ABSTRACT
The Pulverized stem bark of Indigofera
arrectawas exhaustively extracted with methanol and concentrated in vacuo
using rotary evaporator at 40 0C.The
extract was later subjected to solvent partitioning to yield soluble extracts
of n-hexane, ethyl acetate, chloroform, and methanol. Genernal phytochemical
screening of the fractions revealed the presence of secondary metabolites such
as cardiac glycoside,steroid, terpenes flavonoids and tannins. The
antimicrobial activity against S. aureus,S. pyogenes,S.feacalis, S.typhii,
E.coli C. ulcerans,P. vulgaris and C.albicans was tested using the tube
dilution and agar diffusion methods as outlined by the NCCLS. The results of
the antimicrobial activity as indicated by the zonesof inhibition of growth of
microorganism ranged from 20mm to 40mm for the n-hexane extract, 16mm to 21mm
for ethyl acetate extract and 20mm to 27mm for the methanol extract. The MIC
result for the n-hexane, ethyl acetate and methanol extracts ranged from
7.5mg/ml to 15mg/ml. The MIC of 15mg/ml exhibited by the n-hexane extract
against both gram positive and gram negative bacteria indicates broad spectrum
activity of Indigofera arrect. The n-hexane fractions was subjected to
Column Chromatography using silica gel to yield 87 fractions, which were
combined based on their thin layer chromatography analysis and recrystallized
in methanol to give a pure white crystalline powder, which melts at 144oC.
The structure of the isolated compound was established by spectroscopic
analysis and by direct comparison of the data obtained with those reported in literature
to be Stigmasterol (3β,22E-Sigmasta-5,22-dien-3-ol).
TABLE OF CONTENTS
Title page
Abstract
Table of contents
List of abbreviations
CHAPTER ONE
1.0 Introduction
1.1 Definition of
a drug
1.2 Medicinal
plants
1.3 Medicinal
plant research
1.4 Aim
1.4.1 Objectives
1.5 Scope and
limitations of research
1.6 Justification
of the research
CHAPTER TWO
2.0 Literature
review
2.1 Botanical
description of the genus Indigofera arrecta
2.1.1 Other botanical
information
2.1.2 Origin and
geographical distribution
2.2 Chemical
constituents of indigofera arrecta
2.2.1 Traditional
medicinal uses
2.2.2 Uses of some of
the genus
2.3 Production and
international trade
2.3.1 Pharmacological
properties
2.3.2 Adulterations and substitutes
2.3.3 Growth and development
2.3.4 Ecology
2.3.5 Management
2.3.6 Propagation and
planting
2.3.7 Diseases and
pests
2.3.8 Harvesting
2.3.9 Yield
2.4 Handling after
harvest
2.4.1 General
description of Indigofera arrecta (HOCHST
EX.A.RICH )
2.5 Review of some
natural products from plants, tests and
their uses
2.5.1Alkaloids
2.5.2Flavonoids
2.5.3 Saponins
2.5.4 Glycosides
2.5.5 Tannins
2.5.6 Steroids
2.5.7. Terpenoids
2.6 Factors which
can affect the level or the composition of the active ingredients in medicinal
plant
2.7 Some
microorganisms and their effects on the human body
2.7.1 Staphylococci
2.7.2 Streptococci
2.7.3 Candida
2.7.4 Enterobacteriaceae
2.7.5 Klebsiella
2.7.6 Escherichia
coli
2.7.7 Salmonellae
CHAPTER THREE
3.0 Materials and
method
3.1 Materials/reagents/equipment
and analytical procedure
3.1.1 Solvents
3.1.2 Equipment
3.1.3 Reagent
3.1.4 Microbial
media, test organisms and equipment for antimicobial test
3.1.5 The
identification and preparation of plant material
3.1.6 Extraction
procedure for crude extract
3.2 Preliminary
phytochemical screening
3.2.1 Test for
steroids/terpenes
3.2.2 Test for
flavonoids
3.2.3 Test for
alkaloids
3.2.4 Test for
tannins
3.2.5 Test for
anthraquinones
3.2.6 Test for
saponins
3.2.7 Test for
glycoside (fecl3 test)
3.3 Antimicrobial
screening
3.3.1 Preparation of
bacterial test organisms
3.3.2 Preparation of
fungal test organisms
3.3.3 The stock
dilution of the plant extracts
3.3.4 Preparation of
the nutrient agar
3.3.5 Preparation of
the sabouraud dextrose agar media
3.3.6 The punched
agar diffusion method [bryant, 1972]
3.3.7 Preparation of
inoculums of test organisms
3.3.8 Sensitivity
test of the extract using agar diffusion method
3.3.9 Determination
of minimum inhibitory concentration using tube dilution method
3.4 Minimum
bactericidal concentration (mbc)
3.4.1 Chromatographic
purification of extracts
3.4.2 Thin layer
chromatography (TLC)
3.4.3 Column
chromatography
3.4.4 Solvents
system/elution
3.4.5 Gel filtration
chromatography
3.4.6 Thin layer chromatography
of the n-hexane extract
3.4.7 Column
chromatography of n-hexane fraction
CHAPTER FOUR
4.0 Results
4.1 Result of
extraction
4.2 Result of
phytochemical screening
4.3 Results of
antimicrobial activity
4.4 Result of
chromatographic separation
4.5 Column
chromatography of n-hexane fraction
4.6 Isolation of
EB
4.7 TLC analysis
of EB
4.8 Result of
antimicrobial activity of compound EB
CHAPTER FIVE
5.0 Discussion of
result
5.1 Extraction
5.2 Phytochemical
screening
5.3 Antimicrobial
screening
5.4 Physical and
chemical properties of EB
5.4.1 Spectral
analysis
5.4.2 FTIR
5.4.3 1H NMR
5.4.4 13C NMR
5.4.5 DEPT
CHAPTER SIX
6.0 Summary,
conclusion and recommendation
6.1. Summary
6.2 Conclusion
6.3 Recommendation
References
LIST
OF ABBREVIATION
cm Centimeter
cm-1 Per
centimeter
Hz Hertz
ml Milliliter
nm Nanometer
ppm Parts per
million
UV Ultra violet
IR Infrared
λmax Wave length
of maximum absorption
TLC Thin layer
chromatography
δ Chemical
shift in ppm
s Singlet
d Doublet
dd Double
doublet
MHz Mega hertz
% Percentage
Rf Retardation
factor
HMBC Hetero
nuclear multiple bond correlation
HSQC Homo nuclear
single quantum coherence
COSY Correlation
spectroscopy
DEPT Distortionless
enhancement by polarization transfer
MIC
Mimimum inhibitory concentration
MBCMimimum
bactericidal concentration
CHAPTER ONE
1.0
INTRODUCTION
1.1
Definition of a drug
A
drug can be described as any chemical substance that has no nutritional value
when
introduced into the body but
causes some physiological effects within the system (Mbah, 2000). Drugs are
classified under pharmaceuticals. Pharmaceutical drug, according to Dey (2006),
also refers to as medicine or medicament, can be loosely defined as any
substance intended for use in the diagnosis, cure, mitigation, treatment or
prevention of diseases.
Some
pharmaceuticals occur naturally in plants. These can be called phyto
pharmaceuticals. By the strictest definition, they are drugs or chemicals that
may have health related effects but are not considered essential nutrients.
Protein, carbohydrates, fats, minerals and vitamins are regarded as essential
nutrients. Some pharmaceuticals found in plants include gedunin and nimbolide
from Azadirachta indica (Neem) (Khalid and Duddeck, 1993); santonin, a
sesquiterpenoid lactone is found in species of Artemisia which grows in
Asia, quinine and alkaloid occurs in the bark of cinchona tree;
penincillin- a beta-lactam is produced by fungi in the genus Penicillium
(Finar, 2003) and reserpine-an alkaloid is isolated from Rauwolfia
plant.
1.2Medicinal plants
According to
biblical records, Prophet Ezekiel reported that the fruits will serve as food
and their leaves for healing (Ezekiel 47:12). Thus, the use of plants for
medicinal purposes dates back to thousands of years ago as the earliest humans
used various plants to treat illness (Dey, 2006). Medicinal plants
or healing herbs, as they are called, are used in treating and preventing
specific ailments and diseases and as such are considered to play a beneficial
role in health care.
Srivastava et
al., (1996) earlier stated that hundreds of plant species are recognized as
having medicinal values and four out of every five of those plants are
collected from the wild forest while most are from the flora of developing
countries. The medicinal properties or values may be present in one or all the
plants parts like roots, stem, back, leaves, flower, fruit or seeds.
In fact,
with all the progress in synthetic chemistry and biotechnology, plants are
still indispensable source of drugs and natural products on the basis of their
therapeutics (Lawn, 1993).
Some common medicinal plants that
occur in our locality includeAzadirachta indica
(Neem), Ocimum gratissiumpersea americana, Vernonica amygdaline, Astonia boonei,
Zanthoxyllium gilleti and
Bucchslozia coreacae among others.
1.3 Medicinal
plant research
The efficacy of these medicinal
plants is based on the presence of secondary metabolites which belong to a
group of compounds called natural products. Natural products are those chemical
compounds derived from living organisms, plants, animals, insects and the study
of natural products is the investigation of their structure formation,
applications and purpose in the organisms. The drugs or active ingredients
derived from natural products are usually secondary metabolites, for example,
terpenes, flavonoids, saponins and alkaloids and their derivatives. Today,
these compounds must be pure and highly characterized compounds through
scientific research.
Medicinal
plant research starts by people carrying out general screening of plants which
are collected either randomly or based on local reputations as medicinal plants
after botanically identified by a reputable authority or plant taxonomist. This
screening consists mainly of solvent extraction and standard tests of the
extracts for the presence of such class of compounds or secondary metabolites
as alkaloids, saponins and phenolic compounds. This in itself may not lead to
the discovery of any new biologically active compound if carried out
competently and consistently but it could provide data from which plants with
potential biological activity could be selected for further detailed study
(Adjanohoun et al., 1991,Farnsworth,1996).
The extracts
are then fractionated with a view of isolating pure compounds. Modern isolation
techniques include all types of chromatography often guided by bioassays to
isolate the active compounds. The chromatographic procedures include absorption
and partition chromatography in columns, thin layer and recently high
performance liquid Chromatography. The structures of the isolatein modern times
are elucidated primarily by spectroscopic techniques. The compounds can be
identified as already known compounds or entirely new compounds.
1.3.1 AIM
1.
The aim of this work was to justify or otherwise the claimed ethnomedicinal
uses of the plant
1.3.2 OBJECTIVES
I.
Collection, proper botanical identification, drying and
pulverizing of the plant.
II.
Extraction of the powdered plant
material using different solvents based on the elutropic series i.e from non-polar
to most polar.
III.The extract
of this plant
would be subjected
to phytochemical and
antimicrobial
screening
IV. Analytical separations involving several consecutive steps of chromatographic
techniques.
V.
Structural elucidation
of the isolated compound(s) using
spectral techniques
1.4:SCOPE AND LIMITATION OF THE RESEARCH
The scope of this research work
would be:
1.
The phytochemical screening,
antibacterial, antifungal screening, isolation, characterization and structural
elucidation of the active principles of Indigofera arrecta.
1.5 JUSTIFICATION
OF THE RESEARCH
The need to
know the active ingredients in the stem of Indigofera arrecta which are
responsible for the cure/treatment of various ailments such as epilepsy, sores
treatment, diarrhea and ulcer and to have scientific evidence of the claims of
the ethnomedicinal practices of the stem of Indigofera arrecta.
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