Phytochemical Analysis And In-Vitro Antimicrobial Studies Of Leaf Ethanol Extract Of Vernonia Amygdalina

ABSTRACT

The antimicrobial activities of Vernonia amygdalina ethanol and aqueous leaves extracts were investigated against some selected clinical isolates; Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella spp., and Candida albicans. Agar well diffusion technique was employed to determine the Minimum Inhibitory Concentration (MIC) of the extracts (mg/ml) against the tested clinical isolates. The result showed that Vernonia amygdalina leaves extracts possess strong antimicrobial activities against the tested clinical isolates with aqueous leaves extracts exhibiting significantly (P<0.01) better zone of inhibition ranging from (11.4 to 12.5mm) against the tested clinical isolates compared to ethanol leaves extracts which showed moderate activities (P<0.05) ranging from (10.8 12.4mm) at concentration of 100mg/ml, although the differences are not significant (P>0.05).The Minimum Inhibitory Concentration (MIC) of the aqueous and ethanol leaves extract for the different clinical isolates both ranged between 12.5 and 50mg/ml. The Phytochemical screening result revealed the presence of flavonoids, anthraquinones, saponins, alkaloids, tannins, cardiac        glycosides, steroids, terpenoids and cardenolide, identified in the extracts. The results of this study showed that Vernonia amygdalina leaves extracts can be used as potential herbs for drug development for the curing of pathogens tested in this study and these activities could be attributed to the presence of these secondary metabolites.

TABLE OF CONTENTS

COVER PAGE

TITLE PAGE

APPROVAL PAGE

DEDICATION

ACKNOWLEDGEMENT

ABSTRACT

CHAPTER ONE

INTRODUCTION

1.1      BACKGROUND OF THE STUDY

• STATEMENT OF THE PROBLEM
• AIM AND OBJECTIVES OF THE STUDY
• RESEARCH HYPOTHESIS
• RESEARCH QUESTIONS
• SIGNIFICANCE OF THE STUDY
• SCOPE OF THE STUDY
• PROJECT ORGANISATION

CHAPTER TWO

LITERATURE REVIEW

• REVIEW OF THE STUDY
• OVERVIEW OF A BITTER LEAF
• ORIGIN OF BITTER LEAF
• USES OF BITTER LEAF
• PROPERTIES OF BITTER LEAF
• BENEFIT OF BITTER LEAF
• COMPOUNDS ISOLATED FROM VERNONIA AMYGDALINA
• TRADITIONAL USES OF VERNONIA AMYGDALINA
• BIOLOGICAL ACTIVITIES OF VERNONIA AMYGDALINA
• CHEMOPREVENTIVE PROPERTIES OF VERNONIA AMYGDALINA

CHAPTER THREE

METHODOLOGY

• COLLECTION OF SAMPLES
• TEST ORGANISMS
• SAMPLE PREPARATION AND EXTRACTION
• STERILITY PROOFING OF THE EXTRACT
• STANDARDIZATION OF THE BACTERIAL CELL SUSPENSION

3.5.1 Determination of minimum inhibitory concentration (MIC) of the extract on the test organisms

3.5.2  Determination of zone of inhibition

3.6    PHYTOCHEMICAL ANALYSIS OF THE LEAVES EXTRACTS

3.6.1 Test for Flavonoids

3.6.2  Test for Alkaloids

3.6.3  Test for Saponins

3.6.4  Test for Phenol

3.6.5  Test for Glycosides

3.6.6  Test for Tannins

3.6.7  Test for volatile oils

3.6.8 Test for Steroids

3.6.9  Test for Terpenoids

3.6.10            Test for Anthraquinones

3.7      STATISTICAL ANALYSIS

CHAPTER FOUR

4.0  RESULTS AND DISCUSSION

CHAPTER FIVE

• CONCLUSION
• RECOMMENDATION

CHAPTER ONE

1.0                                                               INTRODUCTION

1.1                                                   BACKGROUND OF THE STUDY

With the recent trend of high percentage resistance of multiple- drug resistant microbial strains, efforts have been intensified by researchers to search for possible alternatives (Adetunji et al., 2013). Medicinal plants and traditional preparation with antimicrobial activities have been used extensively in the West African regions. These plants of medicinal important have been proven to be very effective even where treatments with antibiotics failed (Oshim et al., 2016). Vernonia amygdalina commonly called bitter leaf (English), Shuwaka (Hausa), Ewuro (Yoruba), Oriwo (Edo), and Olubu (Igbo) is a perennial shrub belonging to the family Asteraceae (Ghamba et al., 2014; Gashe & Zeleke, 2017). V. amygdalina is a shrub that can grows to 10 m tall with petiole leaf of about 6 mm in diameter and elliptic in shape and grows throughout tropical Africa and has been domesticated in various parts of West Africa including Nigeria, where it is locally used as vegetable in soups (Etim et al., 2012; Habtamu & Melaku, 2018). In some African countries including Nigeria, this plant species is traditionally used to treat many ailments including diabetes (Akah & Okafor, 1992), malaria, helminth infections, fever, (Magadula & Erasto, 2009), promote wound healing (Adetutu et al., 2011) and to treat microbial infections (Noumedem et al., 2013). Also, the Hausa tribe of the northern part of Nigeria used the root and twig of V. amygdalina to treat stomach-ache and gastrointestinal troubles (Akinpelu, 1999). It is also prescribed to nursing mother as it improves lactation (Anibijuwon et al., 2012).

The pharmacological study of V. amygdalina have been reported to demonstrated Antihelmitic and Antimalarial properties (Abosi & Raseroka, 2003), antitumorigenic properties (Izevbigie et al., 2004), analgesic and antipyretic activities (Tijjani et al., 2017), hypoglycemic and hypolipidaemic effect in experimental animals (Nwanjo, 2005). The reported biologically active phytoconstituents from V. amygdalina are alkaloids, flavonoids, terpenes, saponins, coumarins, xanthones, phenolic acids, lignans, steroids, anthraquinones (Tona et al., 2004), edotides (Izevbigie, 2003), and sesquiterpene lactone (Kupchan et al., 1969). Despite the vast traditional used of V. amygdalina, it is still considered among the under exploited crops of economic significance. Thus, this study is aimed at determining the antimicrobial activity and the phytochemical constituents of V. amygdalina collected from Lapai, Niger State, Nigeria.

1.2                                                  PROBLEM STATEMENT

Clinical drug are said to be traceable to the major cause of some chronic disease in most cases when it is wrongly taken and it is also costly. Because of these problems there is an urgent need to substantiate scientifically the roles of contemporary and native medicinal practices. This exercise will open up a new class of drugs which could be cheap, easily affordable, and with less or no side effects. Vernonia amygdalina has been ascertained to provide various culinary and medicinal properties, these medicinal properties exert bacteriostatic and bacteriocidal effect on some bacteria (Effraim et al., 2010). This study was carried out to determine the phytochemical and antimicrobial of properties ethanol extracts of Vernonia amygdalina.

1.3                                    AIM AND OBJECTIVES OF THE STUDY

The aim of the present study was to investigate the phytochemical analysis and antimicrobial studies of leaf ethanol extracts of Vernonia amygdalina. The objectives of the study are:

The specific objectives of this study where:

1. To assess the phytochemical components of the extracts from the leaf of Vernonia amygdalina
2. To investigate the in-vivo anti-microbial activities of extracts from Vernonia amygdalina leaf in laboratory at different concentration

• To study the general benefit of Vernonia amygdalina leaf extract.

1.4                                                   RESEARCH HYPOTHESIS

H₁: Extracts from the leaf of Vernonia amygdalina does contain phytochemical components

H₂: Extracts from the leaf of Vernonia amygdalina shows significant percentage of parasitaemia inhibition.

1.5                                                   RESEARCH QUESTIONS

1. What are the phytochemicals screening of bitter leaf?
2. What are the medicinal benefits of Vernonia amygdalina?

• What is the common name of Vernonia amygdalina and its uses?

1.6                                           SIGNIFICANCE OF THE STUDY

This study have exposed used us to different medicinal values of Vernonia amygdalina leaf extract which is said to be used to cure different bateria. This study will also serve as means of understanding the general benefit of Vernonia amygdalina (better leaf).

1.7                                                   SCOPE OF THE STUDY

This study analyses the phytochemical and antimicrobial properties of the leaf of Vernonia amygdalina. The antimicrobial activities of Vernonia amygdalina ethanol and aqueous leaves extracts were investigated against some selected clinical isolates; Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella spp., and Candida albicans. The Phytochemical screening is also carried out for the presence of flavonoids, anthraquinones, saponins,      alkaloids, tannins, cardiac glycosides,  steroids, terpenoids and cardenolide, identified in the extracts.

1.8                                                         PROJECT ORGANISATION

The work is organized as follows: chapter one discuss the introductory part of the work,   chapter two presents the literature review of the study, chapter three describes the methods applied, chapter four discusses the results of the work, chapter five summarizes the research outcomes and the recommendations.

CHAPTER FIVE

4.1                                    RESULTS AND DISCUSSION

Vernonia amygdalina (bitter leaves) is widely consumed in food and used in traditional medicine practice because of its immense medicinal properties which exert bacteriostatic and bacteriocidal effect on some bacteria (Effraim et al., 2010; Okafor et al., 2019).

This study revealed that Vernonia    amygdalina (bitter leaves) leaves extract possess strong antimicrobial       activities  against the tested clinical isolates with aqueous leaves extracts exhibiting  significantly (P<0.01) better zone of inhibition against the tested clinical isolates compared to ethanol leaves extracts which showed moderate activities (P<0.05) at concentration of 100mg/ml, although the differences are not significant (P>0.05). This is in conformity with the findings as reported by (Mountdipa, et al., 2010), that the leaves extract of Vernonia  amygdalina are effective against amoebic dysentery, gastrointestinal disorder and has antimicrobial and antiparasitic activity. The aqueous leaves extract in this study yielded better zone of inhibition (11.4 12.5mm) which was found to be more effective against all the tested clinical isolates compared to ethanol leaves extract with zone of inhibition (10.8 – 12.4mm), though the difference was not significant (P>0.05). This is also in agreement with the study reported by (Igile, et al., 2015) which revealed that aqueous leaves extract of bitter leaves are used as tonic for the treatment of various illnesses. Several evidences have also shown that herbalist and native doctors in Africa recommend Vernonia amygdalina        aqueous leaves extract for their patient for the treatment of varieties of ailments ranging from emesis, nausea,     diabetes, loss of appetite, dysentery and other gastrointestinal tract problems to sexual transmitted diseases and diabetes mellitus among others due to its broad spectrum of activities (Argheore, et al., 2010).

In addition, this study contradicts the findings as reported by Obi and Onuaha (2010) and Ogueke et al (2016) on their work on Garcinia kola extracts that, ethanol is the best solvent for the extraction of most active principle of medicinal properties in plant.

Moreover, the Minimum Inhibitory Concentration (MIC) of the aqueous and ethanol leaves extracts for the different clinical isolates both ranged between 12.5 and 50mg/ml.

Similarly, the aqueous bitter leaves extracts appeared to display higher zones of inhibition on Escherichia coli, Pseudomonas aeruginosa with zones of inhibition of 12.5 and 12.2mm respectively followed by Klebsiella spp and Candida albicans with 11.8mm zones of inhibition, and Staphylococcus aureus with the lowest zone of inhibition of 11.4mm. The ethanol bitter leaves extracts also showed significantly (P<0.05) wider zone of inhibition on Candida albicans 12.4mm compared to aqueous leaves extracts which showed moderate inhibitory effects. This showed that the plant extract have various medicinal values and the inhibitory effects of the leaves extract against the clinical pathogens can introduce the plant as potential herbs for drug development for the treatment of ailments caused by these pathogens(Suleiman,2011).

The Phytochemical Screening of the Vernonia amygdalina (bitter leaves) aqueous and ethanol leaves extract was also determined in this study. Phytochemical results of the leaves extracts revealed the presence of flavonoids, alkaloids, anthraquinones,  saponins, Tannins, cardiac glycosides, steroids, Terpenoids and Cardenolide. These Phytochemical compounds exert antimicrobial activity through various mechanisms. These secondary metabolites are known to be biologically active and therefore play significant roles in bioactivity of medicinal plants because the medicinal values of medicinal plant lies in these phytochemical compounds which produced a definite and specific action on the human body.

A study conducted by (Oloyede et al., 2011; Boyo et al., 2011) on Vernonia amygdalina (bitter leaves) revealed that the plant contained flavonoids, saponins, anthraquinones and alkaloids.

Similarly, Imaga et al (2013) also detected the presence         of alkaloids, tannins, saponins, cardiac glycosides         and flavonoids as the most preponderant in their study on aqueous bitter leaf extracts. Vernonia amygdalina leaves extracts was also screened to contain Sesquiterpene lactone , flavonoids, Steroids, glycoside and vernonioside         (Igile et al., 2010; Izevbigie, 2013; Cimanga et al., 2014).

Hitherto, secondary metabolites (tannins, saponins, steroids, flavonoids, alkaloids, cardiac glycoside, anthraquinones, terpenoids, etc) exert antimicrobial activity through different mechanism (Igbinosa et al., 2019).

Table.1 Antimicrobial activities of Vernonia amygdalina (bitter leaves) Ethanol leaves extracts on selected clinical isolates

Zone of Inhibition (mm)

Test organisms                                                 Ethanol extracts (mm)

Table.2 Antimicrobial activities of Vernonia amygdalina (bitter leaves)

Aqueous leaves extracts on selected clinical isolates

Zone of Inhibition (mm)

Test organisms                                                Aqueous extracts (mm)

Table.3 Minimum Inhibitory Concentration (MIC) of Vernonia    amygdalina (bitter leaves)

Ethanol and Aqueous leaves extracts on selected clinical isolates

Solvents                                               Test organisms

Mg/ml                             E.coli       P.aeruginosa   Klebsiella spp.     S.aureus             C.albicans

Ethanol extracts                   50               12.5                     50                     25             25

Aqueous extracts                50               25.0                      50                     12.5           25

Table.4 Phytochemical Screening of Vernonia amygdalina (bitter Leaves)

Ethanol and Aqueous leaves Extract

Phytochemical Screening Test                              Ethanol Extracts Aqueous Extracts Test                  Results                                                                                Results

Anthraquinone                                         + +                         + +

Flavonoids                                                 +                           + +

Saponins                                                     +                            +

Tannins                                                       +                            +

Alkaloids                                                    +                            +

Phenol                                                         _                            _

Cardiac Glycosides                                     +                            +

Volatile Oil                                                  _                            _

Cardenolide                                               +                            +

Steroids                                                      +                            +

Terpenoids                                                  +                            +

Keys:   + + = Abundantly Present

+ = Present in low concentration

– = Absent (not detected)

Flavonoids constituent exhibited a wide range of biological activities like antimicrobial, anti-inflammatory, anti- angionic, analgesic, anti-allergic, cystostatic and antioxidant properties, anti- cancer activities (Hodek et al., 2002; Edeoga et al., 2005) Parehk and Chanda (2007) reported that tannins are known to react with protein to provide the typical tannins effect which is important for the treatment of ulcer (Adegboye et al., 2008). Tannins have been found to form irreversible complex with proline-rich protein (Shimada, 2016) resulting in the inhibition of cell protein synthesis. Herbs that have tannins as their component are stringent in nature and are used for treating intestinal disorder such as diarrhea and dysentery (Dharmananda, 2013). This observation therefore supports the use of Vernonia amygdalina in herbal cure remedies.

Li and Wang (2013) reviewed the biological activities of tannins and observed that tannins have anticancer activity and can be used in cancer prevention, thus suggesting that Vernonia amygdalina has potential as a source of important bioactive molecule for the treatment and prevention of cancer. It is possible that steroids occurred as part of aglyconemoietics of other constituents of plant like saponins and alkaloids , steroidal compounds present in Vernonia amygdalina extract are of important and interest due to their relationship with various anabolic hormones including sex hormones (Okwu, 2011). Neumann et al (2014) also confirmed the antiviral properties of steroids, Quilan et al (2010) worked on steroidal extracts from some medicinal plant which exhibited antibacterial activities on some bacterial isolates.

Alkaloids which are one of the largest groups of phytochemical in plant have amazing effect on human and have led to the development of powerful pain killer medicine (Kam and Liew, 2012). One of the most common biological properties of alkaloids is their toxicity against cells of foreign organisms.

In conclusion, this study showed that Vernonia amygdalina (bitter leaves) extract exerted significant (P<0.05) antimicrobial activities against the tested clinical isolates and might be source of active antimicrobial agents for the development of drugs caused by these pathogens. However, the presence of these phytochemical compounds in Vernonia amygdalina (bitter leaves) leaves extract identified in this study could be attributed to the antifungal and antibacterial activities observed.

5.2   RECOMMENDATIONS

1. Based on this study, it is recommended that people should develop the habit of eating bitter leaf because it has immense medicinal values in the treatment of various human pathogens.
2. It is also recommended that the plant should be used for the development of drugs for the curing of pathogens tested in this study.
3. It is recommended that further study should be conducted on the mechanisms of action of other parts of the plant viz; root, stem, bark, e.t.c.