ABSTRACT
Antibiotics have remained the mainstay of drug therapy of infectious diseases worldwide. Their use is, however, limited by their numerous adverse effects and rapid development of microbial resistance. Identification of natural products from plants that may serve as valuable sources of antimicrobial agents for medicinal or agricultural uses seems to be a viable alternative to the conventional antibiotics. To achieve this goal, biological assays should be carried out in order to identify promising plant extracts, guide the separation and isolation, and to evaluate “lead” compounds. Psidium guajava Linn. and Loranthus micranthus Linn. have been employed traditionally in Nigeria and other parts of the globe for the treatment of various human ailments such as wounds, gastrointestinal tract disorders and other forms of infective and non-infective disorders. The main objective of this study was to identify and isolate the antibacterial compounds from the leaves of Psidium guajava and Loranthus micranthus L. The specific objectives were to: (i) carry out phytochemical evaluation and isolation of antibacterial constituents of the plants using standard methods, (ii) elucidate the structures of the isolated secondary metabolites, and (iii) carry out antibacterial assay of the isolated compounds. Fresh leaves of Loranthus micranthus (Linn.) parasitic on the stem of Persea americana were collected at Nsukka while those of Psidium guajava were collected from the bio-resource area of the University of Port Harcourt in June 2010. The leaves were then cleaned, air-dried for 14 days and milled to coarse powder. The powdered materials (800 g each) were defatted with n-hexane (5 L) and extracted in a soxhlet extractor with 90.0 % methanol. The methanol extract was further fractionated to yield the chloroform, ethyl acetate, acetone and methanol soluble fractions. Each of the fractions was screened for antibacterial activity using Agar-well diffusion method. Phytochemical tests were carried out using standard procedures. The fractions that had the best antibacterial activity were subjected to column chromatographic separation and monitored by analytical thin layer chromatography (TLC). The ethyl acetate fraction (PsG-EF) from P. guajava that gave satisfactory bioassay result was subjected to further Sephadex-LH 20 chromatographic fractionation and purification to afford ten fractions (PsG-EF1 to PsG-EF10) which were pooled. Fractions PsG-EF4, PsG-EF5 and PsG-EF7 that had good antibacterial activity were subjected to semi-preparative reverse phase high pressure liquid chromatography (HPLC) purification to isolate the phenolic compounds; I-V. The structures of these compounds were elucidated by analytical and spectral techniques which included: ultra violet (UV), proton nuclear magnetic resonance (1H-NMR), carbon-13 nuclear magnetic resonance (13C-NMR), distortionless
enhancement by polarization transfer (DEPT), proton-proton correlation spectroscopy (1H-1HCOSY), heteronuclear multiple quantum correlation (HMQC), heteronuclear multiple bond correlation (HMBC) and electron spray ionization-mass spectroscopy (ESI-MS) analyses. The isolated compounds were screened against standard strains of Staphylococus aureus (ATCC 25923) and Escherichia coli (ATCC 35219) using broth dilution assay method, and the MIC values determined and compared with ceftriaxone. All data obtained were analyzed by GraphPad Prism® 5 using differences in mean by two-way ANOVA and further subjected to Bonferroni post-tests to compare replicate means. The results were presented as mean ± SEM. Differences between means were considered significant at P<0.05. The results showed that the ethyl acetate fraction (PsG-EF) from P. guajava yielded one known compound (IV) and four novel phenolic compounds (I, II, III & V). The isolated compounds were elucidated as : 2,4-dihydroxy-6-O-βD-glucopyranosyl benzophenone (I); 2,4-dihydroxy-3-methyl-6-O-βD-glucopyranosylbenzophenone (II); 2,4-dihydro xy-3-methyl-6-O-βD-glucopyranosylbenzophenone (4→5″, 6'→1″) benzene-2″,3″,4″,5″-tetraol (III); quercertin-3-O-αL-arabinofuranoside (IV) and 2,4-dihydroxy-6-O-βD-glucopyranosylbenzophenone (4→5″, 6'→1″) benzene-2″,3″,4″,5″-tetraol (V). Compounds I, II, III, and V are new natural products which have not been previously reported in literature for this plant, guava, and the trivial names Guajaphenone A, B, C and D were proposed, while Compound IV has been previously reported as Guaijaverin. The various fractions of Psidium guajava L. exhibited significant (p < 0.05) antibacterial activities while for Loranthus micranthus L., its various fractions showed significantly lower values (p > 0.001) when compared with the control (ceftriaxone) suggestive of a generally weak or negligible antibacterial action. All the isolated compounds from P. guajava were also found to have moderate antibacterial activities against E. coli and S. aureus in comparison with ceftriaxone whlie I and IV showed lower MICs than those of the other isolates against the test organisms.
CHAPTER ONE
GENERAL INTRODUCTION
1.0 PREAMBLE
Over the years, medicines and medicinal agents derived from plants have made large contributions to human health and well-being. This is because they are either used directly as phytomedicines for the treatment of various ailments or they may become the base and the natural blueprint for the development of new drugs (Cseke et al, 2006).
Herbal medicine also called phytotherapy or phytomedicine has been around since the beginning of recorded history. It has also been described as the therapeutic use of medicinal plants referred to as herbs (Thea et al, 2008). Herbal medicine has become an integral part of standard health care, based on a combination of time honored traditional usage and ongoing scientific research. Surging interest in medicinal herbs has increased scientific scrutiny of their therapeutic potential and safety. Some of the medicinal plants are believed to enhance the natural resistance of the body to infections (Atal et al, 1986).
According to the World Health Organisation (WHO), herbal medicines could also be referred to as phytopharmaceuticals sold as over the counter products in modern dosage forms such as tablets, capsules, syrups or liquids for oral use or dietary supplements containing herbal products, also called nutraceuticals available in modern dosage forms, or even referred to as medicines consisting of other crude, semi processed or processed medicines, which have a vital place in primary health care and developing countries like Nigeria.
Traditional medicines are finished drug products intended for self-medication or application that contain, as the active principles, herbal ingredients that have received relatively little attention in world scientific literature, but for which traditional or folkloric use is well documented in herbal references. It may contain chemically defined or herbal based materials in addition to the active principles (Canada, 1989).
The medicinal properties of plant have been investigated in the light of recent scientific development throughout the world due to their potent pharmaceutical activities and low toxicity. Today many countries still rely on the medical values of herbs and use of medicinal plants for their therapeutic practices (Thea et al, 2008). In this same vein, Nigeria which is having a vast heritage of knowledge and expertise in herbal medicines is not an exception.
Finally, it has been variously established that the identification of these natural products from plants that may serve as valuable sources of bioactive agents for medicinal and agricultural uses largely depends on bioactivity-directed isolation (Cseke et al, 2006).
1.1 INFECTIOUS DISEASES AND CONVENTIONAL ANTIBIOTIC THERAP
Infectious diseases, also known as contagious diseases or transmissible diseases, and include communicable diseases, comprise clinically evident illness (i.e., characteristic medical signs and/or symptoms of disease) resulting from the infection, presence and growth of pathogenic biological agents in an individual host organism. In certain cases, infectious diseases may be asymptomatic for much or their entire course. Infectious pathogens include some viruses, bacteria, fungi, protozoa, multicellular parasites, and aberrant proteins known as prions. These pathogens are the cause of disease epidemics, in the sense that without the pathogen, no infectious epidemic occurs.
Transmission of pathogen can occur in various ways including physical contact, contaminated food, body fluids, objects, airborne inhalation, or through vector organisms (Ryan and Ray, 2004). Infectious diseases that are especially infective are sometimes called contagious and can be easily transmitted by contact with an ill person or their secretions. Infectious diseases with more specialized routes of infection, such as vector transmission or sexual transmission, are usually regarded as contagious but do not require medical quarantine of victims. The term infectivity describes the ability of an organism to enter, survive and multiply in the host, while the infectiousness of a disease indicates the comparative ease with which the disease is transmitted to other hosts; and as such, an infection is not synonymous with an infectious disease, as some infections do not cause illness in a host (Ryan and Ray, 2004). Among the almost infinite varieties of microorganisms, relatively few cause disease in otherwise healthy individuals. Infectious disease results from the interplay between those few pathogens and the defenses of the hosts they infect. The appearance and severity of disease resulting from any pathogen depends upon the ability of that pathogen to damage the host as well as the ability of the host to resist the pathogen. Clinicians therefore classify infectious microorganisms or microbes according to the status of host defenses – either as primary pathogens or as opportunistic pathogens:
Primary pathogens cause disease as a result of their presence or activity within the normal, healthy host, and their intrinsic virulence (the severity of the disease they cause) is, in part, a necessary consequence of their need to reproduce and spread. Many of the most common primary pathogens of humans only infect humans; however many serious diseases are caused by organisms acquired from the environment or which infect non-human hosts.
Organisms which cause an infectious disease in a host with depressed resistance are classified as opportunistic pathogens. Opportunistic disease may be caused by microbes that are ordinarily in contact with the host, such as pathogenic bacteria or fungi in the gastrointestinal or the upper respiratory tract, and they may also result from (otherwise innocuous) microbes acquired from other hosts (as in Clostridium difficile colitis) or from the environment as a result of traumatic introduction (as in surgical wound infections or compound fractures). An opportunistic disease requires impairment of host defenses, which may occur as a result of genetic defects (such as chronic granulomatous disease), exposure to antimicrobial drugs or immunosuppressive chemicals (as might occur following poisoning or cancer chemotherapy), exposure to ionizing radiation, or as a result of an infectious disease with immunosuppressive activity (such as with measles, malaria or HIV disease). Primary pathogens may also cause more severe disease in a host with depressed resistance than would normally occur in an immunosufficient host.
Many human diseases are caused by pathogenic organisms resulting sometimes in high mortality figures. Among these pathogens, bacteria account for a reasonable percentage of causative organisms implicated in human infectious diseases. Over the years, infections have been managed by the conventional antibiotics. Antibiotics are microbial metabolites or synthetic analogues inspired by them that, in small doses, inhibit the growth and survival of microorganisms without serious toxicity to the host. They therefore exhibit selective toxicity. In many cases, the clinical utility of natural antibiotics has been through medicinal chemistry manipulations of the original structure leading to broader antimicrobial spectrum, greater potency, lesser toxicity, more convenient administration, and additional pharmacokinetic advantages. Through customary usage, the many synthetic substances that are unrelated to natural products but still inhibit or kill microorganisms are referred to as antimicrobial agents instead (Martin, 1998).
Antibiotics are used to treat infections caused by organisms that are sensitive to them, usually bacteria or fungi. They may alter the normal microbial content
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of the body (e.g. in the intestine, lungs, bladder) by destroying one or more groups of harmless or beneficial organisms, which may result in infections (such as thrush in women) due to overgrowth of resistant organisms. These side-effects are most likely to occur with broad-spectrum antibiotics (those active against a wide variety of organisms). Resistance may also develop in the microorganisms being treated; for example, through incorrect dosage or over-prescription. Antibiotics should, therefore, not be used to treat minor infections, which will clear up unaided. Some antibiotics may, in addition, cause allergic reactions (Hendricks and Nemeth, 2010)
1.2 Limitations of Conventional Antibacterial Agent
1.2.1 Bacterial Resistance
Resistance is the failure of microorganisms to be killed or inhibited by antimicrobial treatment. Resistance can either be intrinsic (exist before exposure to drugs) or acquired (develop subsequent to exposure to a drug). Resistance of bacteria to the toxic effects of antimicrobial agents and to antibiotics develops fairly easily both in the laboratory and in the clinic and is an ever-increasing public health hazard.
In clinical practice, resistance more commonly takes place by Resistance (R) factor mechanisms. In more lurid examples, enzymes are elaborated that attack the antibiotic and inactivate it. Mutations leading to resistance occur by many mechanisms. They can result from point mutations, insertions, deletions, inversions, duplications and transpositions of segments of genes or by acquisition of foreign DNA from plasmids, bacteriophages, and transposable genetic elements.
These mechanisms can convert an antibiotic-sensitive cell to an antibiotic – resistant cell. This can take place many times in a bacterium's already short generation time.
Bacterial resistance generally is mediated through one of three mechanisms:
- Failure of the drug to penetrate into or stay in the cell
- Destruction of the drug by defensive enzymes, or
- Alterations in the cellular targets of the enzymes.
All these call for conservative but aggressive application of appropriate antimicrobial chemotherapy. In many cases, however, a resistant microorganism can still be controlled by achievable, though higher, doses than are required to control sensitive populations. These higher doses must be cautiously employed as they may predispose the patient to antibiotic adverse reactions that can be life-threatening.
1.2.2 Adverse Reactions by Agents
Many patients placed on conventional antibiotics have reported various cases of adverse drug reactions ranging from mild to severe/life-threatening reactions including arrhythmias, hepatotoxicity, acute renal failure, and antiretroviral therapy-induced lactic acidosis (Granowitz et al, 2008). Adverse reactions associated with drug use include allergies, toxicities, and side effects. An allergy is a hypersensitivity reaction to a drug. Many allergies are IgE-mediated and occur soon after drug administration. Examples of IgE-mediated type 1 hypersensitivity reactions include early-onset urticaria, anaphylaxis, bronchospasm, and angioedema. Non-IgE-mediated reactions include hemolytic anemia, thrombocytopenia, acute interstitial nephritis, serum sickness, vasculitis, erythema multiforme, Stevens-Johnson syndrome, and toxic epidermal necrolysis. Toxicity, which is generally due to either excessive dosing or impaired drug metabolism, is a consequence of administering a drug in quantities exceeding those capable of being physiologically ‘‘managed’’ by the host. Examples of toxicity caused by excessive dosing include penicillin-related neurotoxicity (e.g. twitching, seizures) and the toxicities caused by aminoglycosides. Side effects include adverse reactions that are neither immunologically mediated nor related to toxic levels of the drug. An example is the dyspepsia caused by erythromycin. Various forms of frequently encountered toxicities/adverse reactions include anaphylaxis, cardiotoxicity, nephrotoxicity, adverse heamatological and dermatological reactions, neurotoxicity, hepatotoxicity, muscoskeletal tocxicity, electrolyte and glucose abnormalities, fever, antibiotic-associated diarrhea/colitis, etc (Granowitz et al, 2008).
1.3 HIGHER PLANTS AS ANTIMICROBIAL AGENTS
The emergence of pathogenic microbes with increased resistance to established antibiotics provides a major incentive for the discovery of new antimicrobial agents. Antimicrobial screening of plant extracts and phytochemicals then represents a starting point for antimicrobial drug discovery.
Main-stream medicine is increasingly receptive to the use of antimicrobial and other drugs derived from plants, as traditional antibiotics (products of microorganisms or their synthesized derivative) become ineffective and as new, particularly viral, diseases remain intractable to this type of drug. Another driving factor for the renewed interest in plant antimicrobials in the past 20 years has been the rapid rate of (plant) species extinction (Lewis and Elvin-Lewis, 1995). There is a feeling among natural-products chemists and microbiologists alike that the multitude of potentially useful phytochemical structures which could be synthesized chemically is at risk of being lost irretrievably. There is a scientific discipline known as ethnobotany (or ethnopharmacology), whose goal is to utilize the impressive array of knowledge assembled by indigenous peoples about the plant and animal products they have used to maintain health (Rojas et al, 1992). Lastly, the ascendancy of the human immunodeficiency virus (HIV) has spurred intensive investigation into the plant derivatives which may be effective, especially for use in underdeveloped nations with little access to expensive Western medicines.
1.3.1 Medicinal Plants with Antibacterial Activity
Many tropical and non-tropical plants have been evaluated for their antimicrobial activity. For want of space, representatives of diverse plant families with documented antibacterial activity and the organisms tested are tabulated below.
table 1: Some plant species with potential antimicrobial activities
| Common/Botanical name | Family | Bacterial Susceptibility | |
| Celosia argentea L. | Amaranthaceae | K. pneumoniae | |
| Tylophora indica (Burm.f.) Merr. | Asclepiadaceae | K. Pneumoniae | |
| Vernonia anthelmintica (L.) Willd. | Asteraceae | K. Pneumoniae | |
| Balanites aegyptiaca (L.) Del. | Balanitaceae | K. Pneumoniae, S. typhimurium | |
| Spathodea campanulata Beauv | Bignonaceae | K. Pneumoniae | |
| Cassia fistula L. | Caesalpiniaceae | K. Pneumoniae, P. mirabilis | |
| Beta vulgaris L. | Chenopodiaceae | K. Pneumoniae | |
| Spinacia oleracea L. | Chenopodiaceae | K. Pneumoniae, P. mirabilis | |
| Commelina benghalensis L. | Commelinaceae | K. Pneumoniae | |
| Rourea santaloides (Vahl.) | Connaraceae | E. aerogenes; K. Pneumoniae; P. | |
| mirabilis | |||
| Cressa cretica L. | Convolvulaceae | K. Pneumoniae | |
| Lepidium sativum L. | Cruciferae | S. typhimurium | |
| Momordica charantia L. | Cucurbitaceae | E. aerogenes; K. Pneumoniae; P. | |
| mirabilis | |||
| Cyperus scarious R.Br. | Cyperaceae | E. aerogenes; K. Pneumoniae; P. | |
| mirabilis | |||
| Ricinus communis L. | Euphorbiaceae | K. Pneumoniae, P. mirabilis | |
| Arachis hypogaea L. | Fabaceae | E. aerogenes; K. Pneumoniae; | |
| Vigna radiata L. | Fabaceae | K. Pneumoniae; P. mirabilis | |
| Fumaria indica (Haussk.) Pugsley. | Fumariaceae | K. Pneumoniae; P. mirabilis | |
| Ocimum kilimanjaricum L. | Labiatae | E. aerogenes; E. coli; K. Pneumoniae; P. | |
| mirabilis; P. vulgaris. | |||
| Artocarpus hetrophyllus Lam. | Moraceae | E. aerogenes; P. mirabilis | |
| Ficus elastica Roxb. | Moraceae | K. Pneumoniae; P. mirabilis | |
| Piper longum L. | Piperaceae | E. aerogenes; K. Pneumoniae; P. | |
| mirabilis; S. typhimurium . | |||
| Gardenia resinifera Roth. | Rubiaceae | K. Pneumoniae; P. mirabilis | |
| Mesua ferra Linn. | Guttiferae | E. aerogenes; K. Pneumoniae; P. | |
| mirabilis; P. vulgaris. | |||
| Alchornea cordifolia | Euphorbiaceae | K. Pneumoniae; E. coli; B. subtilis; S. | |
| aureus | |||
| Chromolaena odorata | Asteraceae | Propionibacterium canes; | |
| Mycobacterium spps. | |||
(Parekh and Chanda, 2007; Okoye and Ebi, 2007)
1.3.2 Plant Secondary Metabolites Associated with Antimicrobial Effect
Plants have been shown to possess an amazing potential to synthesize aromatic substances, most of which are phenols or their oxygenated-substituted derivatives. These substances have been reported to consist of mostly secondary metabolites, of which at least 12,000 have been isolated, a number estimated to still be less than 10% of the total (Schultes, 1978 Plant secondary metabolites, in many cases, serve as plant defense mechanisms against predation by microorganisms, insects, and herbivores. Some, such as terpenoids give plants their odours; others (e.g. quinones and tannins) are responsible for plant pigmentation. Many of these compounds are also responsible for plant flavour. It is therefore not surprising that useful antimicrobial phytochemicals have been derived from these plant secondary metabolites.
1.3.2.1 Flavones, Flavonoids and Flavonols
Flavones are phenolic structures containing one carbonyl group (as opposed to the two carbonyls in quinones). Addition of a 3-hydroxy group yields a flavonol while flavonoids are also hydroxylated phenolic substances but occur as a C6-C3 unit linked to an aromatic ring. These compounds have been known to be synthesized by plants in response to microbial infection and have equally been found in vitro to be effective antimicrobial substances against a wide array of microorganisms (Dixon et al, 1983; Cowan, 1999). Their activity is probably due to their ability to complex with extracellular and soluble proteins and to complex with bacterial cell walls, often leading to the inactivation of the proteins, loss of function and cell lysis (Stern et al, 1996). More lipophilic flavonoids may also disrupt microbial membrane (Tsuchiya et al, 1996). Example of flavonoid compounds with known antimicrobial activity includes the catechins. These are the most reduced form of the C-3 unit in flavonoid compounds that have been extensively researched due to their occurrence in oolong green teas. Teas have been reported to exert antimicrobial activity and also contain a mixture of catechin compounds which inhibited in vitro activity of Vibrio cholerae 01, Streptococcus mutans, Shigella, and other bacteria and microorganisms (Toda et al, 1989; Batista et al, 1994; Borris, 1996; Sakanaka et al, 1989; Sakanaka et al, 1992; Vijaya et al, 1995). Examples of compounds in these groups with antimicrobial activity include flavone (I), catechin (II), chrysin (III) and quercetin (IV).
1.3.2.2 Alkaloids
These are basically heterocyclic nitrogenous compounds. Many of these compounds found in higher plants have shown promising antibacterial activity. For instance, diterpenoid alkaloids, commonly isolated from the plants of the Ranunculaceae, or buttercup family, are commonly found to have antimicrobial properties (Omulokoli et al, 1997). Some of the highly aromatic planar quaternary alkaloids such as berberine (V) have their mechanism of action attributable to their ability to intercalate with DNA (Phillipson and O'Neill, 1987). Other alkaloids with antimicrobial actions are Harmane (VI), Piperine (VII), etc.
1.3.2.3 Terpenoids and Essential Oils
Essential oils are secondary metabolites that are highly enriched in compounds based on an isoprene structure. The observed fragrance in most plants is contained in their essential oil fraction. They consist mainly of compounds belonging to the chemical group called terpenes. When the compounds contain additional elements, usually oxygen, they are called terpenoids. Terpenoids, even though synthesized from acetate units, differ from fatty acids by their extensive branching and cyclization.
Many plant terpenoids have been found to be active against bacteria, fungi, viruses and protozoa (Amaral et al, 1998; Habtemariam et al, 1993; Himejima et al, 1992; Mendoza et al, 1997; Kubo et al, 1993; Hasegawa et al, 1994; Ghoshal et al, 1996; Rao et al, 1993; Sun et al, 1996; Tassou et al, 1995; 2000). Although the mechanism of their antibacterial action is not yet fully understood, terpenes are speculated to act by disrupting cell membranes due to their lipophilic nature. In fact, it has been reported as far back as 1977 that of all the essential oil derivatives being examined, 30% were inhibitory bacteria while 60% inhibited fungi (Chaurasia and Vyas, 1977). For instance, capsaicin [(VIII); a terpenoid constituent found in Chiles peppers] in addition to its wide range of biological activities in humans, has been shown to clearly inhibit various bacteria to differing extents; and although possibly detrimental to the human gastric mucosa, it is bactericidal to Helicobacter pylori (Cichewicz and Thorpe, 1996; Jones et al, 1997). Also, the ethanol-soluble fraction of purple prairie clover yields a terpenoid called petalostemumol, which showed excellent activity against Bacillus subtilis and Staphylococcus aureus but lesser activity against Gram-negative bacteria as well as Candida albicans (Hufford et al, 1993). In the same vein, two diterpenoid compounds isolated from the roots of Plectranthus hereroensis were found to have good activity against S. aureus, V. cholerae, P. aeruginosa, and Candida spp (Batista et al, 1994); while another diterpene, trichorabdal A, isolated from a Japanese herb by Kadota et al (1997) was found to directly inhibit H. Pylori. Terpeoids like menthol (IX) and artemisin (X) have also shown antimicrobial activity.
1.3.2.4 Tannins
The term ‘tannin' is used to describe a group of polymeric phenolic substances capable of tanning leather or precipitating gelatin from solution. The property is known as astringency. Tannins which are found in almost every plant part are divided into hydrolyzable and condensed tannins. Hydrolyzable tannins are based on gallic acid, usually as multiple esters with D-glucose; while the more numerous condensed tannins (often called proanthocyanidins) are derived from flavonoid monomers (Cowan, 1999). Generally, tannins may be formed by condensation of flavan derivatives which have been transported to woody tissues of plant, or alternatively, by polymerization of quinone units (Geissman, 1963).
Tannins have been shown to act at the molecular levels by complexing with microbial proteins through so-called non-specific forces such as hydrogen bonding and hydrophobic effects, as well as covalent bond formation (Haslam, 1996; Stern et al, 1996). Thus, their mode of antimicrobial action may be related to their ability to inactivate microbial adhesins, enzymes, cell envelop transport proteins, etc. They may also complex with polysaccharide (Ya et al, 1988). Tannins have also shown to act via direct inactivation of microorganisms (eg. low tannin concentrations modify the morphology of germ tubes of Crinipellis perniciosa (Brownlee et al, 1990).
According to various documented studies reviewed by Scalbert (1991), tannins were found to be toxic to filamentous fungi, yeasts and bacteria. Condensed tannins have been described to bind cell walls of ruminal bacteria, preventing growth and protease activity (Jones et al, 1994). Though still speculative, tannins are considered to be wholly or partially responsible for the antibiotic activity of various aqueous and solvent extracts of many tropical and temperate plants scattered across the globe (Taylor et al, 1996). Pentagalloyl glucose (XI; hydrolysable tannin) and procyanidine (XII; condensed tannin) have shown remarkable antimicrobial activities (Cowan, 1999).
1.3.2.5 Miscellaneous Plant Constituents
Other major groups of antimicrobial compounds from plants include the simple phenols and phenolic acids, quinones, coumarins, lectins and polypeptides; and mixtures of all these groups. There abound many documented plant derivatives belonging to these chemical groups that have proven antimicrobial activity.
The common herbs terragon and thyme both contain caffeic acid (XIII; a phenylpropane-derived phenolic compound), which is effective against viruses, bacteria and fungi (Wild, 1994; Brantner et al, 1996). Catechol (XIV) and pyrogallol both are hydroxylated phenols, shown to be toxic to microorganisms; while eugenol (XV; a phenolic compound possessing a C3 side chain at lower level of oxidation but also classified as an essential oil) is considered bacteriostatic against both fungi and bacteria (Duke, 1985). Gallic acid (XVI) has also proven to be toxic to some microorganisms. Quinones are aromatic rings with two ketone substitutions which are ubiquitous in nature and are characteristically highly reactive. In addition to providing a source of stable free radicals, they are known to complex irreversibly with nucleophilic amino acids in proteins, often leading to inactivation of the protein and loss of action (Stern et al, 1996). For these reasons, the potential range of quinone antimicrobial effects is great. Kazmi et al (1994) described an anthraquinone from Cassia italica, a Pakistani tree, which was bacteriostatic for Bacillus anthracis, Corynebacterium pseudodiphthericum and Pseudomonas aeruginosa but bactericidal for Pseudomonas pseudomalliae. Also, Hypericin (XVII), an anthraquinone from St. John’s wort (Hypericum perforatum), has received much attention in the scientific journals lately as an antidepressant. This compound has, however, been reported by Duke (1985) to possess general antimicrobial properties. Cowan (1999) reported that rhein (XVIII) which is an anthraquinone compound has broad antimicrobial effects.
Coumarins (XIX) are phenolic substances made up of fused benzene and α – pyrone rings. They are responsible for the characteristic odour of hay. As a group, coumarins have been found to stimulate macrophages which could have an indirect negative effect on infections (Casley-Smith, 1997). Hydroxycinamic acids, related to coumarins, seem to be inhibitory to Gram-positive bacteria (Fernandez et al, 1996). Also, phytoalexins, which are hydroxylated derivatives of coumarins, are produced in carrots in response to fungal infection and can be presumed to have antifungal activity (Hoult and Paya, 1996). General antimicrobial activity was equally documented in coumarin compounds found in woodruff (Galium odoratum) extracts (Thompson, 1978). Although data about specific antibiotic properties of coumarins are scarce, many reports give reasons to believe that some utility may reside in these phytochemicals (Cowan, 1999; Hamburger and Hostettmann, 1991).
Peptides which are inhibitory to microorganisms were first reported by Balls et al (1942). They are often positively charged and contain disulfide bonds. Their mechanism of action may be the formation of ion channels in the microbial membrane, or by competitive inhibition of adhesion of microbial proteins to host polysaccharide receptors (Zhang and Lewis, 1997; Sharon and Ofek, 1986). Inhibition of bacteria and fungi by these macromolecules (e.g. peptides from the herbaceous Amaranthus) has been documented (De Bolle et al, 1996). Also, thionins, which are peptides commonly found in barley and wheat, consisting of 47 amino acid residues are toxic to yeasts and both Gram-negative and Gram-positive bacteria (Fernandes de Caleya et al, 1972). Fabatin, a recently identified 47-residue peptide from fava beans, appears to be structurally related to γ -thionins from grains and inhibits E. coli, P. aeruginosa and Enterococcus hirae but not Candida or Saccharomyces (Zhang and Lewis, 1997).
The antimicrobial activity of several extracts from plants has been linked to compounds belonging to more than one chemical group. For instance, the chewing sticks which are widely used in many African countries as an oral hygiene aid come from different species of plants, and within one stick, the chemically active component may be heterogeneous (Akpata and Akinrimisi, 1977). Crude extracts of one species used for this purpose, Serindeia werneckei, inhibited the periodontal pathogens Porphyromonas gingivalis and Bacteroides melaninogenicus in vitro (Rotimi et al, 1988). Also, the active component of one of the Nigerian chewing sticks (Fagara zanthoxyloides) was found to consist of various alkaloids (Odebiyi and Sofowora, 1979). Pawpaw (Carica papaya) yields a milky sap, often called latex, which is a complex mixture of chemicals (Cowan, 1999). Chief among them is papain, a well-known proteolytic enzyme. It also contains carpaine (an alkaloid) and terpenoids (Thomson, 1978). All these compounds in papaya have been shown to contribute to the antimicrobial properties of its latex which was found to be bacteriostatic to B. subtilis, Enterobacter cloacae, E. coli, Salmonella typhi, Staphylococcus aureus and Proteus vulgaris (Osato et al, 1993).
1.4 LITERATURE REVIEWS OF PLANTS USED1.
4.1 Loranthus micranthus Linn
1.4.1.1 Taxonomy of L. Micranthus
The botanical profile of Loranthus micranthus is as summarized below:
Kingdom: Plantae
Phylum: Angiosperm
Sub-Phylum: Dicotyledons
Order: Santalales
Family: Loranthaceae
Sub-Family: Lorantheae
Genus: Loranthus
Species: micranthus
The mistletoe plant is an evergreen obligate parasite with over 700 species which depends on its hosts for minerals and water only, as it can photosynthesize its carbohydrate by means of its green leaves (Gill, 1973; Griggs, 1991).
The most common species include: European mistletoe (Viscum album L.); American mistletoe (Phoradendron flavescens); Australian/Argentine mistletoe (Ligaria cuneifolia R et. T); African mistletoe, e.t.c.
Figure 2.0: Loranthus micranthus parasitic on a host tree
1.4.1.2 Description of the Family, Genus and Species of L. micranthus
The Loranthaceae family consists of parasites with green leaves found in both tropical and temperate regions. They are mostly small semi-parasitic shrubs attached to their hosts by suckers or haustoria (usually regarded as modified adventurous roots). The family is fairly large with over 36 genera and 130 species, most of which are quite omnivorous in their choice of hosts, but a few are restricted to one or two. Few members of Loranthaceae family root in the earth (e.g. the Western Australia Christmas tree –Nuytsia floribunda, which grows into a small tree of up to 10 metres high). For most others that root on hosts, there is commonly an outgrowth, often of considerable size and
complicated in shape, where the parasite root joins the host. The roots of the parasites often branch within the tissue of the host (as in Viscum).
The genus, Loranthus, consists of several species scattered in many parts of Africa. It belongs to the sub-family, Lorantheae, which is characterized by the presence of stem without secretory canals and has extraxylary phloem. Their flowers have below the petals an outgrowth from the axis in form of small ring or fringe called calyculus. After some weeks of the seeds germinating on branches of its host, the Loranthus plant produces proper flowers which are generally bright red and conspicuous, although one species produces yellow flowers with red tips. The flowers are soon followed by small, fleshy, drupaceous fruits which are much sought after by birds. The red Loranthus is a common sight in many parts of West Africa, particularly in cocoa and cola plantations, where whole branches are often covered with this medicinal herb.
The African mistletoe species, micranthus, is found mainly in the Southeastern part of Nigeria. It grows on a large number of hosts including kola nut (Kola acuminata), avocado (Persea americana), dogoyaro/neem (Azadirachta indica), oil bean (Pentaclethra macrophylla), ogbono (Irvigia gabonensis), lemons/citrus, etc. It produces sympodial, often dichasical, stem and the leaves are usually evergreen and leathery. The cymose inflorescences are in spikes, with the flowers on the internodes as well as on the nodes. Thus, they have clusters of narrowly tubular flowers that are bright-red which appear as clusters of coloured ‘matches’.
1.4.1.3 Ethnomedicinal Uses and Pharmacological Studies on L. micranthus
Several ethnomedicinal usages have been attributed to Loranthus micranthus. These include: blood pressure control (antihypertensive activity), anti-diabetic activity, anticancer, antimicrobial and in many other metabolic diseases which qualified mistletoe as an “all-purpose herb” (Kafaru, 1993; Obatomi et al, 1994; Oliver-Bever, 1986; Dalziel, 1955). Different research works have been carried out on the several species of the plant to demonstrate and support the existence of many of the ethno medicinal claims (Obatomi et al, 1996; Osadebe and Ukwueze, 2004; Osadebe and Akabogu, 2006; Osadebe et al, 2004, 2010, 2012; Ukwueze and Osadebe, 2012; Agbo et al, 2013; Omeje et al, 2012). Previous works on Loranthus micranthus have equally shown that some of its medicinal activities vary with the particular host tree from which it is harvested (Osadebe and Ukwueze, 2004; Osadebe et al, 2004). Other factors that have been shown to affect the phytochemical composition and pharmacological activities of mistletoe plant include species, harvesting season, etc. (Obatomi et al, 1994; Wagner et al, 1996; Osadebe et al, 2008).
1.4.2 Psidium Guajava
1.4.2.1 Taxonomy of P. guajava
The botanical profile of Psidium guajava is as summarized below:
Kingdom: Plantae
Phylum: Angiosperms
Sub-Phylum: Eudicots (unranked): Rosids
Order: Myrtales
Family: Myrtaceae
Subfamily: Myrtoideae
Tribe: Myrteae
Genus: Psidium
Species: guajava
Binomial name: Psidium guajava L.
Psidium guajava L, a fruit-bearing tree commonly known as guava, of the family Myrtaceae, is a native of tropical America. The French call it goyave or goyavier; the Dutch, guyaba or goeajaaba; the Surinamese, guave or goejaba; and the Portuguese, goiaba or goaibeira. Hawaiians call it guava or kuawa. In Guam, it is abas. In Malaya, it is generally known either as guava or jambu batu (Morton, 1987).
1.4.2.2 Morphology of P. guajava
Cultivated varieties grow about 10 m in height and produce fruits within 4 years. Wild trees grow up to 20 m high and are well branched. The guava tree can be easily identified by its distinctive thin, smooth, copper-colored bark that flakes off, showing a greenish layer beneath. The trees might have spread widely throughout the tropics because they thrive in a variety of soils, propagate easily and bear fruits quickly. The fruits are enjoyed by humans, birds and monkeys, which disperse guava seeds and cause spontaneous dumps of guava saplings to grow throughout the rainforest (Wealth of India, 2003).
Figure 5.0: Fruit of Psidium guajava
1.4.2.3 Ethnomedicinal Uses and Pharmacological Studies of P. guajava
Psidium guajava is a medicinal plant used in tropical and subtropical countries to treat many health disorders. In the indigenous system of medicine, different parts of the plant are used for the treatment of various human ailments such as wounds, ulcers, bowels and cholera (Begum et al., 2002a). Investigations have indicated that its bark, fruit and leaves possess antibacterial, hypoglycaemic, anti-inflammatory, analgesic, antipyretic, spasmolytic and CNS depressant activities ( Begum et al., 2002b). It has indeed been variously reported that Psidium guajava leaf extract has a wide spectrum of biological activities such as anticough, antibacterial, haemostasis (Jaiarj et al., 1999; 2000), antidiarrhoeal and narcotic properties (Lozoya et al.,1990), and antioxidant properties (Qian and Nihorimbere, 2004). According to Lutterodt and Maleque (1998) and Meckes et al., 1996, the leaf extract is used to treat diarrhoea, abdominal pain, convulsions, epilepsy, cholera, insomnia and has hypnotic effect.
The long history of guava use has led modern-day researchers to intensify their study on guava extracts. Its traditional use against diarrhea, gastroenteritis and other digestive complaints has been validated in numerous clinical studies. In a study including 17 Thai medicinal plants on anti-proliferative effects on human mouth epidermal carcinoma and murine leukemia cells using MIT assay, guava leaf showed anti-proliferative activity, which was 4.37 times more than vincristine (Manosroi et al. , 2006).
Bark and leaf extracts were shown to have in vitro toxic action against numerous bacteria. Gallocatechin isolated from the methanol extract of guava leaf showed antimutagenic activity against E. coli (Matsuo et al., 1994). Water and chloroform extracts of guava were effective in activating the mutagenicity of Salmonella typhimurium (Grover and Bala, 1993). The antimicrobial activities of P. guajava and leaf extracts, determined by disk diffusion method (zone of inhibition), were compared to tea tree oil (TTO), doxycycline and clindamycin antibiotics. It was shown that P. guajava leaf extracts might be beneficial in treating acne especially those that have anti-inflammatory activities (Qadan et al., 2005). The active flavonoid compound-quercetin-3-O-alpha-l-arabinopyranoside (guaijaverin) – extracted from guava leaves has high potential antiplaque activity by inhibiting the growth of Streptococcus mutans (Limsong et al., 2004). Guava leaf extract also inhibited the growth of Streptococcus aureus in a study carried out by disc diffusion method (Abdelrahim et al., 2002). In several other studies, guava showed significant antibacterial activity against common diarrhea-causing bacteria such as Staphylococcus, Shigella, Salmonella, Bacillus, E. coli, Clostridium and Pseudomonas. Indeed, the aqueous, alcohol and chloroform extracts have been found to be effective against Aeromonas hydrophila, Shigella spp. and Vibrio spp., Staphylococcus aureus, Sarcinta lutea and Microbacterium phlei (Jaiarj et al., 1999). In a more recent study, the aqueous and ethanol:water extracts of P. guajava leaves, roots and stem bark were found to be active against the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis, but virtually inactive against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa (Sanches et al.,2005).
A double-blind clinical study of the effects of a Phytodrug (QG-5) developed from guava leaf showed a decrease in duration of abdominal pain, which was attributed to antispasmodic effect of quercetin present in leaf extract (Xavier et al., 2002). Guava leaf extracts and fruit juices have also been clinically studied for infantile diarrhea. In a clinical study with 62 infants with infantile rotaviral enteritis, the recovery rate was 3 days (87.1%) in those treated with guava, and diarrhea ceased in a shorter period than controls. It was concluded in the study that guava has ‘good curative effect on infantile rotaviral enteritis' (Wei et al., 2000). Lectin chemicals in guava were shown to bind to E. coli (a common diarrhea-causing organism), preventing its adhesion to the intestinal wall and thus preventing infection and resulting diarrhea (Rodriguez et al., 2001). Guava leaf extract has also shown to have tranquilizing effect on intestinal smooth muscle, inhibit chemical processes found in diarrhea and aid in the re-absorption of water in intestines. In another research, an alcoholic leaf extract was reported to have a morphine-like effect, by inhibiting the gastrointestinal release of chemicals in acute diarrheal disease. This morphine-like effect was thought to be related to a chemical, quercetin. The effective use of guava in diarrhea, dysentery and gastroenteritis can also be related to guava's documented antibacterial properties (Tona et al., 2000). In a study carried out with leaf extract of the plant, inhibition of gastrointestinal release of acetylcholine by quercetin present in extract was suggested as a possible mode of action in the treatment of acute diarrheal disease (Lutterodt, 1992).
Guava fruit and leaf showed antioxidant and free radical scavenging capacity (Hui-Yin and Gow-Chin, 2007). A study of aqueous extract of P. guajava in acute experimental liver injury induced by carbon tetrachloride, paracetamol and thioacetamide, showed its hepatoprotective activity. The effects observed were compared with a known hepatoprotective agent, silymarin. Histological examination of the liver tissues supported hepatoprotection (Roy et al., 2006).
During various episodes of screening of medicinal plants, extract from P. guajava leaves was found to exhibit significantly inhibitory effect on the protein tyrosine phosphatase1B (PTP1B). Significant blood glucose lowering effects of the extract were observed after intraperitoneal injection of the extract at a dose of 10mg/kg in both 1-and 3-month-old Lepr(db)/Lepr(db) mice (Oh et al. , 2005). In a study undertaken to investigate the hypoglycemic and hypotensive effects of P. guajava leaf aqueous extract in rats, it showed hypoglycemic activity. The hypoglycemic effect of plant extract was examined in normal and diabetic rats, using streptozotocin (STZ)-induced diabetes mellitus model (Ojewole, 2005). Also, i.p. treatment with 1g/kg guava juice produced a marked hypoglycemic action in normal and alloxan-treated diabetic mice (Cheng and Yang, 1983). In two randomized human studies, the consumption of guava fruit for 12 weeks was shown to reduce blood pressure by an average 8%, decrease total cholesterol level by 9%, decrease triglycerides by almost 8% and increase HDL cholesterol by 8%; while a randomized, single-blind, controlled trial conducted to examine the effects of guava fruit intake on blood pressure and blood lipids in patients with essential hypertension showed the possibility that an increased consumption of guava fruit can cause a substantial reduction in blood pressure and blood lipids without decreasing HDL-cholesterol level (Singh et al., 1992, 1993). Leaf extract of guava had shown ionotropic effect on guinea pig atrium (Conde-Garcia et al., 2003). Some studies reported that the leaf extract and its derivative identified as quercetin has effect on the intracellular calcium levels in gastrointestinal smooth muscle (Lozoya et al., 1990), in cardiac muscle cell (Apisariyakul et al., 1999) and in neuromuscular junction (Chaichana and Apisariyakul, 1996). In other animal studies, guava leaf extracts have shown central nervous system (CNS) depressant activity (Shaheen, 2000). Guava leaf extract showed anticough activity by reducing the frequency of cough induced by capsaicin aerosol (Jaiarj et al., 1999).
1.4.2.4 The Phytochemistry of Psidium guajava
Guava has been found to be rich in tannins, phenols, triterpenes, flavonoids, essential oils, saponins, carotenoids, lectins, vitamins, fibre and fatty acids. According to Olajide et al (1999), the leaves of P. guajava contain an essential oil rich in cineol, tannins, triterpenes and flavonoids. Various reports of phytochemical screening of Psidium guajava leaf showed tannins in aqueous extract; and anthocyans, alkaloids, flavonoids, tannins and steroids/terpenoids in ethanolic extract.
liv
More than twenty identified compounds from Psidium guajava leaf have been reported (Seshadri and Vasishta, 1965; Osman et al., 1974; Lutterodt and Maleque, 1988). The major components are: β-selinene (XX), β-caryophyllene (XXI), caryophyllene oxide (XXII), squalene (XXIII), selin-11-en-4α-ol (XXIV), guaijavarin (XXV), isoquercetin (XXVI), hyperin (XXVII), quercitrin (XXVIII) and quercetin-3-O-gentobioside; morin-3-O-α-L-lyxopyranoside (XXIX), morin-3-O-α-L-arabopyranoside (XXX); β-sitosterol (XXXI), uvaol (XXXII), oleanolic acid (XXXIII), ursolic acid (XXXIV) and one new pentacyclic triterpenoid: guajanoic acid (Lozoya et al., 1994; Meckes et al., 1996; Arima and Danno, 2002; Begum et al., 2004).
| XX | XXI | |||||||||||||
| XXII | XXIII | |||||||||||||
Guava fruit is higher in vitamin C than citrus fruits (80 mg of vitamin C in 100g of fruit) and contains appreciable amounts of Vitamin A as well and is also a good source of pectin (Sunttornusk, 2002). The bark of guava tree contains considerable amounts of tannins (11-27%), and hence is used for tanning and dyeing purposes. Leucocyanidin (XXXV), luectic acid, ellagic acid (XXXVI) and amritoside (XXXVII) have been isolated from the stem bark.
Other compounds that have been isolated from guava plant include avicularin
(XXXVIII; 3-L-4-4-arabinofuranoside), α-pinene (XXXIX), β-pinene (XL), limonene
(XLI), terpenyl acetate (XLII), isopropyl alcohol (XLIII), longicyclene (XLIV), β-bisabolene, β-copanene, farnesene, humulene, cardinene, curcumene, mallic acids, ursolic, crategolic, guayavolic acids, cineol, etc (Shruthi et al., 2013).
1.5 BIOASSAY-GUIDED CHARACTERIZATION OF ANTIBACTERIAL CONSTITUENTS
FROM HIGHER PLANTS
The driving force behind much phytochemical research is the discovery of new biological active compounds for medicinal or agricultural uses. Biological assays then must be carried out in order to identify promising plant extracts, to guide the separation and isolation, and to evaluate lead compounds. Identification of natural products from plants that may serve as valuable sources of bioactive agents for medicinal and agricultural uses largely depends on bioactivity-directed isolation (Cseke et al, 2006).
The choices of bioassays depend a great deal on the amounts of materials to be tested and the time and effort necessary to carry out the assays. Obviously, an in vivo assay using the organism afflicted (humans or animals) would provide the most meaningful results. However, exploratory screening using whole animals is impractical (or unethical), and various in vitro screening methods have been developed to provide guided separation and identification of lead compounds. The latter have the advantage in that they can be automated with robotics and miniaturized, leading to rapid throughput screening of large numbers of samples. In addition, the in vitro bioassays may provide activity information that is precluded by poor bioavailability using a whole-animal in vivo assay. For instance, natural products that inhibit the growth of tumor cells or bacteria in an in vitro assay may identify promising molecular structures that would benefit from semi-synthetic modifications (Cseke et al, 2006).
1.5.1 Principles of Bioassay
Bioassay (or biological assay) is the estimation of the activity or potency of a drug or other substance (e.g. plant extract) by comparing its effects on a test organism with that of a standard preparation. It is a type of scientific experiment conducted to measure the effects of a substance on a living organism and is essential in the development of new drugs and other scientific monitoring. Bioassays may be qualitative or quantitative.
1.5.2 Screening Methods for Antibacterial Agents from Higher Plants
The discovery of promising plant extracts and the subsequent activity-guided isolation of constituents put specific requirements on the bioassays to be used for that purpose. They have to be simple, rapid, reproducible and inexpensive in order to be compatible with the large number of assays to be performed. Antimicrobial activity of plants can be detected by observing the growth response of various microorganisms to those plant tissues or extracts which are placed in contact with them. Many methods for detecting such activity are available, but since they are not equally sensitive or even based upon the same principle, the results obtained will also be profoundly influenced not only by the method selected, but also by the microorganisms used to carry out the test (Vanden-Berghe and Vlietinck, 1991). In general, biological assays or evaluation can be carried out much more efficiently on water-soluble, pure crystalline substances than on mixtures like plant extracts.
1.5.2.1 Test Organisms and Culture Media
The purpose of any antimicrobial investigation will obviously determine to a great extent the choice of test organisms to be used. For an investigation of a general character, the test organisms selected should be as diverse as possible and preferably representative of all important groups of pathogenic bacteria according to their physical and chemical composition and resistance pattern. Most screening studies on plant extracts, however, have been carried out on one or two bacteria, including strains of Staphylococcus aureus and Escherichia coli, although such findings may not adequately predict an interesting broad-spectrum activity or a selective but pronounced activity against some of the problem-pathogenic bacteria in chemotherapy such as resistant S. aureus, P. aeruginosa, Proteus vulgaris, Klebsiella pneumoniae, Neisseria gonorrhoeae, Candida albicans and others. Most bacteria (and yeasts) can be cultivated on standard Mueller-Hinton agar or diagnostic sensitivity test agar (DST) and American type culture collection (ATCC) or similar standard microorganisms are available. Only few bacteria (e.g. Neisseria gonorrhoeae and Campylobacter fetus) require special growth factors which should be included in the standard medium. In general, standard microorganisms should be preferably used as test bacteria during screening for new antimicrobially-active plant components for ease of reproducibility of results by other researchers. If the interest, however, is in finding new products which are selectively active against problem microorganisms causing certain diseases, e.g. resistant P. aeruginosa, it is clearly appropriate to employ the corresponding isolated pathogenic microorganisms (Rwangabo et al., 1988).
1.5.2.2 Antibacterial Testing
The currently available antimicrobial screening methods fall into three broad groups, including diffusion, dilution and bioautographic methods (Rios et al, 1988). These testing methods will only give an idea of the presence or absence of substances with antimicrobial activity in the plant extracts, as the potency of the active ingredients can only be determined on pure compounds using standardized methodologies. The results obtained using any of the methods, however, are influenced by such factors such as extraction method, inoculum volume, culture medium composition, pH and incubation temperature.
In the diffusion technique, a reservoir (e.g. filter paper disc, porcelain/stainless steel cylinder or hole punched in the media) containing the plant extract to be tested is brought into contact with an inoculated medium (e.g. agar) and, after incubation, the diameter of the clear zone around the reservoir (inhibition zone diameter) is measured. In order to lower the detection limit using this method, the inoculated system is kept at low a temperature during several hours before incubation, which favors diffusion over microbial growth and thus increases the inhibition diameter. In most studies, the inhibition zones obtained are compared with those obtained for antibiotics so as to establish the sensitivity of the test organism to the extract. Advantages of the diffusion methods are the small size of the sample used in the screening and the possibility of testing up to five or six compounds per plate against a single microorganism. For the dilution methods, samples being tested are mixed with a suitable medium, which has previously been inoculated with the test organism. After incubation, growth of the microorganism may be determined by direct visual or turbidimetric comparison of the test culture with a control culture which did not receive an addition of the sample being tested, or by plating out both test and control cultures (Kavanagh, 1963). Usually a series of dilutions of the original sample in the culture medium is made and then inoculated with the test organism. After inoculation, the endpoint of the test (MIC-value) is taken as the highest dilution which will just prevent perceptible growth of the test organism (Vanden-Berghe and Vlietinck, 1991). In comparison, several different test microorganisms may be tested simultaneously on the same dilution as against diffusion methods in which several substances or dilutions of one substance may be tested simultaneously against one test microorganism. The agar dilution method is thus very quick, time saving and also very useful to guide the isolation of antimicrobially active components from plant extracts (Bakana et al, 1987; Rwangabo et al, 1988).
Bioautographic methods are employed to localize antibacterial activity on a chromatoGram. The procedures are based on the agar diffusion technique, whereby the antimicrobial agent is transferred from the thin layer or paper chromatoGram to an inoculated agar plate through a diffusion process. Zones of inhibition are then visualized by appropriate vital stains. Although very suitable for testing highly active antibiotics (MIC-values < 10ug/ml), bioautographic methods might not be very promising for testing plant extracts, which often contain much less potent antimicrobial agents than the currently available antibiotics (Vanden-Berghe and Vlietinck, 1991).
1.6 STRUCTURE ELUCIDATION OF BIOACTIVE PLANT METABOLITE
Chemical compounds, usually derived from plants and other natural sources, have been used by humans for thousands of years to alleviate pain, diarrhea, infection, and various other maladies. Until recently, these ”remedies” were primarily crude preparations of plant material of unknown constitution. The revolution in the synthetic organic chemistry during the nineteenth century produced a concerted effort towards identification of the structures of the active constituents of these naturally derived medicinals and synthesis of what were hoped to be more efficacious agents. By determining the molecular structures of the active components of these complex mixtures, it is hoped that a better understanding of how these components work can be elucidated (Knittel and Zavod, 2008).
1.6.1 Preliminary Analysis
Bioassay-directed fractionation is the process of isolating pure active constituents from some type of biomass (eg. plants, microbes, marine invertebrates, etc.) using a decision tree that is dictated solely by bioactivity (Kinghorn, 2008). A variety of chromatographic separation techniques are available for these purposes, including those based on adsorption on sorbents, such as silica gel, alumina, Sephadex, and more specialized solid phases, and methods involving partition chromatography inclusive of counter-current chromatography. Recent improvements have been made in column technology, automation of high-performance liquid chromatography (HPLC; a technique often used for final compound purification) and compatibility with HTS methodology (Butler, 2004).
1.6.2 Application of Modern Analytical Techniques
Routine structure elucidation is performed using combinations of spectroscopic procedures, with particular emphasis on one and two-dimensional 1H- and 13C-nuclear magnetic resonance (NMR) spectroscopy and mass spectroscopy (MS). 2D NMR spectra, generally, provide more information about a molecule than 1D NMR spectra and are especially useful in determining the structure of a molecule, particular for molecules that are too complicated to work with using 1D NMR. Thus, for the structural elucidation of a compound, a simple 1D NMR (H1 and C13) may not be sufficient. Advanced techniques like correlation spectroscopic methods, when analyzed properly, provide the perfect way to determine the structure of a given formula or an entirely unknown compound. The combination of some or all of these 1D and multi-dimensional NMR spectra are all very useful in identification and characterization of the structure, and orientation of bonds in a molecule (Becker, 2000). Considerable progress has been made in the development of cryogenic and microcoil NMR probe technology for the determination of structures in sub-milligram amounts of natural products (Koehn and Carter, 2005). In addition, the automated processing of spectroscopic data for the structure elucidation of natural products is a practical proposition (Steinbeck, 2004).
Another significant advance is the use of “hyphenated” analytical techniques for rapid determination of the structure of natural products without the need for a separate isolation step, such as liquid chromatograph-nuclear magnetic resonance (LC-NMR) and LC-NMR-MS (Koehn and Carter, 2005; Butler, 2004).
1.7 STATEMENT OF PROBLEM
Antibiotics have remained the mainstay of clinical therapy of infectious diseases worldwide. Many microorganisms, however, are becoming increasingly resistant to most of these agents (Jawetz et al., 1989). Another serious limitation of these chemotherapeutic agents is the numerous adverse effects associated with their administration (Snavely and Hodges, 1984). These limitations have, thus, made it imperative that extensive efforts must be made to uncover new antimicrobial agents from alternative sources whose structures and modes of action may very likely differ from those of microbial sources (antibiotics). Compounds extracted from higher plants have the potential of fulfilling this purpose and that has led to the increasing screening studies on several plant extracts in search of new ‘leads’. Unfortunately, most of these works are preliminary investigations of the pharmacological actions of the crude plant extracts, rather than the isolation, identification or the characterization of the active constituents of these extracts.
1.8 JUSTIFICATION OF THE STUDY
Several works have been carried out to verify and confirm the traditional antibacterial uses of Psidium guajava and Loranthus micranthus growing in the tropical rain forest region of Nigeria, but researches on the isolation, characterization and detailed chemical investigations of their pharmacologically active components have remained inexhaustive. There is, therefore, the need for comprehensive chemical and bio-molecular studies on these active constituents, and hence the justifications for this present research.
1.9 AIMS AND SCOPE OF THE WORK
Aims of the Study
- To carry out a detailed bioactivity-guided phytochemical evaluation of the extracts/solvent fractions of the leaves of Psidium guajava and Loranthus micranthus.
- To identify and isolate the antibacterial compounds from the leaves of the plants.
- To characterize and elucidate the structure of the isolates.
- To compare the antibacterial potentials of these isolates with standard antibiotics with a view to developing derivatives of the ‘lead’ compounds and optimize same for activity with respect to potency and selectivity, especially against bacteria causing opportunistic AIDS infections [ e.g. resistant strains of Pseudomonas aeruginosa, and Staphylococcus aureus; including the multi-drug resistant (MDR) strains of methicillin-resistant Staphylococcus aureus (MRSA), and Mycobacterium avium complex (MAC)].
