ISOLATION AND NMR CHARACTERIZATION OF A SECONDARY METABOLITE FROM THE LEAVES OF EMILIA COCCINEA (SIMS) G.DON AND THEIR BIOACTIVITY STUDIES

ABSTRACT

Emilia coccinea is a medicinal plant of African origin that belongs to the family of Asteraceae. This plant has diverse and potential medicinal uses in traditional medicine for treating tumor, inflammation, cough, rheumatism, fever, dysentery, wounds and in preventing miscarriage. This study was undertaken to investigate the bioactive constituents, vitamin content, mineral composition and antibacterial properties of methanolic extract of Emilia coccinea leaf. The quantitative phytochemical screening of the extract revealed the presence of alkaloids (3.87 ± 0.02), phenols (0.43 ± 0.01), flavonoids (0.57 ± 0.01), saponins (0.39 ± 0.02), tannins (0.15 ± 0.01) with alkaloid registering the highest presence. Stigmasterol was isolated from the leaves of E. coccinea and was characterized using 1H, COSY, DEPT, HMBC and HSQC NMR Spectroscopic techniques. Vitamins detected included thiamin (0.12 ± 0.01 mg/100g), niacin (0.62 ± 0.05 mg/100g), riboflavin (0.34 ± 0.01 mg/100g), ascorbic acid (5.20 ± 0.80 mg/100g) and β-carotene (3.83 ± 0.05 mg/100g), with ascorbic acid and thiamine having the highest and lowest concentrations respectively. Minerals detected included calcium (27.24 ± 0.01 mg/100g), magnesium (5.42 ± 0.01 mg/100g), potassium (430.20± 0.01 mg/100g), phosphorus (140.36 ± 0.03 mg/100g), nitrogen (3.63 ± 0.07 mg/100g). Of the minerals detected, Phosphorus had the highest concentration, while nitrogen was the lowest. E.coccinea leaves extract exhibited significant free radical activity at minimum and maximum concentrations of 2.0 mg/ml and 12.0 mg/ml compared to the ascorbic acid used as a standard free scavenger These components are known to be medicinal as well as exhibiting physiological activities in humans. The antibacterial activity at varying concentrations of 100 mg/ml, 50 mg/ml, 40 mg/ml, 20 mg/ml and 10 mg/ml of the extract was investigated against Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa using agar well diffusion method. The results of the antimicrobial assay showed that the extract inhibited the growth of the test organisms in a concentration dependent manner. The highest growth inhibition was seen against B. subtilis and the lowest growth inhibition was against S. aerus with MIC of 5.00±0.00 mg/ml and 35.07±0.71 mg/ml respectively. Thus, the folkloric usage of this plant for the treatment of microbial infections is justified. The results of this research work revealed that Emilia coccinea has a lot of phytochemicals which could be used as raw material by pharmaceutical industries for drug development. The ability of the extract from this plant to exhibit potent inhibition against these pathogens lends a contributory scientific evidence for the use of this plant in traditional medicine.

TABLE OF CONTENTS

                                                                                  Page

Title page                                                                                                                    i

Declaration                                                                                                                  ii

Certification                                                                                                                iii

Dedication                                                                                                                  iv 

Acknowledgements                                                                                                    v

Table of Contents                                                                                                       vi

List of Tables                                                                                                              xii

List of Figures                                                                                                             xiii

List of Plates                                                                                                               xii

Abstract                                                                                                                      xiii

CHAPTER 1: INTRODUCTION 

1.1       Background of the Study                                                                               1

1.2       Statement of the Problem                                                                               2

1.3       Justification of the Study                                                                               2

1.4       Aim and Objectives of the Study                                                                   3

CHAPTER 2: LITERATURE REVIEW

2.1       Origin and Geographical Distribution of Emilia coccinea                             4

2.2       Description of Emilia coccinea                                                                       5

2.3       Asteraceae Family                                                                                         5

2.4       Ethnomedicinal uses of Emilia coccinea                                                        7

2.5       Pharmacological Activity                                                                                8

2.5.1    Antioxidant activity                                                                                        8

2.5.2    Antidiarrhoel activity and anti-microbial activity                                           9

2.5.3    Neuroprotective activity                                                                                 9

2.6       Economic Importance of Emilia coccinea                                                      10

2.7       Chemical Constituents of Emilia coccinea                                                     10

2.8       Phytochemicals                                                                                               14

2.8.1    Alkaloids                                                                                                         15

2.8.2    Flavonoids                                                                                                      18

2.8.3    Saponins                                                                                                          21

2.8.3    Tanins                                                                                                              23

CHAPTER 3: MATERIALS AND METHODS

3.1       Sample Collection and Preparation                                                                 26

3.2       Plant Preparation                                                                                             26

3.3       Extraction of Plant Material                                                                           26

3.4       Column Chromatography                                                                               27

3.5       Thin layer Chromatography                                                                            28

3.6       Quantitative Phytochemical Determination                                                    29

3.6.1    Determination of alkaloids                                                                             29

3.6.2    Determination of tannins                                                                                30

3.6.3    Determination of saponins                                                                              31

3.6.4    Determination of phenols                                                                               32

3.6.5    Determination of flavonoids                                                                           32

3.7       Determination of Vitamins                                                                             33

3.7.1    Determination of ascorbic acid                                                                       33

3.7.2    Determination of niacin                                                                                  34

3.7.3    Determination of riboflavin                                                                            35

3.7.4    Determination of thiamine                                                                              35

3.7.5    Determination of β-carotene                                                                           36

3.8       Determination of Mineral Elements                                                                37

3.8.1    Digestion of minerals                                                                                      37

3.8.2    Determination of sodium and potassium by flame photometry                     37

3.8.3    Determination of calcium and magnesium by complexiometric titration       38

3.8.4    Determination of phosphorus                                                                         39

3.9       Determination of Proximate Composition                                                      39

3.9.1    Determination of protein content                                                                   39

3.9.2    Determination of crude fibre content                                                             40

3.9.3    Determination of lipid content                                                                        41

3.9.4    Determination of moisture content                                                                 42

3.9.5    Determination of carbohydrate content                                                         42

3.10     Antioxidant Activity Determination                                                              42

3.11     Antimicrobial Analysis                                                                                   43

3.11.1 Obtaining and confirming the test organisms                                                 43

3.11.2  Antibacterial susceptibility                                                                             43

3.11.3 Determination of minimum inhibitory concentration                                      44

CHAPTER 4: RESULTS AND DISCUSSION

4.1       Phytochemical Screening Results                                                                   45

4.2       Proximate composition of Emilia coccinea                                                   47

4.3       Vitamin Content Results                                                                                50

4.4       Mineral Element Composition of Emilia coccinea                                         52

4.5       Column Chromatography Results                                                                   54

4.6       Thin – layer chromatography results                                                               55

4.7       Spectral Analysis Results                                                                                56

4.7.1    1H – NMR spectra result                                                                                56

4.7.2    2 – Dimensional NMR spectral interpretation                                                58

4.8       Antioxidant Activity Results                                                                          67

4.9       Antimicrobial Assay Results                                                                           69

CHAPTER 5: CONCLUSION AND RECOMMENDATION

5.1       Conclusion                                                                                                      72

5.2       Recommendations                                                                                          72

REFERENCES                                                                                                        73

LIST OF TABLES

4.1       Phytochemical Composition of E. coccinea                                                   45

4.2       Proximate Composition of E. coccinea                                                          47

4.3       Vitamin Composition of E. coccinea                                                              50

4.4       Mineral Elements Composition of E.coccinea                                                52

4.5       Column Chromatography Result of E. coccinea leaves extract                     54

4.6       ¹H-NMR Chemical Shift for Fraction A20                                                    56

4.7       DEPT NMR Chemical Shift for Fraction A20                                               58

4.8       Antioxidant Determination of Crude Extract from E. coccinea                    67

4.9       Zone of Inhibition of Methanolic Extract of E. coccinea Leaves Extract     69

4.10     Minimum Inhibitory Concentration of  E. coccinea Leaves  Extract             69

LIST OF FIGURES

4.1       ¹H-NMR for Fraction A20                                                                             62

4.2       ¹³C (DEPT) NMR for Fraction A20                                                               63

4.3       ¹H – ¹H – COSY Spectrum for Fraction A20                                                64

4.4       ¹H – ¹³C HSQC Spectrum for Fraction A20                                                  65

4.5       ¹H – ¹³C HMBC Spectrum for Fraction A20                                                 66

LIST OF PLATES

                                                                                                                        Page

Plate 1: Leaves of Emilia coccinea                                                                              7

CHAPTER 1

INTRODUCTION

1.1       BACKGROUND OF THE STUDY

Plants which form part of our environment generally have medicinal values. These days, most plants are languishing in obscurity due to scarcity of information on the bio-protective properties of these plants. So, the need to study medicinal plants according to (WHO 1978) cannot be overemphasized for a vista of reasons including the widespread use of plants in traditional medicine and bringing the knowledge of  plants to the fore as well as the need for health for all. Since the first earth summit in Rio de Janneiro, there has been a sustained global awareness on the importance of the plethora of biodiversity and natural resources from tropical forests for several purposes (Obute et al., 2002).

Recent research studies (Obadoni et al., 2001) have shown that plants are embodiment of important chemicals, which are bioactive in nature and are mostly essential to health. Among such groups of chemicals are carotenoids, flavonoids, vitamins, dietary fibre, minerals, amino acids, prebiotics, etc. Edeoga et al. (2005) reported that plants derive their medicinal property from these bioactive chemical compounds.

 Yellow tassel (Emilia coccinea) belongs to the family Asteraceae. The specie E. coccinea is an herbaceous plant, annual, weak-stemmed, to 1m high, flowers solitary, terminal, from Guinea to Nigeria, Cameroon and occurring through eastern African and into tropical Asia. It is an attractive plant of easy culture with golden-yellow heads. Emilia coccinea (SIMS)G.DON commonly known as scarlet tassel flower is one medicinal plant that has been used traditionally for medicinal purposes to treat a variety of ailments.   E. coccinea as a lesser known indigenous plant in Nigeria can be refined to serve as either health supplements or medicinal herbal formula targeted at treatment of diseases. It is reportedly used in folkloric medicine for the treatment of tumor, inflammation, cough, rheumatism, fever, dysentery, wounds and in preventing miscarriage (Ojiako et al., 2015). The juice of the edible leaves is reportedly used in treating eye inflammations, night blindness, and ear-aches. Several research works have been done to study the phytochemical components of Emilia coccinea and also on the antimicrobial activity of the plant (Nwachukwu et al.,2017).

1.2       STATEMENT OF THE PROBLEM

Emilia coccinea leaves has been widely used in traditional medicine to cure many ailments in different parts of the world, such as vertigo, ringworm, gonorrhea, measles, cough, convulsion etc. (Edeoga et al., 2005).

However, most of the compounds responsible for the physiological activities of this traditional remedy were not known. This has hindered the standardization and development of this herb and made its recognition, acceptance and utilization remain locally restricted.

This necessitated a probe into the isolation and characterization of the bioactive constituents available in the leaves.

1.3    JUSTIFICATION OF THE STUDY

E. coccinea can provide an alternative source of antimicrobial drugs. However, it can only be developed and standardized if its bioactivity is known and its bioactive principles identified and characterized. Therefore, there is need to investigate E. coccinea in order to understand better its chemical composition, properties and efficacy.

This research will provide a database for the active compounds isolated, the phytochemicals, minerals and vitamins present in the leaves, as well as the antimicrobial activities of E. coccinea leaves.

1.4       AIM AND OBJECTIVES

This research work is aimed at the isolation and characterization of secondary metabolites from the leaves of Emilia coccinea. This is to be achieved through the following objectives:

i.)                  To determine the phytochemical, constituents of E. coccinea

ii.)                 To determine the proximate composition of E. coccinea

iii.)              To determine the vitamins composition of  E. coccinea leaves.

iv.)              To determine the mineral elements composition of E. coccinea.

v.)                To determine the anti-oxidants and the anti-microbial properties of the leaves of E. coccinea

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