6.5 Determination of Total Saponins

The powdered leaves of the plant were placed in a 100cm3 flask containing 60cm3

of 50% ethanol. The mixture was boiled for 15 minutes and filtered; 2g of charcoal

was also added. The solution was boiled and filtered. The filtrate was allowed to

cool and equal volume of acetone was added to complete the precipitation of

saponins. The separated saponins were collected by decantation and dissolved in

boiling ethanol and filtered to remove any insoluble substances. The filtrate was

allowed to cool at room temperature which the result is the precipitation of

saponins. The separated saponins were collected by decantation method and

20cm3 of ethanol was added and filtered. The filtrate was heated in a water bath,

after evaporation, the sample was dried in the oven into a constant weight. The

saponin content was calculated in percentage (Edeoga, et al., 2005).

% Saponins = weight of Saponins residues x 100 ………………………………. (2.3) Weight of Sample

2.7 Fractionation of Ethanol Extract

Crude ethanol extract of leave of Euphorbia balsamifera was not soluble in

water; split method of separation was adopted according to (Abubakar, 2009). n-

hexane was directly added to crude ethanol extract and with vigorous stirring

before filtration and the filtrate is n-hexane fraction, the residue was allowed to dry

and same method was repeated with chloroform, ethyl acetate and finally ethanol

to obtain n-hexane, chloroform, ethyl acetate and ethanol fractions. They were

concentrated at 600C in hot air sterilizing cabinet.

41

2.8 Tests for Antibacterial Activity

Bacteria culture used in this study were obtained from the Microbiology

Departement of Usmanu Danfodiyo University Teaching Hospital (UDUTH),

Sokoto. Clinical bacterial cultures used in this study were Escherichia coli,

Staphylococcus aureus, Micrococcus species and Pseudomonas aeruginosa. All

the cultures were grown in Mueller-Hinton. The innoculum was used for

antibacterial assay.

2.8.1 Antibacterial Assay

The extract and fractions (ethanol extract, ethanol fraction, ethyl acetate fraction,

chloroform fraction and n-hexane fraction) of leave of Euphorbia balsamifera were

tested for antibacterial activities by agar well diffusion assay (Pelezer et al 1993)

Bacteria isolated were prepared to match 0.5 McFarland standards. Using the

micropipette, 100µ of organism was spread over the surface of an agar plate. The

procedure was the same for all test organisms. Using a sterile cork borer of 6mm

diameter, four holes were made, in each of culture plates, each of the four holes

were filled with a given concentration (60mg/ml, 90mg/ml and 120mg/ml) of ethanol

extract, ethanol fraction, ethyl acetate fraction, chloroform fraction, and n-hexane

fraction of fixed volumes (0.1 ml). One hole was punched in the centre of the plate

where 0.5µ steptomycin was added as positive control. The cultures were then

incubated at 37oC for 24 hour. The clear zones of inhibition were observed after the

incubation period (Cheesbrough, 2002). Diameter of the zones of growth inhibition

were measured in millimetre for each concentration of the extract and fractions

42

used, using a meter rule and diameter < 8.0mm indicates low sensitivity while >

8.0mm indicates high sensitivity (Collee et al., 1989).

2.8.2 Minimum Inhibition Concentration (MIC)

The MIC of the extract and fraction were determined on solid medium (Nutrient

agar) using method of (Siddiqui and Ali 1997). Standardized suspension of the test

organism was inoculated into a series of sterile tubes of nutrient broth containing

two-fold dilution of extract and fraction of leave and incubated at 37oC for 24h.The

MICs were read as the least concentration that inhibited the growth of the test

organisms

2.8.3 Minimum Bactericidal Concentration (MBC)

The minimum bactericidal concentrations were determined by first selecting tubes

that showed no growth during MIC determination; a loopful from each tube was

subcultured onto extract free agar plates, incubated for another 24h at 37oC. The

minimum bactericidal concentration was considered as the lowest concentration

that could not produce a single bacterial colony (Richard, et al., 2004)

2.9 Thin Layer Chromatography

Commercially pre-coated TLC silica gel plate was used; a line was drawn with a

pencil 2cm at the bottom from one end of the plate. The sample(s) were dissolved

in little chloroform and was spotted on the line drawn on the plate coated with silica

gel by capillary tube and then allowed to dry. The dry plates were placed into a

chromatographic tank containing benzene and ethyl acetate in a ratio of 3:1 as

43

mobile phase and the tank was covered. The solvent rose up on the plate by

capillary action, when the solvent front was just about 2cm to the upper end of the

plate, the plate was removed and a line was drawn to mark the position of the

solvent front. The plates were allowed to dry. Visualization and identification of the

spots that indicate constituents of fraction was done by using a ultra- violet lamp at

a wave length of 254nm, kept in iodine chamber, spray with sulphuric acid and

heat at 1050 C. The Rf value of the spots were measured using meter rule.

2.10 Column Chromatography

100cm3 burette was used as a column with 50g of silica gel as a stationary phase

while mobile phase was petroleum ether hundred percent, followed by 9:1 ratio of

petroleum ether and ethyl acetate as eluting solvents. The column was parked by

wet packing method, after packing was allowed overnight, 0.5g of concentrated

chloroform fraction was dissolved in chloroform solution and soaked with cotton

wool, was placed on top of silica gel in the column. Between the cotton and the top

of silica gel there was disc made of filter paper and the bottom of the column there

was also another cotton wool. 2.4cm3 per minute each were collected in collection

bottles range from 1 to 25. The column fraction’s profiles were monitored by TLC to

confirming the similarities of elutes based on the number and colour (s) of the

spot(s).

2.11 Isolation of Bioactive Compounds Using Preparative TLC

The fraction from the column chromatography was evaporated by using

rotary evaporator, further subjected to preparative TLC for the isolation of bioactive

44

compounds. The sample was dissolved in chloroform for analysis; a line was

drawn with pencil about 2cm from the bottom of the plate. Plate with thickness of

1mm were prepared using stationary phase silica gel and mobile phase benzene:

ethyl acetate 3:1 the glass plate was allowed to dry then the dry plate was placed

in the developing tank of mobile phase. The solvent rose up on the plate by

capillary action, when the solvent front was just about 2cm to the upper end of the

plate. The plate was removed from the tank and mark the solvent front with a

pencil and the plate is dry by using a flow of dry nitrogen and the band is visualize

by using UV light, mark them lightly with a pencil. The edge of a spatula was used

to scrape the bands off onto a lengthwise folded piece of clean white paper, the

scrapings was dissolved in 3cm3 of methanol and silica gel was settled at the

bottom of the beaker, while the isolate was decanted, solvent was removed by

rotary evaporation and the purification is done by recrystallisation process, that is,

dissolving isolate in methanol, filtered and allow the solution to crystallise and

dried. Finally the melting point of crystal was taken and the crystal was kept for

spectroscopic analysis (Mohanlall, et al., 2011).

2.12 Ultra-violet (UV)

Fourier Transform UV-visible spectrophotometer experiment were

performed using Shimadzu (Model 20550) instrument controlled by UV-visible

solution software set at different wavelength from 200nm to 280nm. Cell containing

chloroform was first prepared to serve as a blank then; the crystal was dissolved in

chloroform followed by adding of 2cm3 hydroxylamine and 5cm3 of standard

45

phenolphthalein solution were also added. Window sample were scan and value of

absorption maxima and blank were recorded.

2.13 Infrared Spectroscopy IR

Fourier Transform Infrared spectroscopy (FTIR) experiment were performed

using Shimadsu (Model 8400S) instrument controlled by IR solution software set at

spectra resolution of 4cm-1. A KBr disc was first prepared to serve as blank then,

the crystal was grounded with KBr and pressed to make the sample disc (window).

The prepared sample window was scanned between 400-4000m-1 20 times and

the means were printed.

2.14 GC-MS (Gas Chromatography-Mass Spectroscopy)

Gas Chromatography-Mass Spectroscopy GC-MS experiment was

performed using Shimadzu (G890N Modern). The instrument settings were column

oven and injection temperature at 60oC and 25oC respectively, injection mode was

split, the total flow and column flow were set at 6.1ml/min and 1.55ml/min

respectively, the flow control mode set at linear velocity. The ion source and

interference temperature were set at 200oC and 250oC and the solvent cut time

was set at 2.5minutes and total run time of 47minutes. The sample for analysis

was prepared by dissolving in ethyl acetate. The solution was injected into the Gas

-Chromatographic column through the injection point, the individual components

eluted from the column and then enter the electron analyzer (Mass -

spectrophotometer) detector. They are bombarded with stream of electrons

causing them to break apart into fragment which is readout and compared with

different compounds in the spectrum library

46

CHAPTER THREE

RESULTS AND DISCUSSION

3.1 Results

3.1.1 Percentage Yields of the Plant

The result of the percentage yields of the leaves, stem and root of

Euphorbia balsamifera extracts are presented in Table 3.1

Table 3.1: Percentage Yields of the Plant

Sample Solvent

Used

Volume of

Solvent

Weight of

Sample

Weight of

Extracts

(%) Yields

Leaves

Stem

Root

Ethanol

Ethanol

Ethanol

1200cm3

1200cm3

1200cm3

300g

300g

300g

35.28g

25.51g

29.82g

11.76

8.5

9.94

47

3.1.2 Phytochemical Screening

The crude extracts of leaves, stem and root of Euphorbia balsamifera plant

were screened for the following metabolites: cardia glycosides, saponin, tannis,

terpenoids, anthraquinones, steroids flavonoids, alkaloids and volatile oil are

shown in Table 3.2

Table 3.2: Preliminary Phytochemical Screening of Ethanolic Extracts of

Euphorbia balsamifera leaves, stem and root

Chemical Composition Ethanolic extracts Leaves Stem Root

Cardiac glycosides ++ + + Saponin ++ + + Tannins ++ ++ + Terpenoids + + + Anthraquinones + + + Steroids a) Lieberman's Test + + + b) Salkowski Test + + + Flavonoids a)Sodium hydroxide + + + b) Lead Acetate Test + + + Alkaloids a) Mayer's reagent + + + b) Wagner's reagent + + + Violite oil ++ + +

Key + = Present ++ = Appreciably Present

3.1.3 Melting point

The crude ethanol extract of leaves of Euphorbia balsamifera plant was

analyzed using melting point apparatus, was melted at range of 1400C – 145 0C.

48

3.1.4 Quantitative Estimation of Phytochemical Contents

The results of quantitative estimation of alkaloids, tannins, flavonoids and

saponins contents of the chloroform extract of the leaves of Euphorbia balsamifera

plant are presented in Table 3.3

Table 3.3: Quantitative Estimation of Phytochemical Contents of the

Chloroform Extract of the Leaves of Euphorbia balsamifera

Phytochemical Percentage (%)

Alkaloids 9

Tannins 11

Flavonoids 8

Saponins 8

49

3.1.5 The Yields of Fractionated Components

The results of the yields of fractionated components using various solvents

on the leaves extracts of Euphorbia balsamifera plant are shown in Table 3.4

Table 3.4: Results of the Yields of Fractionated Components using various

Solvents

Fraction Weight

n-hexane 0.2g

Chloroform 0.5g

Ethyl acetate 0.25g

Ethanol 0.32g

50

3.1.6 The Antibacterial Activities

The antibacterial activities of different concentrations of chloroform fraction

of leaves against Escherichia coli, Staphylococcus aureus, Micrococcus species

and Pseudomonas aeruginosa are presented in Table 3.5

Table 3.5: Zone of Inhibition Results (mm)

Conc. mg/ml

Escherichia coli (mm)

Staphylococcus aureus (mm)

Micrococcus species (mm)

Pseudomonas aeruginosa (mm)

Ethanol extract

60 07 09 07 10

90 17 19 18 14 120 20 18 21 19 Ethanol fraction

60 11 14 11 10

90 17 19 18 14 120 18 19 19 21 Ethyl acetate fraction

60 13 07 10 09

90 13 16 15 10 120 20 19 21 17 Chloroform fraction

60 18 15 19 21

90 21 20 20 15 120 24 20 25 20 n-hexane fraction

60 07 08 10 11

90 12 14 10 13

120 15 18 20 19 Control 0.5µ streptomycin

20 24 21 27

51

3.1.6.1 The Minimum Inhibitory Concentration (MIC)

The results of MIC on Escherichia coli, Staphylococcus aureus Micrococcus

species, and Pseudomonas aeruginosa are shown in Table 3.6

Table 3.6 Minimum Inhibition Concentration of Tested Organism

Fractions Escherichia coli (mm)

Staphylococcus aureus (mm)

Micrococcus species (mm)

Pseudomonas aeruginosa (mm)

n-hexane 10 8 8 7

Chloroform 12 6 5 7

Ethyl acetate 7 6 7 9

Ethanol 8 7 10 8

3.1.6.2 Minimum Bactericidal Concentration (MBC)

The results of MBC on Escherichia coli, Staphylococcus aureus

Micrococcus species, and Pseudomonas aeruginosa are shown in Table 3.7

Table 3.7 Minimum Bactericidal Concentration of Tested Organism

Fractions .

Escherichia coli (mm)

Staphylococcus aureus (mm)

Micrococcus species (mm)

Pseudomonas aeruginosa (mm)

n-hexane 8 7 11 9 Chloroform 7 10 12 11 Ethyl acetate 10 11 10 9 Ethanol 8 10 9 10

52

3.1.7 Thin Layer Chromatography of Chloroform Fraction of Leaves

The results of TLC carried out on the chloroform fraction of the leaves of

Euphobia balsamifera using the following solvent system benzene: ethyl acetate

(3:1) are shown in Table 3.8 below

Table 3.8 TLC of Chloroform Fraction

Fraction Solvent System Number of component

Rf values

Chloroform Benzene : Ethyl acetate (3:1)

6

0.61

0.59

0.76

0.86

0.89

0.91

53

3.1.8 Results of Ultra Violet/Visible Analysis

The results of ultra-violet/Visible spectroscopy analysis on component is

presented in Table 3.9

Table 3.9: Ultra Violet /Visible Spectroscopy Analysis Results

Wavelength (nm) Inference

338.50 Polynuclear aromatic compounds

279.50 Six membered double bond extended

conjugation or six membered enone double

extended conjugation

265.50 Conjugated benzene derivatives eg phenyl

benzonic acid

54

3.1.9 Infra-red Spectroscopy Analysis

The results of infra-red spectroscopy analysis of isolated chloroform fraction

of leave of Euphorbia balsamifera plant is shown in Table 3.10

Table 3.10: Infra-red Spectroscopy Analysis Results

Frequency (cm-1)

Assigned Functional Group

Inference

1689.70 C=Ostr Carbonyl group

2816.16 CHstr Overtone bands

1012.66 C-Nstr Amine group

3443.05 O-Hstr OH for carboxylic acid

1689.70 C=Nbd Imines

1192.06 C-Ostr Anhydrides/acid

2816.10 (CH3)n Alkyls

55

3.1.10 GC-MS Analysis

GC-MS analysis of isolated chloroform fraction of leave of Euphorbia

balsamifera plant was carried out using (G89ON Modern). Peak identification was

carried out by comparism of the mass spectral obtained with mass spectral data

available on NIST Version shown in Table 3.11 below

Table 3.11: GC-MS Analysis Results

Peak

No

R.

Time

Area % Base

Peak

Molecular

Weight

Compound

1 22.00 3.19 129 214 Dodecanoic acid

2 24.40 1.84 185 228 Tetradecanoic acid

3 25.89 3.65 116 284 Octadecanoic acid

4 26.57 2.75 213 220 (6H-Indolo[3,2,1-

de][1,5]naphthyridin-6-one)

5 27.95 3.05 129 274 Octadecanoic acid 2-hydroxyl

1,3-propanedlyl ester

6 28.06 8.43 183 274 Dodecanoic acid 2,3-

dihydroxypropyl ester

7 28.38 7.48 55 282 Oleic acid

8 28.56 4.48 284 442 Ascorbic acid 6

octadecanoate

9 29.83 5.63 129 568 Hexadenoic acid 1,2,

ethanediyl ester

10 29.96 3.12 212 326 Dinobulon

11 31.60 25.27 129 340 Docosanoic acid

12 31..83 15.03 129 284 Octadecanoic acid

13 32.02 4.08 129 207 Ethylacridine

14 34.51 12.00 207 562 Oleic acid elcosyl ester

56

3.2 Discussion

Table 3.1 showed the results of extraction of 300g of ground leaves, stems

and root. The leaves had the highest yield (35.28g) representing 11.76% followed

by roots with 29.82g having 9.94% and the least is stems with 25.51g representing

8.5%. This implies that leaves are the most soluble components in the solvent.

The result of phytochemical screening is shown in Table 3.2 which indicates

that the leaves of this plant are very rich in many secondary metabolites. The

results also agreed with the work of (Kamba and Hassan 2010) who reported that

the leaves, stems and the root of Euphorbia balsamifera are rich in secondary

metabolites. Tannins were known for their oxidation inhibiting activity which had

been known for a long time (Calvi et al., 1995). Tannins were found to be present

in high quantity. These are in line with the earlier report by (Gill, 1992) who said

that herbs that contain tannin as their main components are astringent in nature

and are used for treating intestinal disorder. The presence of tannins in the plant

could be responsible for its traditional use in treating intestinal disorder such as

diarrhea, dysentery and inflammatory condition of the digestive tract (Burkill, 1985).

Tannins are widely used in herbal medicine to treat wounds and to arrest bleeding

that is probably the reason why the plant is used in treating skin irritation (Nguyi,

1988). Flavonoids are phenolic compounds that protect plants from the damaging

effect caused by ultra-violet light and microbial infection (Allan and Miller, 1996).

Flavonoids also possess many pharmacological properties such as antioxidant

activities and anti-inflammatory activities. The potent antioxidant activity of

flavonoids; their ability to scavenge hydroxyl radicals’ superoxide anions and lipid

57

peroxide radicals may be the most important function of flavonoids (Alan and

Miller, 1996). These could be the reason why the plant can withstand many climatic

conditions and also its use for pharmacological purposes (Linuma et al., 1994).

Saponins are well known for their antioxidant properties (Beak et al., 1996).

Saponins exhibit cytotoxic effect and growth inhibition against a variety of cells,

making them have anti-inflammatory and anti-cancer properties (`Akindahusi and

Salawu 2005). They have a property of precipitating and coagulating red blood

cells (Okwu and Josiah 2006). Saponins have been reported to have antibacterial,

antifungal and antiviral activities (Soetan et al., 2006). The presence of saponins in

the plant could be responsible for their traditional use in the treatment of skin

diseases and venereal diseases. The presence of steroids has been reported by

(Okwu, 2001) that steroids were found to be present in the leave of the plant; this is

not surprising because the plant’s leaves and latex are used to promote fertility and

increase milk production.

Cardiac-glycosides are non-reducing substances that are composed of

sugar and non sugar parts. They are used therapeutically mainly in the treatment

of cardiac failure due to their anti-arrhythmic effects (Harbone, 1984). Also the

presence of Cardiac- glycosides could be responsible for the traditional use of

plant as a diuretic and laxative (Burkill, 1985)

Terpenoids are generally essential oils obtained from sap, tissue and different

parts of plants and trees. The ethanol extract of Euphorbia balsamifera leaves,

stem or root has high oily, gum and mucilage (mucilage is a type of soluble fibre of

viscous nature). There is a growing interest in natural terpenoids because of their

58

wide spectrum of biological activities. These include bactericidal, fungicidal,

antiviral, cytotoxic, analgestic, anticancer, spermicidal, cardiovascular and anti-

allergic activities. The presence of terpenoids in the plant could be responsible for

their traditional use in the treatment of skin diseases and insect bites (Edeoga and

Gomina 2002)

Alkaloids are naturally occurring organic bases. Table 3.2 shows their

presence in all plant parts. Alkaloids are use as anaesthetic agent. The presence

of anthraquinones in the plant could be responsible for the use of Euphorbia

balsamifera as a diuretic and laxative in Chinese medicine (Burkill, 1985)

The result of antibacterial assay is consistent with the report of (Gill 1992)

who earlier confirmed the antibacterial effects of the ethanol extract of Euphorbia

balsamifera. The result of antibacterial activity shows that the inhibitory activities

exhibited by the extract agree with the report of (Kamba and Hassan 2010) and

(Gill, 1992), all of whom linked antimicrobial properties of plants to the presence of

bioactive secondary metabolites. It was demonstrated that the activity of the extract

was concentration dependent. An increase in the concentration of the fraction

yielded higher activity as shown by the diameter of zone of inhibition (Table 3.5).

Highest activities were observed with the standard antibiotic streptomycin (control)

probably because the antibiotic was in the pure form (Prescott et al., 2002). The

fact that organism may need higher concentration of extracts to inhibit or kill them

may be due to their cell wall components (Banso, 2009). The results in Table 3.6

indicate that the minimum inhibition concentration (MIC) of the leaves fractions of

Euphorbia balsamifera plant ranges between 5mg/ml and 10mg/ml. The effect of

59

the plant fractions on the MIC for the test microorganisms is in line with the report

that microorganisms varied widely in the degree of their susceptibility (Aboaba and

Efuwape, 2001). The fraction finally prevent the growth and killed the organism

completely at MBC of 7mg/ml. The MIC and MCB is normally used to evaluate the

efficacy of the agent and show that the fraction can inhibit the growth of some

microorganism. Several studies had documented the scientific basis that bioactive

constituents inhibit the growth of various microorganism at different concentration

(Kambal and Hassasn) (Simone et al., 1998) plant. The activity of the plant extract

against both gram positive and gram negative bacteria is an indication of the

presence of broad spectrum antibiotic compound in the plant (Parekh and Chanda,

2007). The large zone of inhibition indicates the potency of the active principle of

the plant in Table 3.5. The large zone of inhibition exhibited by the extract and

fractions of Euphorbia balsamifera on Escherichia coli, Staphylococcus aureus,

Micrococcus species, Pseudomonas aeruginosa suggest that the plant can be

used in the treatment of infections commonly associated with the bacteria.

The chloroform fraction showed the highest activity from the sensitivity test

and was subjected to TLC to ascertain the number of compounds it has based on

the number of spot. TLC result presented Table 3.8 shows that chloroform has six

spot with clear separation as shown in Appendix 4. The chloroform fraction was

further subjected to column chromatography for isolation in to pure compound.

Three compounds were isolated; the fraction with clear separation was finally

subjected to preparative TLC for isolation in to a pure compound.

60

IR absorptions bands in Table 3.10 appeared at 3443.05 cm-1 for (O-H

stretching) in acid, alcohol or phenol. The absorption band for(C-H stretching) is

observed at 2816. 6cm-1, 1192.06cm-1 (C-O stretching) due to anhydrides, 1012.66

cm-1 (C-N stretching) due to amine group, 1689.70cm-1 (C=N bending) due to

alcohol group, 1689.70cm-1 (C=O stretching) due to carbonyl group attached to

carboxylic acid. 2833.52cm-1 (C-H stretching ) due to overtone of CH in the

compound, 1422.55cm-1 (C=C bending) due to aromatic ring, 1018.45cm-1 (C-N

stretching) due to amine group, 1622.19cm-1 (C=N bending) due to Imines group,

2954.08cm-1 (CH3 bending) due to alkyls groups.

Summary of the GC-MS analysis of the bioactive compound isolated from

the chloroform fraction of the leaves is given in Table 3.11. The GC chromatogram

gave 14 peaks (Appendix 7) of varying intensities and the chemical constituents

are fatty acid, and alkaloid. The major peak is peak 11 with retention time 31.60

minutes with molecular ion at m/z 340, base peak 129 the compound is docosanoic

acid, (Burkil,1985) reported the presence of the compound in plant extract.

Docosanoic acid is a fatty acid, it regulates blood pressure, is used in the treatment

of wounds and preventing infection and have antioxidants and antimicrobial

properties (Lim, 2013) which is the one of uses of the plant to treat wound and skin

infection (Burkill, 1985). Peak 3 that has retention time 26.57 minutes and gave

the mass spectra of unknown compound with molecular ion at m/z 220, base

peak at m/z 213 when it was compared to the NIST02 Reference Spectral Library

in MS spectral it suggested that the compound is alkaloid (6H-Indolo[3,2,1-

de][1,5]naphthyridin-6-one) with molecular formula C14 H8N2O. (Kamba and

61

Hassan 2010) reported the presence of alkaloid in the extract. Plant contains

indoles are used to treat snake bites, has antimicrobial activities against gram

negative and positive bacteria, use to treat sexual dysfunction and anti-asthmatic

drug (Biswal and Sahoo 2012) which are the some of the uses of the plant for

curing insect bites, asthma, venereal disease (Gill,1992) and has anti-inflammatory

property (Natarajan et al., 2005)

Figure 3.1 6H-Indolo [3, 2, 1-de][1,5]naphthyridin-6-one

The above UV, I.R and GC- MS spectral data and their comparison with those

described in the literatures showed the compounds isolated to be consituents of

(6H-Indolo[3,2,1-de][1,5]naphthyridin-6-one) and fatty acids

62

CHAPTER FOUR

CONCLUSIONS AND RECOMMENDATIONS

4.1 Conclusions

From the analysis carried out in this research work, the results offers a

scientific basis for the traditional uses of plant extract, which phytochemical

analysis revealed the presence of saponins, tannins, cardiac glycosides,

flavonoids, steroids, anthraquinones, alkaloids and terpenoids in the leaves, stems

and root of the plant and also showed varying degrees of antibacterial activities on

the microorganisms tested. The plant understudy gave a promising result of its

potential application in treatment of diseases caused by Escherichia coli,

Staphylococcus aureus, Micrococcus species and Pseudomonas aeruginosa, this

agreed with the work of (Natarajan et al., 2005) that Euphorbia balsamifera

possess antibacterial activities against some gram positive and gram negative.

This is why the plant could be employed in the treatment of microbial induced

ailments.

Three compounds were isolated from chloroform fraction as bioactive but

only one is characterized which point out to be simple compound (6H-Indolo [3, 2,

1-de][1,5]naphthyridin-6-one) and fatty acids. The presence of bioactive compound

justified the use of plant for various ailments. Euphorbia balsamifera is listed as

one of the medicinal plants commonly use in Africa (Ndukwe et al., 2005).

63

4.2 Recommendations

There is need for further studies on the plant parts in order to take the work to

beneficial level to humanity therefore; the researchers should carry out further

investigations on the following:

1. To isolate the remaining bioactive compounds, identify and characterize

them as addition to the plant chemical compounds bank.

2. The isolated and characterized compounds from the plant should be

explored for their medicinal application.

3. To investigate the toxicity of the plant for direct use in the treatment of

infections commonly associated with the above tested microorganisms.

64

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APPENDICES

Appendix 1

Chemical Name Formula Grade % Purity Manufacturer

Ferric Chloride FeCl3 LR 99.5 Sigma

Lead acetate (CH3COO)2Pb LR 99.5 Sigma

Ammonia Solution NH3 LR 35 Sigma

Sodium hydroxide NaOH LR 96 Sigma

Hydrochloric Acid HCl LR 36 Sigma

Sodium Carbonate Na2CO3 LR 99.5 Sigma

Bismuth nitrate Bi(NO3)3.5H2O Analar Sigma

Nitric Acid HNO3 LR 70 Sigma

Potassium iodide KI 99.5 BDH

Mercuric Chloride HgCl2 97 BDH

Acetic Acid CH3COOH Sigma

Iodine (crystals) I2 Sigma

Sulphuric Acid H2SO4 99.8 Sigma

Benzene C6H6 Sigma

Acetic anhydride (CH3CO)2O 99.5 Scietific & Chemical Supplies Ltd.

Nutrient agar LR

Ethanol C 2H5OH Analar 99.8 Sigma

Chloroform CHCl3 Analar 99.8 Sigma

Ethyl Acetate CH3COOC2H5 Analar 99.5 Sigma

n- Hexane C6H14 Analar 99.5 Sigma

Petroleum Ether Analar 99.8 Sigma

Silica Gel 60 – 120 mesh

SiO2 LR _ Qualikems

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Appendix 2: Computation of Extracts yield

Weight of extract x 100 Weight of sample Leave 35.8 x 100 = 11.76% 300

Stem 25.51 x 100 = 8.50% 300

Root 29.82 x 100 = 9.94% 300

Calculation of Rf Value using benzene: ethylacetate

Rf 1 = 4.0 = 0.61 6.5

Rf 2 = 4.5 = 0.59 6.5

Rf 3 = 5.0 = 0.76 6.5

Rf 4 = 5.6 = 0.80 6.5

Rf 5 = 5.8 = 0.89 6.5

Rf 6 = 5.9 = 0.91 6.5

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Appendix 3: Zone of Inhibition

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Appendix 4: TLC Plates of chloroform fraction

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Appendix 5: UV Spectra

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Appendix 6: IR Spectra

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Appendix 7

GC-MS Spectra

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