(POSTGRADUATE SCHOOL) PHYTOCHEMICAL SCREENING AND ANTIBACTERIAL STUDIES

88 Pages·2017·0.8 MB·English

by  Kamputz Kutz

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.

i
DEDICATION
This dissertation is dedicated to my late husband Femi Bello Bashir. May your soul
rest in peace, amen.

ii
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

iii
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.
iv
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
v
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
vi
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

vii
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

viii
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

ix
LIST OF PLATE
Plate1.1 a photograph showing section of Euphorbia balsamifera plant ..................3
x