PHYTOCHEMICAL AND ANTIMICROBIAL SCREENING OF THE STEM BARK EXTRACTS) OF INDIGOFERA ARRECTA HOCHST EX A. RICH(FABACEAE)
Abstract
The powdered bark of Indigofera arrecta was exhaustively extracted with methanol and vacuum concentrated at 40 °C using a rotary evaporator. The extract was later subjected to solvent partitioning to obtain soluble extracts of n-hexane, ethyl acetate, chloroform and methanol. General phytochemical screening of fractions revealed the presence of secondary metabolites such as cardiac glycosides, steroids, terpenes, flavonoids and tannins. Antibacterial activity against Staphylococcus aureus, S. pyogenes, S.faecalis, S.typhi, E.coli C. ulcerans, P. vulgaris, and C. albicans were tested using tube dilution and agar diffusion methods as described by NCCLS. Antimicrobial activity results ranged from 20 mm to 40 mm for n-hexane extract, 16 mm to 21 mm for ethyl acetate extract, and 20 mm to 27 mm for methanol, as indicated by zones of inhibition of microbial growth. did. extract. MIC results for n-hexane, ethyl acetate, and methanol extracts ranged from 7.5 mg/mL to 15 mg/mL. The MIC of 15 mg/ml for the n-hexane extract against both Gram-positive and Gram-negative bacteria indicates that Indigofera arrecta has a broad activity spectrum. 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
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. According to the strictest definition, these are drugs or chemicals that can affect health, but are not considered essential nutrients.Essential nutrients are proteins, carbohydrates, fats, minerals and vitamins. . Some medicinal products found in plants include gedunin and nimbolide from Azadirachta indica (neem) (Khalid and Duddeck, 1993). Santonin, a sesquiterpenoid lactone, is found in Artemisia species growing in Asia, and quinine and alkaloids are found in the bark of cinchona trees. Penicillin-β-lactams are produced by Penicillium fungi (Finar, 2003), and reserpine-alkaloids are isolated from the Rauwolfia plant.
1.2 Medicinal plants
According to the biblical record, the prophet Ezekiel reported that the fruit was edible and the leaves healing (Ezekiel 47:
12). The use of medicinal plants therefore dates back thousands of years when the first humans used various plants to treat disease (Dey, 2006). Also called medicinal plants or medicinal herbs, they are used to treat and prevent certain ailments and ailments and are believed to be beneficial in health care.
Srivastava et al., (1996) previously noted that hundreds of plant species have medicinal properties, and four out of five of these plants are native to wild forests, while most are found in developing countries. I pointed out that it is native to plants. A medicinal substance or value may be present in any or all parts of a plant, such as roots, stems, backs, leaves, flowers, fruits or seeds. Indeed, despite all the advances in synthetic chemistry and biotechnology, plants are still an essential source of therapeutic-based medicines and natural products (Lawn, 1993).
Common medicinal plants found in our area include Azadirachta indica (neem), Ocimum gratissimum persea americana, Vernonia amygdalina, Alstonia boonei, Zanthoxylum gilletii, and Buchholzia coriacea.
1.3 Medicinal plant research
The effectiveness of these medicinal plants is due to the presence of phytochemicals belonging to a group of compounds called natural compounds. Natural products are compounds derived from living organisms, plants, animals and insects, and the study of natural products is the study of their structure formation, uses and purposes in living organisms. Pharmaceuticals or active ingredients obtained from natural products are usually secondary metabolites such as terpenes, flavonoids, saponins, alkaloids and their derivatives. Today, these compounds must be pure and highly characterized compounds through scientific research.
The study of medicinal plants involves the general screening of plants that have been botanically identified by a respected authority or botanical taxonomist and then collected at random or based on local reputation as medicinal plants. begins with the implementation of This screening primarily consists of solvent extraction and standard testing of extracts for the presence of classes of compounds or secondary metabolites such as alkaloids, saponins and phenolic compounds. This alone, if done competently and consistently, may not lead to the discovery of new biologically active compounds, but further study may lead to the discovery of plants with potential biological activity. (Adjanohoun et al., 1991, Farnsworth, 1996).
The extract is then fractionated with the aim of isolating the pure compounds. Modern separation techniques include all types of chromatography often guided by bioassays to separate active compounds. Chromatographic methods include absorption and partition chromatography on columns, thin layer and more recently high performance liquid chromatography. The structure of modern isolates is mainly elucidated by spectroscopic techniques. A connection can be identified as a known connection or a completely new connection.
1.3.1 Purpose
1. The purpose of this study was to justify or refute the plant’s alleged ethnomedical uses.
1.3.2 Purpose
I. Plant collection, identification of suitable plants, drying and powdering.
II. Extraction of plant powders with different solvents based on elution series, i. H. non-polar to most polar.
III. Extracts of this plant are subjected to phytochemical and antibacterial screening
Ⅳ An analytical separation involving several sequential steps of chromatographic techniques.
V. Structural Elucidation of Isolated Compounds Using Spectral Techniques
1.4:
Research scope and limitations
The scope of this study is:
1. Phytochemical screening, antibacterial, antifungal screening, isolation, characterization and structure elucidation of active ingredients of Indigofera arrecta.
1.5 Research Justification
The Need to Know the Active Ingredients of Indigofera arrecta Strains