ABSTRACT
Globimetula oreophila(Loranthaceae) growing on Citrus aurantifolia is a mistletoe used widely in various cultures for the treatment of cancer, hypertension, diabetes, arthritis, heart problems, insomnia, infertility and pneumonia.Shade-dried powdered plant materialwas extracted using three methods; cold maceration, Microwave assisted extraction (MAE) and Polarity based extraction (PBE). The extractions afforded nine extracts: Crude ethanol (CEE=21.40g) from maceration; hexane (HEX=5.0g), Chloroform (CHL= 5.6 g), ethyl acetate (EA=6.9g) and ethanol (EE=26g) from MAE; and ethyl acetate (EA2=7.0g), chloroform (CHL2=7.5g), chloroform-ethanol (CE=1.14g) and ethanol (EE2=15.6g) from PBE..Repeated gel filtration of extract CE on sephadex LH-20 column eluting with methanol resulted in the isolation of two flavonoids;7-methoxyquercetin and Quercetin. The structures of these compounds were elucidated on the basis ofspectroscopic techniques (UV, FTIR, 1D and 2D NMR) and comparison with literature data. Antimicrobial Studies was done using the agar well diffusion method which shows that CEE and EE1 had strong antimicrobial activity against all the tested organisms with zones of inhibition ranging from 10-27mmbut has no activity againstE. coli.All the extracts showed strong activity against K. pneumoniae except extract HC which has no activity against all the isolates. The isolated compounds showed activity against all the bacterial strains except E. coli and VRSA.The extracts and isolated compounds showed potent synergistic activity with ciprofloxacin against the tested organisms.The findings of the study suggests that all the extracts tested have strong antimicrobial activity which might be due to the isolated phytochemical constituents.
TABLE OF CONTENTS
Title Page —————————————————————————————————-i Declaration—————————————————————————————————ii Certification————————————————————————————————- iii Acknowledgement—————————————————————————————— iv Abstract——————————————————————————————————– v Table of Contents——————————————————————————————- vi List of Figures————————————————————————————————- x List of Tables————————————————————————————————- xii List of Plates————————————————————————————————- xiii Abbreviations———————————————————————————————- xiv CHAPTER ONE ——————————————————————————————– 1
1.0 Introduction ——————————————————————————————— 1
1.1 Infectious Diseases ————————————————————————————– 1 1.2 Combination Therapy ——————————————————————————— 3
1.3 Statement of Research Problem ……………………………………………………………………………….. 4
1.4 Justification …………………………………………………………………………………………………………….. 4
1.5 Aim …………………………………………………………………………………………………………………………. 5
1.6 Objectives of the Study …………………………………………………………………………………………….. 5
CHAPTER TWO ………………………………………………………………………………………………………….. 6
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2.0LITERATURE REVIEW …………………………………………………………………………………………. 6
2.1 Loranthaceae …………………………………………………………………………………………………………… 6
2.1.1 Ethnomedical uses of mistletoes (Loranthaceae Family) ……………………………………………… 7
2.2 Globimetula oreophila ………………………………………………………………………………………………. 7
2.2.1 Taxonomy ……………………………………………………………………………………………………………… 8
2.2.2 Ethno-medicinal uses of Globimetula oreophila and other species in the Genus …………….. 8
2.2.3 Biological activities found in the genus Globimetula …………………………………………………… 8
2.2.4 Phytochemistry of Globimetula oreophila and the genus Globimetula …………………………… 9
2.2.5 Flavonoids ……………………………………………………………………………………………………………… 9
2.2.6 Compounds isolated from species of Globimetula …………………………………………………….. 11
2.3 The host plant-Citrus aurantifolia (Rutaceae) ……………………………………………………………… 12
2.3.1 Citrus aurantifolia ………………………………………………………………………………………………… 12
2.3.2 Taxonomy ……………………………………………………………………………………………………………. 13
2.3.3Ethnomedical Uses of Citrus aurantifolia …………………………………………………………………. 14
2.3.4 Pharmacological Activities of Citrus aurantifolia ……………………………………………………… 15
2.3.5 Phytochemical Constituent of Citrus aurantifolia ……………………………………………………… 18
CHAPTER THREE …………………………………………………………………………………………………….. 23
3.0 MATERIALS AND METHODS …………………………………………………………………………….. 23
3.1 Materials ……………………………………………………………………………………………………………….. 23
3.1.1 Solvents/Reagents and Chromatographic Materials …………………………………………………… 23 3.1.2 Test Organisms————————————————————————————- 23
3.2Collection of Plant Material. ……………………………………………………………………………………. 23
3.3Extraction ………………………………………………………………………………………………………………. 23
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3.3.1 Cold Maceration……………………………………………………………………………………………………. 24
3.3.2 Microwave assisted extraction (MAE) …………………………………………………………………….. 24
3.3.3 Polarity Based Extraction (PBE) …………………………………………………………………………….. 24
3.4Preliminary Phytochemical Investigation…………………………………………………………………. 26
3.4.1 Test for Steroids/Terpenes ……………………………………………………………………………………… 26
3.4.2Test for flavonoids …………………………………………………………………………………………………. 26
3.4.3Test for Alkaloids ………………………………………………………………………………………………….. 27
3.4.4Test for tannins ……………………………………………………………………………………………………… 28 3.4.5 Test for Anthraquinones—————————————————————————– 28
3.4.6Test for saponins ……………………………………………………………………………………………………. 28
3.5Chromatographic Procedure …………………………………………………………………………………… 29
3.5.1Thin Layer Chromatography (TLC) …………………………………………………………………………. 29
3.5.2 Gel Filtration Chromatography ……………………………………………………………………………….. 29
3.6Spectroscopic techniques: ……………………………………………………………………………………….. 30
3.7Antimicrobial assays ……………………………………………………………………………………………….. 30
3.7.1 Determination of antimicrobial activity ……………………………………………………………………. 30
3.7.2 Combination Effect ……………………………………………………………………………………………….. 31
CHAPTER FOUR ……………………………………………………………………………………………………….. 32
4.0 RESULTS ……………………………………………………………………………………………………………… 32 4.1 Extraction——————————————————————————————– 32
4.2 Preliminary Phytochemical Investigation………………………………………………………………… 33
4.3 Thin Layer Chromatography …………………………………………………………………………………. 34
4.4 Gel Filtration chromatography of Chloroform- ethanol extract (CE) ………………………. 38
4.5 Isolation of SD1 and SD2 ………………………………………………………………………………………… 40
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4.6 Analysis of SD1 ………………………………………………………………………………………………………. 41
4.6.1 TLC analysis of SD1 ……………………………………………………………………………………………… 42
4.6.2Chemical test on compound SD1 ……………………………………………………………………………… 42
4.6.3Melting Point of SD1 ……………………………………………………………………………………………… 42
4.6.4Spectroscopic analysis of SD1 …………………………………………………………………………………. 42
4.7 Analysis of compound SD2 ……………………………………………………………………………………… 57
4.7.1 TLC analysis of SD2 ……………………………………………………………………………………………… 57
4.7.2Chemical test on compound SD2 ……………………………………………………………………………… 58
4.7.3Melting Point of SD2 ……………………………………………………………………………………………… 58
4.7.4Spectroscopic analysis of SD2 …………………………………………………………………………………. 58
4.8 Antimicrobial Assays ……………………………………………………………………………………………… 74 4.8.1 Antimicrobial Activity—————————————————————————- 74 4.8.2 Combination Effect——————————————————————————- 78
CHAPTER FIVE ………………………………………………………………………………………………………… 80
5.0 Discussions …………………………………………………………………………………………………………….. 80
CHAPTER SIX …………………………………………………………………………………………………………… 88
6.0 SUMMARY, CONCLUSION AND RECOMMENDATIONS …………………………………. 88
6.1 Summary ……………………………………………………………………………………………………………….. 88
6.2 Conclusion …………………………………………………………………………………………………………….. 88
6.3 Recommendations ………………………………………………………………………………………………….. 89
REFERENCES ……………………………………………………………………………………………………………. 90 APPENDIX ———————————————————————————————– 101
CHAPTER ONE
1.0 Introduction Plant kingdom has served as an inexhaustible source of pharmaceuticals from time immemorial (Parikh et al., 2005). The occurrence of natural products with medicinal properties has led to the widespread use of herbal remedies across the world. This is as a result of the development of several drugs and chemotherapeutics from medicinal plants (Gyawali, 2010). More studies have been conducted on the components and the pharmacological properties of these plants (Pieter and Vlietinck, 2005; Soares et al., 2005; Jung et al., 2006). There are over 10 dozens of drugs which are all single entities extracted from higher plants or modified further synthetically that are currently in use globally (Newman et al., 2000). Interest in medicinal plants as a re-emerging health aid has been fuelled by the rising costs of prescription drugs in the maintenance of personal health and well-being, and the bio-prospecting of new plant-derived drugs (Lucy and DaSilva, 1999). The primary benefits of using plant-derived medicines are that they are relatively safer than synthetic alternatives, offering profound therapeutic benefits at affordable costs (Iwu, et al., 1999). 1.1 Infectious Diseases Infectious diseases account for approximately one-half of all deaths in tropical countries (Iwu, et al., 1999); the World Health Organization estimates that nearly 50,000 people die each day throughout the world from infectious diseases (WHO, 2004). Different antibiotics exercise their inhibitory activity on different pathogenic organisms. However, the development and spread of resistance to currently available antibiotics is a worldwide concern.
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Antibiotics are defined as natural compounds, produced by microorganisms, with selective antibacterial activity that does not have any strong side effects on human (Chanda and Rakholiya, 2011). Their mechanism of action is either through killing the bacteria (bactericidal effect) or by inhibiting bacterial growth (bacteriostatic effect). The discovery of antibiotics had eradicated the infections that once ravaged humankind. But their indiscriminate use has led to the development of multidrug-resistant pathogens. For example, around 90–95% of Staphylococcus aureus strains worldwide are resistant to penicillin (Casal et al., 2005) and in most of the Asian countries 70–80% of the same strains are methicillin resistant (Chambers, 2001).
The increasing phenomenon of acquisition of resistance among microorganisms to antimicrobial drugs is attributed to the indiscriminate and improper use of current antimicrobial drugs (Usha et al., 2010). Clinically important bacteria are characterized not only by single drug resistance, but also by multiple antibiotic resistance (Levy, 2002). Drug resistance presents an ever increasing global health threat that involves all major microbial pathogens and antimicrobial drugs (Stuart and Bonnie, 2004; Olayinka et al., 2009); these are difficult to treat and are responsible for a variety of infectious diseases. The rate of emergence of antibiotic resistant bacteria is not matched by the rate of development of new antibiotics to combat them (Prescott and Klein, 2002). Antibiotics that work today may not work tomorrow. It is essential to investigate newer drugs to which there is lesser resistance (Sarkar et al, 2003). As resistance to old antibiotics spreads, the development of new antimicrobial agents has to be expedited if the problem is to be solved. However, record of rapid, widespread emergence of resistance to newly introduced
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antimicrobial agents indicates that even new families of antimicrobial agents will have a short life expectancy (Coates et al., 2002). The steadily increasing bacterial resistance to existing drugs is a serious problem, and therefore there is a dire need to search for new classes of antibacterial substances, especially from natural sources. Sometimes the use of single antibiotic does not produce the desired effective inhibitory effects and to overcome this, a combination of drugs often exercises their synergistic effect which surpasses their individual performance. An alternative therapy to treat antibiotic resistant microorganisms is the use of plant extracts. There are several reports on the antimicrobial activity of different plant extracts that were effective antimicrobics(Vaghasiya et al., 2008; Fisgin et al., 2009; Jeong et al., 2009; Darwish and Aburjai, 2010; Yoon et al., 2011). However, the problem of drug resistance is on the increase. The need of the hour is to develop still newer, useful and important antimicrobial agents (Sharma and Kumar, 2006; Negi and Dave, 2010); or new ways to treat the resistant microorganisms. An alternative approach is the use of combination therapy i.e. synergism between known antibiotics and bioactive plant extracts.
1.2 Combination Therapy
Combination therapy can be used to expand the antimicrobial spectrum, to prevent the emergence of resistant mutants, to minimize toxicity and to obtain synergistic antimicrobial activity, it could be an alternative to monotherapy for patients with invasive infections that are difficult to treat, such as those due to multi-resistant species and for those who fail to respond to standard treatment (Kamatou et al., 2006).
As high level acquired resistance to conventional antibiotics is frequent, it is reasonable to use combination therapy in order to achieve bactericidal synergism. The combination can
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be of different plant extracts or plant extracts with standard antibiotics or antibiotics with some chemicals. Such combinations against resistant bacteria will have different mechanisms of action and it may lead to new choices for the treatment of infectious diseases. Synergism is defined as a positive interaction created when two agents are combined and together they exert an inhibitory effect (on the targeted organisms) that is greater than the sum of their individual effects. Plant antimicrobials have been found to be synergistic enhancers in that though they may not have any antimicrobial properties alone, but when they are taken concurrently with standard drugs they enhance the effect of that Drug (Kamatouet al., 2006). 1.3 Statement of Research Problem Infectious diseases account for approximately one-half of all deaths in tropical countries (Iwu, et al., 1999). This may be due to poverty, increasing incidence of multiple drug resistance and side effects associated with currently available antimicrobials. The increasing phenomenon of acquisition of resistance among microorganisms to antimicrobial drugs is attributed to the indiscriminate and improper use of current antimicrobial drugs (Usha et al., 2010). 1.4 Justification
This work was conducted because of the dire need to search for newer and more potent antimicrobial agents to which there is lesser resistance in a world where there is an alarming increase in multidrug resistance by microorganisms.There are many advantages of using antimicrobial compounds from medicinal plants, such as fewer side effects, better patient tolerance, less expensive, acceptance due to long history of use, and being
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renewable in nature (Gur et al., 2006) and also higher plants represent a potential source of novel antibiotic prototypes (Parekh and Chanda, 2007). To the best of our knowledge, there is no reported work on the pharmacological activity of Globimetula Oreophila growing on Citrus aurantifolia. Also there is no report on the establishment of the phytochemical constituents of the plant as well as isolation and characterization of some bioactive compounds from the plant. 1.5Aim The aim is to isolate and characterize some of the bioactive compounds in the plantGlobimetula oreophilaand investigate their combination effect with Ciprofloxacin. 1.6 Objectives of the Study The aim will be achieved through the following objectives:
i. Preliminary Phytochemical screening of the extracts.
ii. Separation, purification and isolation of the bioactive constituents using chromatographic techniques.
iii. Characterization and structural elucidation of the isolated compound(s) using spectroscopic techniques (IR, UV 1H and 13C NMR and MS).
iv. Determination of the antimicrobial activity of the plant extracts and isolated compounds.
v. Investigation of the effect of extracts and isolated compound(s) on antibacterial activity of ciprofloxacin.
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