PHYTOCHEMICAL AND ANTIMICROBIAL SCREENING OF THE STEM BARK EXTRACTS) OF INDIGOFERA ARRECTA HOCHST EX A. RICH(FABACEAE)

ABSTRACT

The Pulverized stem bark of Indigofera arrecta was 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. General 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.faecalis, S.typhi, 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 zones of 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 arrecta. 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-Stigmasta-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 Wavelength of maximum absorption

TLC Thin layer chromatography

δ Chemical shift in ppm

s Singlet

d Doublet

dd Double doublet

MHz Megahertz

% Percentage

Rf Retardation factor

HMBC Heteronuclear multiple bond correlation

HSQC Homonuclear single quantum coherence

COSY Correlation spectroscopy

DEPT Distortionless enhancement by polarization transfer

MIC Minimum inhibitory concentration

MBC Minimum 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.

1.2 Medicinal 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 include Azadirachta indica (Neem), Ocimum gratissimum persea americana, Vernonia amygdalina, Alstonia boonei, Zanthoxylum gilletii and Buchholzia coriacea 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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