ABSTRACT
Vitex doniana is used in herbal medicine for the treatment of diseases and infection such as wounds, burns, epilepsy, diarrhea and diabetes. These claims are yet to be fully documented through scientific evidence, hence the uncovering of the secondary metabolites present in the leaves, stem and root barks of V. doniana and assessment of their antioxidant, antimicrobial and antidiabetic potentials. Phytochemicals, vitamins, minerals and proximate compositions were determined using standard methods. GC/MS technique was employed in the determination of the volatile constituents of the chloroform crude extracts of leaves, stem and root barks of V. doniana. The overall range of bioactive component range were for alkaloids (1.68-6.39%), flavonoid (2.18-5.02%), saponin (0.86-4.12%), tannin (0.58-2.50%) and phenol (0.293-2.20%). The plant vitamins content comprise ascorbic acid (0.011-0.021%), niacin (0.000128-0.000249%), riboflavin (0.000144-0.000260%), thiamin (0.000165-0.000497%) and β-caotenoid (0.0566-0.1227%). The plant parts are good sources of K, Ca, Mg, P, Fe, Cu, Se and Zn. V. doniana leaf crude extract showed a maximum of 0.041trypsin inhibitory unit (TIU), 25% inhibition against Gram-positive human pathogen, 50% inhibition with the Gram-negative microorganisms. The antidiabetic experiment with 25 albino rats showed no significant change in the induced diabetes after prolonged period treatment, though there was weight gain. By GC/MS analyses the compounds identified were thirteen from the leaves, nine from the stem bark and six from the root bark in the class of alkane hydrocarbon, tocopherols, alkaloids, ether, steroids, ester of unsaturated fatty acids and sulphur compounds all of which possess physiological uses, for example, [6] 11-[Cyclohexyl ethanamide] Olean 9/11, 13/14-diene, revealed in its fragmentation pattern the amide function responsible for activity. The identified compounds revealed bio-protective potentials of V .doniana . In conclusion the leaves, stem bark and root bark of V. doniana have potentials for pharmacological and physiological functions for good eye health maintenance, treatment of diabetes, fertility, postpartum bleeding, infection, anemia, and immunity owing to high value of ascorbic acid in synergy with α-tocopherol and selenium
TABLE OF CONTENTS
Title page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table of Contents vi
List of
Tables ix
List of
Figures x
List of
Plates xi
Abstract xii
CHAPTER 1:
INTRODUCTION
1.1 Background of the Study 1
1.2 Statement of the Problem 3
1.3 Justification of the Research 3
1.4 Aim and Objectives 3
1.5 Scope of the Study 4
CHAPTER 2: LITERATURE REVIEW
2.1 Origin and Ecology of Vitex doniana 5
2.2 Scientific Classification of Vitex doniana 5
2.3 The Genus Vitex 6
2.4 Morphology of the Plant Vitex doniana 9
2.5 Ethnomedical
Uses of Vitex doniana 9
2.6 Biological Activity of Vitex doniana 10
2.7 Economic
Importance of Vitex doniana 11
2.8 Chemical Compounds Isolated from Vitex species 11
2.8.1 Compounds isolated from Vitex negundo 11
2.8.2 Compounds isolated from Vitexc anescens 12
2.8.3 Compounds isolated from Vitex doniana 13
2.9 Phytochemicals 15
2.9.1 Flavonoids 15
2.9.2 Saponins 16
2.9.3 Steroids 16
2.9.4 Tannins 17
2.9.5 Alkaloids 17
2.9.6 Phenols 17
2.9.7 Terpenes 18
2.9.7.1 Classes of terpenes 18
2.9.8 Glycoside 19
2.9.9 Lipids 20
2.9.9.1 Classes of lipids 21
2.9.9.2 Sterols 22
2.9.9.3 Phospholipids 23
2.9.9.4 Health effect of lipids 23
2.9.10 Vitamin 24
2.9.10.1
Classification of vitamins 25
2.9.10.2
Fat-soluble vitamins 25
2.9.10.3
Water-soluble vitamins 25
2.9.10.4
Health effects of vitamins 27
2.10 Atomic Absorption Spectroscopy 28
2.11 Mass Spectrometry 28
CHAPTER
3: MATERIALS AND METHODS
3.1 Materials 31
3.2 Methods 31
3.2.1 Experimental procedure 31
3.2.1 Collection and identification of plant
material 32
3.2.3 Preparation of plant sample 32
3.2.4 Extraction of plant material 32
3.2.5 Quantitative determination of phytochemical 34
3.2.6 Concentration
of chloroform extract 34
3.2.7 Vitamins determination 38
3.2.8 Mineral element determination 41
3.2.9 Proximate
composition determination of Vitex
doniana 47
3.2.10 Antioxidant activity determination: By trypsin
inhibitor analysis 50
3.2.11 Antimicrobial determination 51
3.2.12
Anti-diabetic determination 52
CHAPTER
4: RESULTS AND DISCUSSION
4.1 Phytochemical Results of Vitex doniaana Leaves, Stem Bark and Root
Bark 56
4.2 Vitamin
Composition of Vitex doniana Leaves, Stem and Root barks 59
4.3 Mineral
Elements Results of Vitex doniana Leaves, Stem and Root Barks 62
4.4 Proximate
Results of Vitex doniana Leaves, Stem and Root Barks 66
4.5 Structure
Elucidation of Compounds 70
4.5.1 Vitex doniana leaves 71
4.5.2 Vitex doniana stem structure analysis 98
4.5.3 Vitex doniana root bark structure analysis 110
4.6 Characteristic
Structural Functions of Peak Compounds 125
4.6.1 Key
structures and activity relationship of the keto-functions in [9]
and
[10] 126
4.6.2 Antinutritional
factor of V. doniana leaves: trypsin
inhibitor and
antioxidant
activity 127
4.6.3 Antidabetic
activity 128
4.6.4 Antimicrobial
activity 134
4.7 Evaluation
of Metabolites of V. doniana for
Medicinal Uses 136
CHAPTER
5: CONCLUSION AND RECOMMENDATION
5.1 Conclusion 138
5.2 Recommendations 138
5.3 Contributions to Knowledge 139
5.4 Suggestion for Further Studies 140
REFERENCES
APPENDICES
LIST
OF TABLES
2.1: Composition of Visionplus 27
4.1 Phytochemical Results of V. doniana Leaves, Stem and Root Barks 56
4.2: Vitamin Composition of V. doniana Leaves, Stem and Root Barks 59
4.3: Mineral Element Content of V. doniana 62
4.4: Proximate Composition of Vitex doniana 66
4.5: Phytochemicals
Identified from GC-MS Analyses of the Leaves,
Stem
Bark and Root Bark of Chloroform Extract of V.
dioniana 119
4.6: Phytochemicals
Identified from GC-MS Analyses of the Stem
Bark
of Chloroform Extract of V. dioniana 120
4.7: Phytochemicals
Identified from GC-MS Analyses of the Root Bark
of
Chloroform Extract of V. dioniana 121
4.8: Nature
and Characteristic Properties of the Hydrocarbons 123
4.9: Antinutritional
Factor of V. doniana Leaves: Trypsin
Inhibitor 128
4.10: Antidiabetic
Study of Activity of Chloroform Extract of
V.
Doniana Leaves
with albino rats 130
4.11: Antimicrobial
Activity of the Chloroform and Aqueous Leaf
Extract
of V. doniana 133
LIST OF FIGURES
4.1: Chromatogram
of V. doniana 71
4.2: GC/MS
Spectrum of Compound [6] 75
4.3: Fragmentation
Pattern of Compound [6] 76
4.4: GC/MS
Spectrum of Compound [7] 78
4.5: Fragmentation
Pattern of Compound [7] 79
4.6: GC/MS
Spectrum of COMPOUND [10] 80
4.7: Fragmentation
Pattern of Compound [10] 81
4.8: GC/MS
Spectrum of Compound [12] 82
4.9: Fragmentation
Pattern of Compound [12] 83
4.10 GC/MS
Spectrum for Compound [16] 84
4.11: Fragmentation
Pattern of Compound [16] 85
4.12: Fragmentation
Pattern of Compound [21] 86
4.13: GC/MS
Spectrum of Coumpound [22] 87
4.14: Fragmentation
Pattern of Compound [22] 88
4.15: 12-[(4-Methoxy
2-en 3-γ-Butyrolactone)] 4,4,8,10,14,17,20,20 Octamethyl
1,4,4a,5,6,6a,6b,7a,7b,8,8b,9,10,11,12,12a,14,14a octadecahydro
24 pic Ursan 12-en-3-one 91
4.16: Chromatogram
of V. doniana Stem Bark 98
4.17: GC/MS Spectrum of Compound
[19] 102
4.18: GC/MS spectrum of Compound [21] 105
4.19: GC/MS spectrum of Compound [22] 106
4.20: Chromatogram
of V. doniana Root Bark 110
4.21: GC/MS
Spectrum of Compound [26] 113
4.22: GC/MS Spectrum of Compound
[27] 17-[2-(4-Methyl 3-propenyl
cyclohexene 1-yl
4-propyl thiazole) lanost 3-ol 17-[2-(4-Methyl
3-propenyl
cyclohexene 1-yl 4-propyl thiazole) lanost 3-ol 115
LIST OF PLATES
1: V. doniana
Leaf 8
2: V. donianastem Bark 8
3: V.
doniana Root Bark 8
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND
OF THE STUDY
Owing
to the abundance of diverse native naturally occurring wild edible plants, all civilizations have always had traditions
of using plant to promote healing. Though,
herbal or traditional medicine tends to look primitive and unscientific
compared to synthetic drugs, the truth, however, is that the use of plants to
heal or combat illness or diseases is perhaps as old as humankind. On the other
hand, the biocides are derived from phytotoxic plant species classified as
botanicals that contain secondary metabolites as their active ingredient with diverse
chemical structures, and different mechanism of action (Amanze, 2010). So the
use of herbs for medicine, and pesticides has made plants and plant products to
remain as the basis of development of modern drugs and biocides and other plant
products (Gills, 1992; Uzoma, 2004).
Various plant parts have been
generally used for treatment of intractable diseases or disease conditions in
man from history (Uzoma, 2004). Although a significant number of modern
medicines include synthetic substances, many still contain drugs naturally
produced by plants, and as recent as 50 years ago the vast majority of
medicines used in the treatment of human diseases and ailments were
plant-produced (Stern, 1994). Those useful plants associated with medicinal
uses are tagged medicinal plants (Lewis and Elvin-Lewis, 1977). Man has used
plants to treat common infectious diseases. The use of herbal medicine to treat
illnesses has grown in the current technological era. Throughout the world,
fruits; stem bark and leaves are used to treat a variety of diseases and
advance humankind. Any particular plant that has been used and noted
as capable of boosting immunity, reduce incidences of cancer in regular users,
treat anaemia, gonorrhea, diarrhea, jaundice, leprosy, dysentery, liver
diseases, or improve fertility as the case may be, because its biological
activities has been ethnobotanically reported, such is therefore a medicinal
plant (Stern, 1994). Nonetheless, the demand from both public and medical
establishments makes it mandatory that a study which will lead to the
scientific explanation of the plant’s therapeutic capabilities be conducted for
it to gain increasing credibility and acceptance. Therefore, some of the
traditional medicines are still included as part of the habitual treatment of
various maladies (Rossier, 2006).
A medicinal plant’s root bark extract, juice from
leaves, and seed paste are all useful having efficacious secondary metabolites
and their derived products. The continuing emergence of drug-resistant-microbes
and the increasing evolutionary adaptations by pathogenic organisms to commonly
used antimicrobials have reduced the efficacies of several antimicrobial agents
currently in use. This is another way of saying that natural novel sources such
as plants remain and continue to be necessary where and when an ailment
develops resistance to treatment; plants are basis for many synthetic drugs (Sofowora,
1982; Maureer-Grimes et al., 1996;
Pascual et al., 2002), and natural remedy
is excellent when the herbal treatment is a preventive measure which is the
last strategy in fighting diseases. The effectiveness of a herbal treatment
depends largely on the presence of the various complex chemical substances of
different compositions which are found in the plant’s secondary metabolite
(Prajapati et al., 2003) and function
as defence against herbivores, microbes and competing plants (Wink, 1988).
The present study was carried out to isolate and
characterize bioactive compounds from the leaves, stem bark and root bark of V. doniana Sweet and to evaluate some of
the biological activities of their extracts.
1.2 STATEMENT OF THE PROBLEM
With morethan 270 species in the Vitex genus, Vitex doniana is a recognized species of plant. According
to phytochemical reports on Vitex species, they are abundant in terpenoids,
flavonoid glycosides, iridoids and ketosteroids (Ono et al., 2000). Iridoid glycosides with the names agnuside,
eurostoside, nishindaside and isonishidaside have been isolated from leaves and
fruit of some Vitex species as a result of research. Among the pharmacological
effects of the isolates include anti-inflammatory and analgesic activities. The
isolates’s pharmacological effects include analgesic and anti-inflammatory properties.
There is however, a dearth of published data regarding the V. doniana leaves, stem bark and root bark potential for
bioprotection. Data related to the chemical constituentss of bioactive
components from V. doniana are
scarce. Also, the claims made by local traditional herbalists on the use of V. doniana in herbal medicine for the
treatment of ailments are without scientific evidence and authentication
1.3 JUSTIFICATION OF THE STUDY
The leaves of V.
doniana are consumed not only as food but also traditionally employed
extensively to advance fruitfulness and stop postpartum bleeding in women and
to treat eye troubles. The leaves are also touted as materials for the
treatment of diabetes by local herbalists.The stem bark in powdered state are
used in tea infusion to eliminate backache while the root bark decoction is
used toimprove fertility. All these are based on personal communication and are
not documented, hence the need for this research.
1.4 AIM AND OBJECTIVES
This research is aimed at uncovering the
bioactive secondary metabolites present in the leaves, stem bark and root bark of
V. doniana and assessing some of
their bio-protective potentials. This was achieved through the following
specific objectives, to:
i)
determine the bioactive
constituents of the leaves, stem bark and root bark of V. doniana using GC/MS technique.
ii)
determine the proximate,
mineral and vitamin contents of the leaves, stem bark and root bark of V. doniana.
iii)
evaluate the
antimicrobial potency of the crude chloroform extracts of V. doniana.
iv)
evaluate the
anti-diabetic property of the crude extracts with a view to relating the
structure of identified peak compounds to functional activity.
v)
determine the antioxidant
activity of the crude extracts.
vi)
establish ethno-botanical
basis for the use of V. doniana leaves,
stem and root barks in the treatment of diseases and infections compared with
standard vision plus drug.
1.5 SCOPE OF THE STUDY
This coverage of this study targeted identification of
the peak compounds from the leaves, stem bark and root bark of V. doniana by Agilent GC/MS analytical
method backed up by fragmentation pattern and spectra of known standard
compounds.The antioxidant, antimicrobial and antidiabetic activities of the
secondary metabolites of the leaves and the mineral elements as well as the
proximate compositions of the leaves, stem bark and root bark of vitex species
common in South Eastern Nigeria were investigated. The bioprotective properties
and the uses of the leaves, stem bark and root bark were discussed with a view
to establishing the basis or otherwise of use of the plant in
ethnomedicine.
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