Plants as a potential source of antibiotics

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CHAPTER 2 LITERATURE REVIEW

Introduction

Plants have served as a source of new pharmaceutical products and inexpensive starting materials for the synthesis of many known drugs. Natural products and their derivatives represent more than 50% of the drugs in clinical use in the world (Cowan, 1999, Sofowora, 1984) (Table 2-2). Although the first chemical substance to be isolated from plants was benzoic acid in 1560, the search for useful drugs of known structures did not begin until 1804 when morphine was separated from Papaver somniferum L. (Pium). Since then many drugs from higher plants have been discovered, but less than 100 with defined structures are in common use. Less than half of these (Table 2-1) are accepted as useful drugs in industrialized countries (Farnsworth, 1984). Considering the great number of chemicals that have been derived from plants as medicine, scientific evaluation of plants used traditionally for the treatment of bacterial infection seems to be a logical step of exploiting the anti-microbial compounds, which may be present in plants. Plant-based anti-microbials represent a vast untapped source of medicines With enormous therapeutic potential (Cowan, 1999). They are supposedly effective in treatment of infectious diseases while simultaneously mitigating many of the side effects that are often associated with synthetic anti-microbials (Iwu et al., 1999)

Antibiotic resistance

Resistance to anti-microbial agents is recognized at present as a major global public health problem. Infective diseases account for approximately one-half of all deaths in tropical countries. In industrialized nations, despite the progress made in the understanding of microorganisms and their control, incidents of epidemics due to drug resistant microorganisms and the emergence of hitherto unknown disease-causing microbes, pose enormous public health concerns (Iwu et al., 1999).
Almost since the beginning of the antibiotic era, bacterial resistance has been seen as the major obstacle to successful treatment (Iwu et al., 1999). Microbial resistance to antibiotics in the clinic emerged soon after their first use in the treatment of infectious disease, and continue to pose a significant challenge for the health care sector. Resistance has now firmly emerged as a problem in the wider community. At the end of the 1960s the Surgeon General of the United States stated that: “we could close the book on infectious diseases.” At the time he uttered these words the emergence of resistance did not seem to affect therapeutic options. Although S. aureus had become resistant to benzylpenicillin and showing resistance to thethincillin, it remained sensitive to gentamicin and infections could therefore still be treated. At the start of the next century, things looked very different. Already at least three bacterial species, capable of causing life-threatening illness (Enterococcus faecalis, Mycobacterium tuberculosis and Pseudomonas aeruginosa), had become resistant to every one of the 100 antibiotics, available except for vancomycin (Iwu, 1999). Vancomycin is the antibiotic of last resort for treatment of resistant infections and within the past year scientists have found strains of Streptococcus pneumoniae and S. aureus to be resistant to this antibiotic. This is attested by the spread, with associated deaths, of infection by methicillin-resistant Staphylococcus aureus and the increased prevalence of drug-resistant S. pneumoniae in patients suffering from pneumonia. Anti-microbial resistance is driven by inescapable evolutionary pressures and is therefore predictable and inevitable. The emergence in the past year of vancomycin-resistant S. aureus an event that has been anticipated for the past decade with great dread, punctuates this assertion. Hardly any group of antibiotics has been introduced to which some bacterium has not developed resistance (Iwu et al., 1999).
Recent reports have shown a marked increase in antibiotic resistance of food-poisoning bacteria due to non-rational and excessive use of antibiotics as therapeutic agents or as growth promoters in livestock. Another factor of resistance potentially lies in the use of antibiotic resistant genes as selection markers in genetically modified organisms (GMOs) (http://www.biosafety. ihe.de 1999). The main safety issue of concern is the release of these resistant genes to sensitive organisms when these GMOs are introduced into the environment.
Due to emergence of drug resistant bacteria, the search for new antibacterial compounds with improved activity is necessary (Harold and Heath 1992a). Many indigenous plants are used in treating bacterial related diseases. Only a small fraction of these indigenous plants has been investigated (Carr and Rogers 1987).
Understanding of the mechanism of action of resistance development remains the foundation of new cycles of antibiotic discovery. Such events demonstrate that antibiotic management and new discovery must continue in the face of these pressures.

Natural products in drug discovery

Medicinal plants use is widespread (Farnsworth, 1991). The production of medicines and the pharmacological treatment of diseases began with the use of herbs (Tyler, 1997). Life saving and essential drugs from medicinal plants such as morphine, digoxin, aspirin, emetine, and ephedrine were introduced into modern therapeutics several centuries ago. However, plants have been used as drugs for over millenia by human beings. Plants historically have served as models in drug development for some major reasons: The first being that each plant is a unique chemical factory capable of synthesizing large numbers of highly complex and unusual chemical substances. In the United States of America alone, about 25% of popularity in the use of plant-derived preparations (Farnsworth and Morris, 1976). It has also been estimated by the World Health Organization (WHO) that about 80% of the population of the developing countries rely exclusively on plants to meet their health care needs (Farnsworth et al., 1985).
The second reason involves biologically active substances derived from plants have served as templates for synthesis of pharmaceuticals. Such compounds may have poor pharmacological and toxicological profiles. While the reason concerns the fact that highly active secondary plant constituents have been instrumental as pharmacological tools to evaluate physiological processes (Farnsworth, 1984). There are numerous illustrations of plant-derived drugs.
Despite the expense involved in the development of a drug today, at least US$230 million and a time span between 10 – 20 years (Farnworth, 1984), nature remains the most reliable and most important source of novel drug molecules. Nature provides 80% of all pharmacological and therapeutic lead compounds and the NCI estimates that over 60% of the compounds currently in pre-clinical and clinical development in its laboratories are of natural origin. Thus higher plants remain an important and reliable source of potentially useful chemical compounds not only for direct use drugs, but also as unique prototypes for synthetic analogues and as tools that can be used for a better understanding of biological processes (Farnsworth, 1984).
Literally thousands of phytochemicals with inhibiting effects on microorganisms have shown in-vitro activity. One may argue that these compounds have not been tested in vivo and therefore activity cannot be claimed, but one must take into consideration that many, if not all, of these plants have been used for centuries by various cultures in the treatment of diseases. Another argument could possibly be that at very high concentrations, any compound is likely to inhibit the growth of microorganisms. Firstly, if this is the case, the high concentrations required would no doubt have serious side effects on the patient unfortunate enough to contract an illness. Secondly, these compounds are compared with those of standard antibiotics already available in the market. This means that the concentrations used must compare favourably to those that have already passed the test. A summary of useful anti-microbial phytochemicals is given in Table 2-2 (Cowan, 1999).
Asiaticode, an anti-microbial compound isolated from Centella asiatica (used traditionally in skin diseases and leprosy), has been studied in normal as well as delayed-type wound healing. The results indicated significant wound healing in both models. Another compound, cryptolepine, isolated from Crytolepis sanguinolenta and active against Campylobacter species, has been used traditionally in Guinea Bissau in the treatment of hepatitis and in Ghana for the treatment of urinary and upper respiratory tract infections and malaria.

Plants as a potential source of antibiotics

The use of medicinal plants is widespread (Farnsworth, 1994). The production of medicines and the pharmacological treatment of diseases began with the use of herbs (Tyler, 1997). Life saving and essential drugs from medicinal plants such as morphine, digoxin, aspirin, emetine, and ephedrine were introduced into modern therapeutics several centuries ago. However, plants have been used as drugs for over millenia by human beings.
Other than for purposes of scientific inquiry, plants historically have served as models in drug development for three reasons: (a) Each plant is a unique chemical factory capable of synthesizing large numbers of highly complex and unusual chemical substances. In the United States of America alone, about 25% of prescription drugs contain active principles that are still extracted from higher plants and there is increasing popularity in the use of plant-derived preparations (Farnsworth and Morris, 1976). It has also been estimated by the World Health Organization (WHO) that about 80% of the population of the developing countries rely exclusively on plants to meet their health care needs (Farnsworth et al., 1985). (b) The biologically active substances derived from plants have served as templates for synthesis of pharmaceuticals. Such compounds may have poor pharmacological and toxicological profiles. (c) Many highly active secondary plant constituents have been instrumental as pharmacological tools to evaluate physiological processes (Farnsworth, 1984).
There are numerous illustrations of plant-derived drugs. Some selected examples are presented in Table 2.
The isoquinoline alkaloid emetine obtained from the underground part of Cephaelis ipecacuanha and related species has been used for many years as an amoebicidal drug as well as for the treatment of abscesses resulting from Escherichia histolytica Another important drug of plant origin with a longhistory of use is quinine. This alkaloid occurs in the bark of the cinchona tree. Apart from its usefulness in the treatment of malaria, it can be used to relieve nocturnal leg cramps (Iwu et al., 1999).
Similarly, higher plants have also played important roles in cancer therapies. Recent examples include, combretastatins from Combretum caffrum (Pettit and Shigh, 1987). In the last two decades a series of stilbenes and dihydrostilbenes (the combretastatins) with potent cytototoxic activity, and acidic triterpenoids and their glycosides with molluscicidal, antifungal, antimicrobial activity, have been isolated from species of Combretum (Rogers, 1989b). Other antineoplastic agents include taxol and several derivatives of camptothecin from Taxus brevifolia and Camptotheca acuminate, respectively.

Plants and antibacterial production

An antibiotic has been defined as a chemical compound derived from or produced by living organisms, which is capable, in small concentrations of inhibiting the growth of micro-organisms (Evans, 1989). This definition limited antibiotics to substances produced by microorganisms but the definition could now be extended to include similar substances present in higher plants. Plants have many ways of generating antibacterial compounds to protect them against pathogens (Kuc, 1990). External plant surfaces are often protected by biopolymers e.g. waxes, and fatty acid esters such as cutin and suberin. In addition, external tissues can be rich in phenolic compounds, alkaloids, diterpenoids, steroid glycoalkaloids and other compounds, which inhibit the development of fungi and bacteria (Kuc, 1985). Cell walls of at least some monocotyledons also contain antimicrobial proteins, referred to as thionins (Carr and Klessig 1989).
Plant cells containing sequestered glycosides release them when ruptured by injury or infection. These glycosides may have antimicrobial activity against the invading pathogens or may be hydrolyzed by glycosidases to yield more active aglycones. In the case of phenolic compounds, these may be oxidized to highly reactive, antimicrobial quinones and free radicals (Kuc, 1985; Dean and Kuc, 1987). Thus, damage to a few cells may rapidly create an extremely hostile environment for a developing pathogen. This rapid, but restricted disruption of a few cells after infection can also result in the biosynthesis and accumulation of phytoalexins, which are low molecular weight anti-microbial compounds, which accumulate at sites of infection (Kuc, 1985; Carr and Klessig, 1989; Dean and Kuc, 1987). Some phytoalexins are synthesized by the malonate pathway others by the mevalonate, or shikimate pathways, whereas still others require participation of two or all three of the pathways. Phytoalexins are degraded by some pathogens and by the plant; thus they are transient constituents and their accumulation is a reflection of both synthesis and degradation rates.
Biopolymers are also often associated with the phytoalexin accumulation at the site of injury or infection. These biopolymers include: lignin, a polymer of oxidized phenolic compounds; callose, a polymer of β-1, 3-linked glucopyranose; hydroxyproline-rich glycoproteins, and suberin. They provide both mechanical and chemical restriction of development of pathogens (Kuc 1985; Carr and Klessig, 1989; Rao and Kuc, 1990).
The macromolecule produced after infection or certain forms of physiological stress includes enzymes, which can hydrolyse the walls of some pathogens including chitinases, β-1,3-glucanases and proteases (Carr and Klessig, 1989). Unlike the phyoalexins and structural biopolymers, the amounts of these enzymes increase systemically in infected plants even in response to localized infection. These enzymes are part of a group of stress or infection-related proteins commonly referred to as pathogenesis-related (PR) proteins. The function of many of these proteins is unknown. Some may be defense compounds while others may regulate the response to infection (Carr and Klessig, 1989; Boller, 1987; Rao and Kuc, 1990).
Another group of systemically produced biopolymer defense compounds comprises the peroxidases and phenoloxidases (Hammerschmidt et al., 1982; Rao and Kuc, 1990). Both can oxidize phenols to generate protective barriers to infection, including lignin. Phenolic oxidation products can also cross-link to carbohydrates and proteins in the cell walls of plants and fungi to restrict further microbial development (Stermer and Hammerschmidt, 1987). Peroxidases also generate hydrogen peroxide, which is strongly antimicrobial. Associated with peroxidative reactions after infection is the transient localized accumulation of hydroxyl radicals and super oxide anion, both of which are highly reactive and toxic to cells.
Plants therefore have several mechanisms to counter anti-microbial attack. Some of the anti-microbial compounds in plants may be exploited for use against bacterial diseases in man. Plants have developed an arsenal of weapons to survive attacks by microbial invasions. These include both physical barriers as well as chemical ones, i.e. the presence or accumulation of anti-microbial metabolites. These are either produced in the plant (prohibitins) or induced after infection, the so-called phytoalexins. Since phytoalexins can also be induced by abiotic factors such as UV irradiation, they have been defined as ‘antibiotics formed in plants via a metabolic sequence induced either biotically or in response to chemical or environmental factors (Grayer et al., 1994).
When an infection or damage to a plant takes place, a number of processes are activated and some of the compounds produced become activated immediately whereas phytoalexins take two three days to be produced. Sometimes it is difficult to determine whether the compounds are phytoalexins or prohibitins and moreover, the same compound may be a preformed anti-microbial in one species and a phytoalexin in another (Grayer et al., 1994). Since the advent of antibiotics in 1950s, the use of plant derivatives as anti-microbials has been virtually non-existent but that pace is rapidly on the increase as we begin to realize the need for new and effective treatments. The worldwide spending on finding new anti-infective agents is expected to increase 60% as from 1993 and plant source are especially being investigated (Grayer et al., 1994).

DECLARATION 
ACKNOWLEDGEMENTS 
LIST OF ABBREVIATIONS USED 
ABSTRACT
PAPERS PREPARED FROM THIS THESIS
CONFERENCES AND PROCEEDINGS
CHAPTER 1. INTRODUCTION
Introduction
1.2 Hypothesis
1.3 Aim of research
CHAPTER 2: LITERATURE REVIEW
2.1 Introduction
2.2 Antibiotic resistance
2.3 Natural products in drug discovery
2.4 Plants as a potential source of antibiotics
2.5 Plants and antibacterial production
2.6 The familly Combretaceae
2.7 Ethnopharmacology of Combreteceae
2.8 Phytochemistry/chemistry and biological activity of Combretaceae
2.9 Methods developed and results obtained in the phytomedicine programme
CHAPTER 3: PLANT COLLECTIONS, EXTRACTION AND ANALYSIS
Introduction
3.2 Material and Methods
3.3 Results
3.4 Discussion and conclusion
3.5 Summary
CHAPTER 4: BIOLOGICAL ASSAYS FOR PRELIMINARY SCREENING 
4.1 Introduction
4.2 Materials and methods
4.3 Results
4.4 Discussion and conclusion
4.5 Summary
CHAPTER 5: PRELIMINARY SEPARATION AND ISOLATION OF BIOACTIVE COMPOUNDS
5.1 Introduction
5.2 Materials and methods
5.3 Results and discussions
5.4 Summary
CHAPTER 6: INSTRUMENTAL ANALYSIS AND STRUCTURAL ELUCIDATION OF ISOLATED COMPOUNDS
6.1 ntroduction
6.2 Nuclear magnetic resonance spectroscopy [NMR ]
6.3 Mass spectrometry
6.4 IR
6.5 Results and Discussions
6.6 Summary
CHAPTER 7: BIOLOGICAL CHARACTERIZATION OF ISOLATED COMPOUNDS
7.1 Introduction
7.2 Material and methods
7.3 Results and Discussion
7.4 Summary
CHAPTER 8: GENERAL CONCLUSION
8.1Introduction
8.2 Evaluation on the best preliminary fractionation procedure
8.3 Isolation and chemical characterization of antibacterial compounds
8.4 Biological characterization of plant species and isolated compounds
8.5 Evaluation of how well phytochemistry agrees with taxonomy based on anatomy
CHAPTER 9: REFERENCES
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