PHYTOCHEMICAL ANALYSIS AND ANTIMICROBIAL PROPERTIES OF CRUDE EXTRACTS OF VERNONIA AMYGDALINA DEL. ON MICROBIAL ISOLATES IMPLICATED IN TOMATO PASTE SPOILAGE.

ABSTRACT

The antimicrobial activities and preliminary phytochemical screening of crude aqueous extract of Vernonia amygdalina Del. was carried out against isolates obtained from spoilt tomato paste which included Escherichia coli, Pseudomonas aeruginosa, Micrococcus spp, Acinetobacter spp, Lactobacillus spp, Aspergillus spp, Rhizopus spp and Penicillium spp. The extract exhibited antibacterial and antifungal activity against the test organisms. The phytochemical screening of Vernonia amygdalina showed the presence of tannins, cardiac glycosides, saponins, flavonoids, steroids, terpenes, anthraquinones and alkaloids. V. amydalina exhibited antimicrobial action action against test organisms judged by their various zones of inhibition; P. aeruginosa (9.50mm), Micrococcus spp (16.50mm), Escherichia coli. (12.33mm), Acinetobacter spp (10.33mm), Lactobacillus spp (13.50mm), Aspergillus spp (12.50mm), Rhizopus spp (10.33mm) and Penicillium spp (14.50mm). The crude extracts exhibited strong potency against the bacteria and fungi with Penicillium spp and Micrococcus spp being the most susceptible when compared to the control (Chloramphenicol). Since this crude extract was able to inhibit the growth of the organisms associated with tomato paste spoilage, it could as well be used for the preservation of tomato paste.

TABLE OF CONTENTS

Title page i

Certification ii

Dedication iii

Acknowledgement iv

Table of Contents v-vi

List of Tables vii

Abstract viii

CHAPTER ONE

1.1 Background of Study 1

1.2      Aim and Objectives             4

CHAPTER TWO

2.0 LITERATURE REVIEW 5

2.1 The Nature of Food Spoilage 6

2.2 Significance  of   Agents  of  Tomato  Spoilage 9

2.3 Plant antimicrobials in food applications 9

2.4 Application of Plant Antimicrobials in Shelf-Life Extension

of Food 10

2.5 Vernonia amygdalina Del 11

2.6 Nutrient Composition/Nutritional Uses 12

CHAPTER THREE

3.0 MATERIALS AND METHODS 13

3.1 Collection of Plants Materials 13

3.2 Preparation of Plant Material and Plant Extracts 13

3.2.1 Cold Aqueous Extract 13

3.2.2 Preparation of media 13

3.2.3 Isolation of Bacteria 13

3.2.4 Isolation of Fungi 14

3.2.5 Purification (Subculture) of Bacterial Isolates 14

3.2.6 Purification of Fungal Isolates 14

3.3 Characterization and Identification of Bacterial Isolates 15

3.3.1 Antibacterial Assay 15

3.4 Phytochemical Screening of the Leaves Extracts 15

3.4.1 Test for Flavonoids 16

3.4.2 Test for Alkaloids 16

3.4.3 TestforSaponins 16

3.4.4 Test for Phenol 16

3.4.5 Test for Glycosides 16

3.4.6 TestforTannins 17

3.4.7 Test for volatile oils 17

3.4.8 Test for Steroids 17

3.4.9 Test for Terpenoids 17

3.4.10 Test for Anthraquinones 17

3.4.11 Test for Cardenolides Keller-KillianiTest 18

CHAPTER FOUR

4.0 RESULTS 19

CHAPTER FIVE

5.0 DISCUSSION 25

5.1      CONCLUSION 27

5.2 RECOMMENDATION 28

References 29

LIST OF TABLES

Table Title Page

1 Morphological, biochemical and sugar fermentation profile of

Bacterial isolates 20

2 Isolation and identification of fungal isolates 21

3 Mean Zone of inhibition of crude aqueous

Extract of V. amygdalina leaves against bacterial isolates                         22

4 Mean Zone of inhibition of crude aqueous

Extract of V. amygdalina leaves against fungal isolates                               23

5 Phytochemical Screening of Vernonia amygdalina (bitter leaves)

Ethanol and Aqueous leaves Extract 24

CHAPTER ONE

1.1 Background of Study

It has been noted that, despite the use of plant extracts in ethno-medicine, African cuisines and recently in plant protection, that their phytochemical composition and active ingredients have not been fully documented (Okwu and Njoku, 2009). Corroborative studies estimated that only 4 -10% of the 250,000 plant species constituting the biodiversity of the world’s flora have been examined chemically for antimicrobial activity (Pallant, 2010). A huge potential therefore exists in this regard especially in the tropical areas like Nigeria; with vast untapped rainforest, for these fungi-toxic plant species to be extensively examined chemically. Scientific analysis of plants it has been observed follows a logical pathway beginning with a lead from the natives. Hence, Cowan (1999) reported further that initial aqueous and alcoholic screening of plants extracts for antimicrobial activities are followed by other organic extraction methods for determination of their phytochemical com-positions. According to Wessells and Hopsons (1988), marvelous assortments of chemicals which are noxious to pathogens and even pests, have been found to be present in plants. These antimicrobial constituents (phytochemicals) include alkaloids, flavonoids, saponins, tannins, phenols, terpenoids, glycosides, anthraquinones, coumarins, polyphenols, Phlobatannin and steroids (Wessells and Hopsons, 1988; Edeoga et al., 2005; Okwu and Njoku, 2009; Soladoye and Chukwuma, 2012).

Natural products, such as plants extract, either as pure compounds or as standardized extracts, provide unlimited opportunities for new drug discoveries because of the unmatched availability of chemical diversity. According to the World Health Organization (WHO), more than 80% of the world's population relies on traditional medicine for their primary healthcare needs. The use of herbal medicines in Asia represents a long history of human interactions with the environment. Plants used for traditional medicine contain a wide range of substances that can be used to treat chronic as well as infectious diseases (Sasidharan et al., 2011). Due to the development of adverse effects and microbial resistance to the chemically synthesized drugs, men turned to ethnopharmacognosy. They found literally thousands of phytochemicals from plants as safe and broadly effective alternatives with less adverse effect. Many beneficial biological activity such as anticancer, antimicrobial, antioxidant, antidiarrheal, analgesic and wound healing activity were reported. In many cases the people claim the good benefit of certain natural or herbal products. However, clinical trials are necessary to demonstrate the effectiveness of a bioactive compound to verify this traditional claim. Clinical trials directed towards understanding the pharmacokinetics, bioavailability, efficacy, safety and drug interactions of newly developed bioactive compounds and their formulations (extracts) require a careful evaluation. Clinical trials are carefully planned to safeguard the health of the participants as well as answer specific research questions by evaluating for both immediate and long-term side effects and their outcomes are measured before the drug is widely applied to patients. According to the World Health Organization (WHO), nearly 20,000 medicinal plants exist in 91 countries including 12 mega biodiversity countries. The premier steps to utilize the biologically active compound from plant resources are extraction, pharmacological screening, isolation and characterization of bioactive compound, toxicological evaluation and clinical evaluation (Sasidharan et al., 2011).

Medicinal plants are plants which have a recognized medical use. They range from plants which are used in the production of mainstream pharmaceutical products to plants used in herbal medicine preparations. Herbal medicine is one of the oldest forms of medical treatment in human history, and could be considered one of the forerunners of the modern pharmaceutical trade. Medicinal plants can be found growing in numerous settings all over the world (Joy et al., 1998).

The exploitation of plants by man for the treatment of diseases has been in practice for a very long time. Herbal drug constitutes a major part in all the traditional system of medicines (Higa et al., 1994). Screening of compounds obtained from plants for their pharmacological assay has indeed been the vast source of innumerable therapeutic agents representing molecular diversity engineered by nature. It is therefore necessary and urgent to fight against emerging and reemerging infectious diseases. Further, newer strains are being continuously discovered which are refractory to the current arsenal of drugs (Erturk et al., 2006). The World Health Organisation (WHO) estimated that 80% of the population of developing countries rely on traditional medicine mostly plant drugs, for their primary health care needs. Medicinal plants are being natural, non-narcotic, having no side effect. Demand for medicinal plants is increasing in both developing and developed countries. Over the past few decades; there has been much interest in natural materials as source of new antibacterial agents. Different extracts from traditional medicinal plants have been tested. Many reports show the effectiveness of traditional herbs against microorganisms as a result plants have become one of the bases of modern medicine (Evans et al., 2002). Natural product of higher plants may give a new source of antibacterial agents with possibly a novel mechanism of action. The selection of crude plant extract for screening the antibacterial activity has the potential of being more successful in the initial steps than screening of pure compounds (Subhas et al., 2002).

1.2          AIM AND OBJECTIVES

The broad aim of this research work was to determine the microorganisms associated with tomato spoilage and their susceptibility to crude aqueous extract of Vernonia amygdalina Del.

To achieve this certain objectives were outlined, they include:

1. To determine qualitatively and quantitatively, the phytochemicals present in V. amygdalina Del.

2. To isolate and identify bacterial and fungal species associated with tomato spoilage.

3. To determine the inhibitory activity of aqueous extract of V. amygdalina on bacterial and fungal isolates from spoilt tomato.

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