ISOLATION AND CHARACTERIZATION OF BIOACTIVE COMPOUNDS FROM STEMBARK EXTRACT OF UAPACA PILOSA HUTCH

ABSTRACT

Uapaca pilosa (Hutch.) a plant used in some parts of Africa in the treatment of dysentery, menstrual pain, fever, constipation, erectile dysfunction, skin infections, female sterility, pile, rheumatism, emetic, tooth-troubles and fatigue. The dried plant was extracted, the extract was subjected to phytochemical investigation using standard method revealed the presence of alkaloids, flavonoids, anthraquinones, tannins, saponins, steroids, terpenoids and glycocides. Extensive silica gel column chromatography of the ethylacetate fraction of the stem bark extract, the most active of all the fractions, led to the isolation of two compounds GF1 and GF2. Their identities were determined by analysis of their spectral data using FTIR, 1D and 2D NMR. The structures of the compounds were supported by comparing their spectral data with the literature. GF1 was found to be betulin while GF2 was found to be beta-sitosterol. The antimicrobial screening of the crude extract and fractions using agar well diffusion method showed activity against Staphylococcus aureus, Shigella dysenteriae, Salmonella typhii, Bacillus subtilis and Escherichia coli. The Zone of Inhibition of the plant extract against selected microorganisms ranges from 13mm to 17mm against

Staphylococcus aureus, 10mm to 14mm against Bacillus subtilis, 12mm to 15mm against Shigella dysenteriae, 15mm to 18mm against Escherichia coli and 10mm to 11mm againstSalmonella typhii. The MIC and MBC for the extract, fractions and isolated compounds were also determined. The range of Minimum Inhibitory concentration is between 6.25 mg/mL to 25 mg/mL for Staphylococcus aureus, 25 mg/mL for Shigella dysenteriae, 6.25 mg/mL for Bacillus subtilisand 12.50 mg/mL for

Escherichia coli while the Minimum Bactericidal Concentration range between 12.50 mg/mL for Staphylococcus aureus, 50 mg/mL for Shigella dysenteriae, 12.50 mg/mL for Bacillus subtilis and 25 mg/mL for Escherichia coli. This study on the stem bark

extract from Uapaca pilosa, used traditionally in some parts of Africa as a medicinal plant for the treatment of various ailments has confirmed that it has antimicrobial activity against the microbes that cause some of these diseases.

TABLE OF CONTENTS

Title Page

Abstract

Table of Contents

CHAPTER ONE

1.0       INTRODUCTION

1.1       Statement of the Research Problem

1.2       Aim of the Research

1.3       Objectives of the Research

1.4       Justification of the Research

CHAPTER TWO

2.0       LITERATURE REVIEW

2.1       The Euphorbiaceae Family

2.2       The Uapaca genus

2.3       Uapaca pilosa

2.4       Taxonomy of the Plant

2.5       Traditional Uses of Uapaca pilosa

2.6       Medicinal Importance of Other Uapaca Species

2.7       Some Compounds Isolated from Uapaca Species

2.8       Some Compounds Isolated from Euphorbeceae Family

CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1.0    Materials

3.1.1    Equipment

3.1.2Thin Layer Chromatography (TLC)

3.2.0    Methods

3.2.1    Extraction of Plant Material.

3.2.2    Preliminary Phytochemical Screening

3.2.2.1.            Test for Reducing Sugars (Molischs test)

3.2.2.2 Test for Tannins (Ferric Chloride test)

3.2.2.3 Test for Flavonoids (Shinoda test).

3.2.2.3.1 Magnessium Chips test

3.2.2.3.2 Sodium Hydroxide test

3.2.2.4 Test for Anthraquinones

3.2.2.4.1 Free Anthraquinones

3.2.2.4.2 Combined Anthraquinones

3.2.2.5Test for Saponins (Frothing test)

3.2.2.6 Test for Glycoside (FeCl3 test)

3.2.2.7 Test for cardiac glycoside (kella-killani test)

3.2.2.8 Test for Steroids/Triterpenes

3.2.2.8.1 Liebermann-Buchard test

3.2.2.8.2 Salkowski test

3.2.2.9. Test for Alkaloids

3.2.3.0Antimicrobial Studies of Extracts and Isolated components

3.2.3.1 Preparation of the Plants Extracts for antimicrobial screening

3.2.3.2Preparation of culture media

3.2.3.3Susceptibility Test

3.2.3.4Minimum Inhibitory Concentration (MIC)

3.2.3.5 Minimum bactericidal concentration (MBC)

3.2.3.6 Minimum fungicidal concentration (MFC)

3.3.0 Column Chromatography30 3.3.1Chromatographic Separation

3.3.1.1Column Chromatography of ethyl acetate Fraction of Uapaca pilosa

3.3.1.2Preparative Thin Layer Chromatography of the Sub-fractions (SF1 and SF2)

3.4 Melting Point Determination

3.5 Spectral Analysis32

CHAPTER FOUR

4.0 RESULTS

4.1 Result of Extraction of the Stembark of Uapaca pilosa

4.2. Result of Phytochemical screening

4.3       Result of antimicrobial activity of the plant extracts

4.4       Result of Chromatographic Separation

4.5       Column Chromatography of Ethyl acetate fraction

4.6       Thin Layer Chromatography Analysis of Isolated Compounds

4.7       Result of Thin layer Chromatography analyses of GF1 and GF2

4.9       Spectroscopic Analyses of GF1 and GF2

4.10     Antibacterial Activity of Isolated Compounds

4.10.1 Antimicrobial Activity of GF1 and GF2

CHAPTER FIVE

5.0       DISCUSSION

5.1       Extraction of the stem bark of Uapaca pilosa

5.2       Phytochemical Screening of the Stem bark of Uapaca pilosa

5.3       Antimicrobial Screening of Stem bark of Uapaca pilosa

5.4       Isolation, Purification and Characterisation of Isolates from Uapaca pilosa

5.4.1 Isolation and Characterisation of GF1

5.4.2 Isolation and Characterisation of GF2

CHAPTER SIX

6.0       SUMMARY, CONCLUSION AND RECOMMENDATIONS70

6.1       Summary

6.2       Conclusion

6.3       Recommendation

References

CHAPTER ONE

1.0              INTRODUCTION

Over the years the world traditional medicine has been known to take its source from higher plants and their extracts in the treatment of diseases and infections (Sofowora, 1983). Until 19^th century, when the development of chemistry and synthetic organic chemistry started, medicinal plants were the sources of active materials used in healing and curing human diseases. Before the advent of modern methods of producing drugs, medicinal plants such as Allium sativum, Azadirchata indica and Citrus limonum were used in treating both malaria and typhoid fever. Also some plant leaves were used in treating skin rashes and to heal wounds. Likewise, modern pharmaceuticals rely heavily on these medicinal plants for their raw materials such as cocoa leaves and opium plant from papaver species for analgesics. The active principles of plants differ from one plant to another due to the diversity in biological activities (Sofowora, 1983; Kubmarawa et al., 2007; Krishnaiah et al., 2009).

Traditional medicinal practice has been established for centuries in many parts of the world. Numerous plants and herbs are used globally by traditional medicine practitioners. The practice is known to vary from one country to another (Sofowora, 1984). Extracts from the various plant parts (leaves, stem bark and roots) of various higher plants are used in herbal medicine production (Sofowora, 1983, 1984, 1993). Plants` extracts are given singly or as concoctions for the treatment of various ailments. In actual sense more than 75% of the world population depend on these various forms of concoctions and herbal decoctions for the treatment of infections (Robenson and Zhang, 2011). Phytochemical constituents are the basic raw materials source for the establishment of pharmaceutical industries (Mothana and Lindequist, 2005; Wojdylo et al., 2007). The constituents present in the plant play vital roles in the crude drugs identity. Phytochemical screening is very important in identifying new sources of therapeutically and pharmacologically important compounds like alkaloids, anthraquinones, flavonoids, phenolic compounds, saponins, steroids, tannins and terpenoids (Akindele and Adeyemi, 2007).

Some plants such as Aloe vera, Alliium sativum, Maranta arundinacea, Pimpinella anisum and Arnica montana widely distributed in Africa, Asia and Southern part of North America have been reported to be the basis of treatment in human diseases and also as useful components in the development of new active components (Boudreau and Beland, 2006; Bunyapraphatsaraet al., 1996; Alan et al., 1995). The World Health Organization (WHO) estimates that 80 % of the world‟s population relies mainly on herbal medicine for primary healthcare (Hong et al., 2010). In China, traditional medicine is largely based on around 5000 plants which were used in treating 40 % of urban patients and 90 % of rural patients (Abdel-Azim et al., 2011). In industrialized countries, plants have contributed more than 7000 compounds used in the pharmaceutical industries including ingredients in heart drugs, laxatives, anti-cancer agents, hormones, contraceptives, diuretics, antibiotics, decongestants, analgesics, ulcer treatments and anti-parasitic compounds (Simo, 2012). About 25 % of all prescription drugs dispensed by Western pharmacists is likely to contain ingredients derived from plants (Simo, 2012). These include: Laevodopamine from tropical legume Mucuna deeringiana, used for treating Parkinson disease (dos Santos et al., 2012). Picrotoxin derived from Anaminta cocculus, a tropical climbing plant from south East Asia, is used as a nervous system stimulant and in cases of barbiturate poisoning (Abebe and Haramaya, 2013). Reserpine, extracted from the root of the serpent-root, Rauwolfia serpentine, is used for lowering blood pressure, as a tranquilizer and in India as a remedy for snake bites (Unnikrishnan, 2004). Eucalyptol obtained from species of eucalyptus, is a well-known antiseptic used in throat medicines, cough syrups, ointments, liniments, as inhalant for bronchitis and asthma. Eucalyptus is used throughout the world and is regarded as a universally available product (Eschler et al., 2000). Cultivation has replaced wild collection for the supply of some essential drugs used in modern medicine. The Madagascar rosy periwinkle (Cathrathus roseus) is widely cultivated in Spain and Texas for its alkaloids vinblastine and vinscristine, which are used for treating childhood leukaemia and hoolgkin disease (Bauer et al., 1996).

The best known example is probably aspirin, chemically related to a compound that was first extracted from the leaves and bark of willow tree, Salix alba and a herb meadow sweet, Filipino dula malaria. The anti-malarial drug quinine, extracted from the bark of a South American tree, Cinchona ledgeriana, was first brought to Europe (where malaria was widespread) in early 17^th century by Jesuit priests (Fruhstorfer et al, 2001). It was once remarked that Oliver Cromwell died of malaria because he refused to be treated with a “Jesuit” medicine. Synthetic guanine has now been developed for drug use, but the bark is still in use to treat certain heart arrhythmias and commercially sold as a bitter flavouring agent well known in tonic water (Fruhstorfer et al, 2001).

Also, the bark of yohimbe, Pausinnystalia yohimbe is used extensively in traditional healthcare system in West Africa (Robber and Tyler, 1999).

1.1              Statement of the Research Problem

About half of the number of death recorded in the tropical countries are largely due to infectious diseases (Iwu et al., 1999). This can be linked to the increasing bacterial resistance to antibacterial drugs (Ojiako, 2014). Hence there is need to develop a more convenient and very active therapeutic antimicrobial agents.