ISOLATION AND CHARACTERIZATION OF BIOACTIVE COMPOUNDS FROM STEM BARK EXTRACT OF Uapaca pilosa Hutch

 

Abstract

Uapaca pilosa (Hutch.) Used in parts of Africa to treat dysentery, menstrual cramps, fever, constipation, erectile dysfunction, skin infections, female infertility, hemorrhoids, rheumatism, vomiting, dental problems, and fatigue. The dried plant was extracted and the extract was subjected to phytochemical analysis using standard methods, revealing the presence of alkaloids, flavonoids, anthraquinones, tannins, saponins, steroids, terpenoids and glycosides. became. Extended silica gel column chromatography of the ethyl acetate fraction of the stem bark extract was the most active of all fractions, leading to the separation of the two compounds GF1 and GF2. Their identities were determined by analysis of 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 betulin and GF2 was beta-sitosterol. Antimicrobial screening of crude extracts and fractions using the agar-well diffusion method showed activity against Staphylococcus aureus, Shigella, Typhi, Bacillus subtilis and Escherichia coli. The zone of inhibition of plant extracts against selected microorganisms was 13 mm to 17 mm against Staphylococcus aureus, 10 mm to 14 mm against Bacillus subtilis, 12 mm to 15 mm against Shigella, and 12 mm to 15 mm against Escherichia coli. 15 mm to 18 mm and 10 mm. mm to 11 mm against Salmonella typhi. His MICs and MBCs of extracts, fractions, and isolated compounds were also determined. The minimum inhibitory concentration range was 6.25 mg/mL to 25 mg/mL for Staphylococcus aureus, 25 mg/mL for Shigella, 6.25 mg/mL for Bacillus subtilis, and 12.50 mg/mL for E. coli. is 12.50 mg/ml for Staphylococcus aureus, 50 mg/ml for Shigella, 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 Euphorbiaceae Family

CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1.0 Materials

3.1.1 Equipment

3.1.2 Thin 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 (Molisch’s 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 Magnesium 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

3.2.2.5 Test for Saponins (Frothing test)

3.2.2.6 Test for Glycoside (FeCl3 test)

3.2.2.7 Test for cardiac glycosides (keller-killani test)

3.2.2.8 Test for Steroids/Triterpenes

3.2.2.8.1 Liebermann-Burchard test

3.2.2.8.2 Salkowski test

3.2.2.9. Test for Alkaloids

3.2.3.0 Antimicrobial Studies of Extracts and Isolated components

3.2.3.1 Preparation of the Plants Extracts for antimicrobial screening

3.2.3.2 Preparation of culture media

3.2.3.3 Susceptibility Test

3.2.3.4 Minimum Inhibitory Concentration (MIC)

3.2.3.5 Minimum bactericidal concentration (MBC)

3.2.3.6 Minimum fungicidal concentration (MFC)

3.3.0 Column Chromatography

3.3.1 Chromatographic Separation

3.3.1.1 Column Chromatography of ethyl

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

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 19th 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, Azadirachta 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. Active compounds in plants vary from plant to plant due to the diversity of bioactivities (Sofowora, 1983; Kubmarawa et al., 2007; Krishnajah et al., 2009).

Traditional medicine has been established in many parts of the world for centuries. Numerous plants and herbs are used by practitioners of traditional medicine around the world. Practices are known to vary from country to country (Sofowora, 1984). Extracts from various plant parts (leaves, stem bark, and roots) of various higher plants have been used in the manufacture of medicinal herbs (Sofowora, 1983, 1984, 1993). Plant extracts are administered individually or as preparations to treat various ailments. In fact, over 75% of the world’s population rely on these various forms of preparations and herbal decoctions to treat infections (Robinson and Zhang, 2011). Phytochemical constituents are the basic raw material sources for building the pharmaceutical industry (Mothana and Lindequist, 2005; Wojdylo et al., 2007). Components present in plants play an important role in the identification of herbal medicines. Phytochemical screens are of great importance for identifying new sources of therapeutically and pharmacologically important compounds such as alkaloids, anthraquinones, flavonoids, phenolic compounds, saponins, steroids, tannins and terpenoids (Akindele and Adeyemi, 2007). Some plants, such as Aloe vera, Allium sativum, Maranta arundinacea, Pimpinella anismum, and Arnica montana, are the basis for the treatment of human diseases and have been reported as useful ingredients in the development of new drugs (Boudreau and Beland et al. , 2006; Bunyapraphatsara et al., 1996; Alan et al., 2006).

Legume Mucuna deeringiana for the treatment of Parkinson’s disease (dos Santos et al., 2012). Picrotoxin, extracted from Anamyrta cocculus, a tropical vine native to Southeast Asia, is used as a nervous system stimulant and for barbiturate poisoning (Abebe and Haramaya, 2013). Reserpine, extracted from the root of the snake root Rauwolfia serpentina, is used as a sedative to lower blood pressure and in India as a treatment for snakebites (Unnikrishnan, 2004). Eucalyptol, extracted from eucalyptus species, is a well-known antiseptic used as a throat remedy, cough syrup, ointment, ointment, and inhaler for bronchitis and asthma. Eucalyptus is used worldwide and is considered a globally available product (Eschler et al., 2000). Cultivation has replaced wild gathering for the supply of several important pharmaceuticals used in modern medicine. Madagascar rose periwinkle (Catharanthus roseus) is widely cultivated in Spain and Texas for the alkaloids vinblastine and vincristine used to treat childhood leukemia and Hodgkin’s disease (Bauer et al., 1996).

Perhaps the best-known example is aspirin, which is chemically related to the compound first extracted from willow leaves and bark. The antimalarial drug quinine, derived from the bark of his Cinchona ledgeriana, a South American tree, was first brought to Europe (where malaria was prevalent) in the early 17th century by Jesuit priests (Fruhstorfer et al., 2001). It was once noted that Oliver Cromwell died of malaria because he refused “Jesuit” treatment. Although synthetic guanine is currently being developed as a drug, the bark is still used to treat certain cardiac arrhythmias and is marketed as a known bitter in tonic water (Fruhstorfer et al., 2001).

Yohimbe bark, Pausinystalia yohimbe, is also widely used in traditional medicine systems in West Africa (Robber and Tyler, 1999).

Presentation of research topic
About half of the recorded deaths in tropical countries are mainly due to infectious diseases (Iwu et al., 1999). This may be related to increased bacterial resistance to antimicrobials (Ojiako, 2014). Therefore, there is a need to develop more convenient and highly active therapeutic antibacterial agents. Purpose of research
The aim of this research work is to isolate and characterize bioactive components present in plants.

Research goal
phytochemical screening of raw plant extracts,
antibacterial screening of raw plant extracts,
Isolation and identification of phytochemicals present in extracts and antimicrobial screening of isolated/identified compounds.
research justification
Uapaca pilosa (Hutch) has been used in traditional medicine in many tropical regions to treat protozoal, bacterial, and fungal infections. To our knowledge, studies of phytochemical and antimicrobial activity of Uapaca pilosa have not been evaluated. Therefore, there is a need to validate the ethnomedical uses of plants.

 

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