ABSTRACT
This study is about the isolation, characterization and anti-inflammatory activity of the secondary metabolites of the stem bark of Allophylus africanus Beauv (Sapindaceae). The plant has a wide distribution in West Africa, with Nigeria (Mambila Plateau, Calabar) inclusive. It has been used in communities for both its medicinal and economic importance. The stem bark of Allophylus Africanus was extracted by percolation using chloroform as solvent and allowed to concentrate by evaporation at room temperature. TLC was carried out after column chromatography and an Rf value of 0.87 was obtained at a solvent mixture of hexane: chloroform at ratio 1:4 from the finger column chromatography carried out on fraction AF3. Characterization using spectra analysis which include FT-IR, 1H-NMR, 13C-NMR, H-H COSY, H-C COSY, 135 DEPT and Mass Spectra of A. africanus stem bark extract AF3 led to the elucidation of the structure of the proposed compound and proffer scientific basis for its use in the treatment of inflammatory disorders. FT-IR spectrum indicated that the compound could be an aromatic derivative with C-C stretching found at frequency 1465. Aromatic protons were seen at the chemical shift values of 7.25 ppm and Olefinic protons at 5.124 ppm, 5.146 ppm and 6.162 ppm appeared as doublets of doublets of doublets. COSY suggested the presence of –C=C-O-CH2-OCH2- moiety. Phytochemical screening revealed a predominance of tannins, saponins, flavonoids and alkaloids with little glycoside and no terpenes and steroid seen in the stem bark of A. africanus. The anti-inflammatory potency of the stem bark of Allophylus africanus P. Beauv were determined in vivo for suppressive and curative activities in rats using different doses of the extract (300 mg/kg & 600 mg/kg). The A. africanus extracts significantly reduced the increase in paw diameter induced by carrageenan at all doses (300 mg/kg & 600 mg/kg) as against normal saline treated group. The results suggest that the extract possesses considerable anti-inflammatory activity this shows that the plant possesses considerable therapeutic potential and thus provides some scientific basis for the traditional use of the stem bark in the treatment of inflammation and inflammatory disorders.
TABLE OF CONTENTS
Title Page ………………………………………………
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Declaration …………………………………………
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Certification ….......................................
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iii
Acknowledgements iv
Dedication
…………………….……...… …………… ……….……….............v
Table of Contents……………………………… …………………………………. vi
List of Tables.………………………...…………… ……..…………… xii
List of Figures…………………………..…………………….. ……… …...… xiii
Acronyms xiv
List of Plates xvi
Abstract …………………….…….…………………………
……..…… ….... xvii
CHAPTER 1: INTRODUCTION 1
Background of the
Study 1
1.1 Statement
of Research Problem 3
1.2 Justification
of The Study 3
1.3 Aims and
Objectives of the Study 3
CHAPTER 2:
LITERATURE REVIEW 5
2.1 Introduction of
Family Sapindaceae 5
2.1.2 Identification and Characteristics sapindaceae family into genera 5
2.1.3 Morphological characterisation 5
2.1.4 Key to genera of African sapindaceae 6
2.1.5 Classification
of sapindaceae family into genera 11
2.2 Distribution
and Description of Allophylus Species
12
2.2.1 Occurrence
and description of Allophylus species
in Africa 13
2.2.2 Occurrence
and description of common allophylus
species in Nigeria 14
2.2.3
Allophylus africanus 14
2.2.4 Description
of A. Africanus 15
2.3 Chemical
Constituents of Allophylus Species 15
2.3.1 Bioactive
component 16
2.3.2 Phytochemical
constituents 17
2.4 Biological
Activities of Allophylus Species 19
2.5
Some Selected Family of Sapindeciae 19 2.5.1
Allophylus serrattus 19
2.5.2 Sapindus mukorossi 21
2.6 Inflammation 26
2.6.1 Concept of inflammation 26
2.6.2 Types of inflammation 26
2.6.3 Causes of Inflammation 27
2.6.4 Inflammatory
disorders 27
2.6.5 Drug treatment 28
2.6.6 Plants as sources of anti-inflammatory agents 28
2.7 Phytochemicals
with Their Medicinal Activity 29
2.7.1 Alkaloids 29
2.7.2 Saponins 30
2.7.3 Flavonoids 31
2.7.4 Tannins: 32
2.7.5 Steroids: 32
2.7.6 Phenols: 33
2.7.7 Terpenoids: 33
2.7.8 Glycosides:
33
2.8 Chromatography 34
2.9 Types of Chromatography 40
2.9.1 Column chromatography 41
2.9.2 Gas
chromatography 42
2.9.2.1 Analysis
of a mixture by gas chromatography GC 43
2.9.3 Thin layer
chromatography 44
2.9.3.1 Analysis
of a mixture by thin layer chromatography TLC 45
2.9.3.2
Identification by TLC 45
2.9.3.3 Preparing
to run TLC 46
2.9.4
Application areas of chromatography in medicine 51
2.10 Nuclear Magnetic Resonance 51
2.10.1
COSY 54
2.10.2 C-H-COSY
OR HETCOR 55
2.10.3 HMQC 55
2.10.4 HSQC 55
2.10.5 HMBC 55
2.10.6 EXSY 56
2.10.7 NOESY,
ROESY 57
2.10.8 HOESY 57
2.10.9 HMBC
Spectra 58
2.10.10 NMR Spectroscopy
of Carbohydrates 58
2.10.11 Mass Spectroscopy (MS): 59
CHAPTER 3: MATERIALS AND
METHODS 62
3.1 Materials 62
3.2 Plant Collection,
Identification and Preparation 63
3.2.1 Sample Preparation 63
3.2.2 Extraction of the Crude
Plant Materials 63
3.2.2.1 Extraction
of Allophylus africanus 63
3.2.2.2. Concentration of chloroform Extract of the Stem
Bark of
Allophylus africanus 64
3.3 Qualitative
Analysis 64
3.3.1 Preliminary
Phytochemical Screening 64
3.3.1.1 Preparing
Dragendorff Reagent 64
3.3.1.2 Flavonoids 64
3.3.1.3 Tannins 65
3.3.1.4 Saponins 65
3.3.1.5 Phenols 65
3.3.1.6 Alkaloids 65
3.3.1.7 Cardiac
Glycoside 65
3.3.1.8 Terpenes 65
3.3.2 Animals 66
3.3.3 Drugs
administration 66
3.3.4 Groupings 66
3.3.5 Pharmacological
Screening 66
3.3.5.1 Acetic
Acid-Induced Writhing in rat 66
3.3.5.2 Carrageenan-Induced
Paw Oedema in Rats 67
3.4 Statistical
Analysis 67
3.5 Thin
Layer Chromatography 67
3.6 Column
Chromatography 68
3.7 Finger
Chromatography 71
3.8 Spectra
Analysis 72
3.9 Anti-Inflammatory
Activities 72
3.9.1 Invivo osmotic
fragility effect compared to that of aspirin 72
CHAPTER 4: RESULTS AND
DISCUSSION 75
4.1 TLC
Results on Some Column Fractions 75
4.2 Spectral
Analysis of Isolated Extract AF3 From Stem Bark of
Allophylus africanus. 75
4.2.1. FT-IR analysis of the isolated chloroform extract (AF3) of stem
bark of Allophylis
africanus. 75
4.2.2 Proton-NMR of AF3 78
4.3 PROTON-PROTON
COSY Spectrum 80
4.4 CARBON-PROTON–
HETCOR 82
4.5: 13 CARBON DEPT 84
4.6 Mass Spectra Profile of The Isolated
Chloroform Extract of
Allophylus africanus. 88
4.7 PHYTOCHEMICAL
ANALYSIS OF STEM BARK OF
Allophylus africanus 94
4.8 Acetic
Acid-Induced Writhing Test 96
4.9 Carrageenan-Induced
Paw Oedema in Rats 96
CHAPTER 5: CONCLUSION AND RECOMMENDATION 102
5.1 Conclusion 102
5.2 Recommendations 103
References
LIST OF TABLES
2.0 Classification of sapindaceae 12
2.1 Medicinal uses of Allophylus serrattus 21
2.2 List of saponins
isolated from Sapindus mukorossi 23
2.3 Terms in Chromatography 36
2.4 The commonly used chromatographic
techniques 38
2.5 Common Problems in TLC 48
3.1 Solvent system employed in the chloroform extract of
the stem bark extract of
Allophylus africanus. 69
3.2 Solvent system
employed in the Finger column chromatography on the
selected and pooled
column chromatography of the isolates from stem bark
of Allophylus africanus 71
4.1 IR absorption of the isolate
from Allophylus africanus. 77
4.2 Absorption Regions gotten from the isolate 79
4.3 Chemical
Shifts of the isolates and their Signals. 86
4.4 Chemical
Shifts of the isolates and their Signals 87
4.5 Qualitative
phytochemical screening of Allophylus
africanus 94
4.6 Effect of chloroform extracts of Allophylus africanus on paw
circumference of wistar
rats. 99
LIST OF FIGURES
2.2 Some chemical compounds isolated from various Allophylus Species 18
2.3 Structure of Sapimusaponins A-B
and O-P. 24
2.4 Structure of Sapimusaponins
C-E. 24
2.5 Structure of Sapimusaponins F-J,
Q-R. 24
2.7 Gas Chromatography analysis 44
2.9 1-dimensional NMR spectra of carbon-13
and hydrogen. 53
2.10 Examples of 2-dimensional NMR spectra. 56
3.1 Flow chart
of methodology 74
4.1 FT-IR Spectrum of the isolated chloroform extract (AF3)
of Allophylus africanus. 76
4.2
Proton NMR (1H-NMR) of AF3 78
4.3
Proton-Proton COSY (H-H COSY) of AF3 80
4.4
Proton-Carbon HETCOR NMR for the isolated chloroform extract of stem
bark of Allophylus africanus from AF3. 82
4.5 13C-DEPT (135) NMR for the isolated
chloroformic extract of stem
bark of Allophylus africanus from AF3 84
4.6 Expanded 13C-DEPT
(135) NMR for the chloroform extract of stem
bark of Allophylus africanus from AF3. 85
4.7 Mass spectrum of the isolated ethanol
extract of Allophylus africanus. 88
4.8 Base Peak 89
4.9 Suggested
fragmentation pattern of the mass spectrum analysis of
isolated compound of Allophylus
africanus. 92
4.10 Proposed
structure from isolated compound of Allophylus
africanus. 93
4.12 Carrageenan-Induced Paw Oedema in Rats 97
4.13 % Heamolysis 97
4.14 % Inhibition of
Oedema/Inflamation (15 min) 98
4.15 % Inhibition of
Oedema/inflammation (30 min) 100
4.16 % Inhibition of
Oedema/inflammation (1 hr) 101
4.17 % Inhibition of
Oedema/inflammation (2 hrs) 101
ACRONYMS
%: Percentage.
3D: Three Dimension
4D: Four Dimension
ANOVA: Analysis
of Variance.
ASA: Acetyl
Salicylic Acid.
B.P: British Pharmacopoeia.
CAM: Complementary and Alternative Medicine.
CM: Centimeter.
DAMPs: Damage-Associated Molecular Patterns.
DOSY: Diffusion
Ordered Spectroscopy.
DPPH: 1,1 – Diphenyl-2-2-picrylhydrazyl.
EXSY: Exchange
Spectroscopy
FAA: Formalin Acetic Acid.
Fig: Figure
FT-IR: Fourier Transform Infrared Spectroscopy
G: Gram
GC – MS: Gas Column Spectrophotometry.
H2SO4:
Sulphuric acid.
HCl: Hydrochloric acid.
HETCOR: Heteronuclear Correlation
HMBC: Heteronuclear
Multi-Bond Connectivity
HMQC: Heteronuclear
Multi-Quantum Coherence
HOESY: Heteronuclear
Overhauser Effect Spectroscopy
HPLC: High Performance Liquid Chromatography
HSQC: Heteronuclear Single Quantum Coherence
IR:
Infrared
KG: Kilogram.
L: Liter.
LD50: Median lethal dose.
ML: Milliliter.
MHz: Mega Hertz
MM: Millimeter.
NMR: Nuclear Magnetic Resonance
N/S: Normal saline.
NOE: Nuclear Overhauser Effect
NOESY: Nuclear Overhauser Effect Spectroscopy
NSAIDs: Non-steroidal anti-inflammatory drugs.
PAMPs: Pathogen-associated molecular patterns.
PPM: Parts per million
PRRs: Pattern recognition receptors.
Rf: Retention Factor.
SEM: Standard Error of Mean.
TOCSY: Total Correlation Spectroscopy
TLC: Thin layer chromatography.
UV: Ultraviolet Light.
W/W: Weight per weight.
WHO: World Health Organization.
LIST OF PLATES
Plate 2.1:
Photographs of characteristic features of Sapindaceae 9
Plate
2.6: Column Chromatography 42
Plate 3.1: Allophylus africanus Stem Bark and Leaves 73
Plate 3.2: Allophylus africanus Stem Bark 73
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