CHAPTER ONE
1.1 INTRODUCTION AND LITERATURE REVIEW
Plants remain the most common source of anti-microbial agents. Their usage, as traditional health remedies, is the most popular for 80 % of world population in Asia, Latin America and Africa. Herbs are reported to have minimal side effects. In recent years, pharmaceutical companies have spent a lot of time and money in developing natural products extracted from plants, to produce more cost effective remedies that are affordable to the population.
1.2 Background to the study
The rising incidence in multidrug resistance amongst pathogenic microbes has further necessitated the need to search for newer antibiotic sources 1. Several members of the genus Bridialare being used traditionally for wide variety of ethno-pharmacological properties. The plant of BridialmacranthaNees (Lauraceae), commonly known as Gulmau, is a large tree grows up to 27 m in height, found in Bihar and Deccan peninsula (Western Ghats of Maharashtra, India). The bark is used in pleurisy, asthma and rheumatism2. The leaves are also used externally in the treatment of ulcers3,4. The bark of BridialmacranthaNees has a pleasant odour, is cheap substitute for cinnamomuminers. The bark is a rich source of mucilage5. Anti-inflammatory activity of bark has also been reported6. As a result of indiscriminate use of antimicrobial drugs in the treatment of infectious diseases, microorganisms have developed resistance to many antibiotics. There is need to develop alternative antibioticdrugs from natural origin. One approach is to screen localmedicinal plants which represent rich source of novelantimicrobial agents.
1.3 Aims and Objectives of the study
The present study was carried out toinvestigate the antimicrobial properties of Bridialmacranthaextracted by various solvents.
Specific objective are
- To investigate the Inhibitoryeffect of methanol extract of Bridialmacrantha on disease causing
- To investigateInhibitoryeffect of ethyl acetate extract of Bridialmacranthaon disease causing organisms.
- To investigate Inhibitory effect n-hexane extract of Bridialmacranthadisease causing organism.
1.4 Scope of the study
The scope of the study covers inhibitory effect ofBridialmacrantha of three different extracts on microorganism.
1.5Significance of the study
The study is of benefit to all Nigeria citizens and to fellow researchers. All citizens of Nigeria will benefit from the study as the study will elucidate that benefit of bridialmacranthaas a medical plant. The study will serve as source of reference material for fellow researchers for related study.
2.1 LITERATURE REVIEW
The plant of
MachilusmacranthaNees (Lauraceae), commonly known
as Gulmau, is a large tree grows up to 27 m in height,
found in Bihar and Deccan peninsula (Western Ghats of
Maharashtra, India). The bark is used in pleurisy, asthma
bridialmacrantha(Nees) Kosterm belongs to the family Lauraceae. It is a tree growing up to 30m in length and is mainly distributed in Western peninsula, Ceylon and India etc. In India, the plant is found in various states such as Karnataka, Bihar, Maharashtra and Assam up to an altitude of 2100m. It is called gulmavu in Kannada. The plant is reported to contain several phytochemicals such as norlignans, alkaloids, β-sitosterol and others. The plant in particular leaves and bark has several traditional uses(Tatiya AU, Saluja,2011) The leaves and bark are traditionally used in the treatment of arthritis, traumatic injury, edema, and wounds(Shrisha,2011). In northcentral Western Ghats of India, the paste orpoultice made from stem/bark is applied externally to treat bone fracture(Upadhya, Hegde,Bhat , Hurkadale,2012). The bark is used for treating asthma, tuberculosis and rheumatoid arthritis. In HallakkiGowda tribe of Western Ghats of Karnataka, India, the powdered bark is mixed with egg-white and applied on the affected part in form of poultice as an anti-inflammatory and anti-rheumatic agent(Tatiya AU, Saluja,2011).The plant Machilusmacrantha is shown to exhibit several bioactivities. The leaf and stem bark extracts were shown to exhibit inhibitory activity against bacteria and fungi(Shrisha&Raveesha,2012).Various extracts of stem bark showed anti-inflammatory and anti-arthritic activity in rats(Kulkarni& Gokhale,2013). Extracts and fractions of the bark were shown to possess antiinflammatory, antinociceptive and membrane stabilizing activity(Tatiya AU, Saluja,2011)In the present study, wedetermined antibacterial and radical scavenging potential of leaf and bark extract of Bridialmacrantha.
2.2 Pathogenic Organisms
The progressive increase in antimicrobial resistance among enteric pathogens in developing countries is becoming a critical area of concern. Treatment of diarrhoea caused by bacteria with antibiotics often results in drug resistance in both enteric and non-enteric diarrhoea-causing bacteria (Fhogartaigh and Edgeworth, 2009). This is most likely related to the frequent unrestricted use of over-the-counter drugs without medical supervision.
Antibiotic resistance to trimethoprim-sulfamethoxazole, ampicillin, chloramphenicol, and tetracycline was noted among Campylobacter spp, Shigella, Salmonella and E. coli (Isenbargeret al. 2002). High resistance to antimicrobials, such as erythromycin, ciprofloxacin, vancomycin, and fusidic acid by Campylobacter spp. isolated from human diarrhoeal stools was demonstrated in Vhembe district, South Africa (Samieet al. 2012). Samieet al. (2012) demonstrated that unsafe drinking water was the cause of diarrhoea among HIV-infected patients in Makhado municipality of the Limpopo province of South Africa. Acinetobacterlwoffii, Vibrofluvialis, Enterobactercloacae, Shigellaspp., Pseudomonas spp. and Yersinia enterocoliticawere isolated from water samples taken and were found to be highly resistant to cefazolin (83.5%), cefoxitin (69.2%), ampicillin (66.4%), and cefuroxime (66.2%). Intermediate resistance was observed against gentamicin (10.6%), cefepime (13.4%), ceftriaxone (27.6%), and cefotaxime (29.9%).
Antibiotics such as amoxicillin/clavulanate and co-trimoxazole, sometimes used in the treatment of infectious diarrhoea of bacterial origin, may paradoxically cause hepatotoxic reactions. Other side effects of antibiotics include cutaneous reactions, gastrointestinal disorders and yeast overgrowth (Andrade et al. 2011).
At present, the continued development of new antimicrobials, other than antibiotic ones, particularly those used for the treatment of salmonella spp, E.coli and candida albicanseteis critically important and for such the use of bridialmancrantha in the treatment of some of the organism becomes vital in today medical treatment.
- 3ANTIMICROBIALS
In an attempt to combat the various forms of disease that have continued to plague humans from time immemorial to this day, different types of antimicrobials have been developed to fight the pathogens responsible for these diseases. Antimicrobials, which are substances that kill or inhibit the growth of microorganisms, could be in the form of antibiotics, which are products of microorganisms or synthesised derivatives (Cowan 2010), antimicrobial peptides produced by complex organisms as well as some microbes (Jenssenet al. 2016) and medicinal plants, which appear to be the focus of mainstream medicine today (Cowan 2010).
2.3. 1Types And Sources Of Antimicrobials
Different types of antimicrobials exist: antibiotics, anti-viral, anti-fungal, anti-protozoan etc. Antibiotics are used in the treatment of bacterial infections and can be obtained from either natural source like from bridal man or synthetic sources. Examples of those with a natural origin are phenyl propanoids (chloramphenicol), polyketides (tetracycline), aminoglycosides (streptomycin, gentamycin), macrolides (erythromycin), glycopeptides (vancomycin) and second-generation β-lactams (cephalosporins). Those from synthetic sources are sulphonamides, quinolones and oxazolidinones. Most antibiotics exert their action either by inhibition of the bacterial cell wall or protein synthesis. Exceptions are the quinolones that inhibit DNA synthesis, and the sulphonamides that inhibit the synthesis of metabolites used for the synthesis of deoxyribonucleic acid (DNA) (Singh and Barrett 2006). Most anti-viral, anti-fungal, anti-protozoa and anti-cancer drugs however are obtained from synthetic sources.
Because of the re-occurring resistance of pathogenic microorganisms to antibiotics, as well as the side effects presented by these antibiotics, investigation of other sources of antimicrobials, such as medicinal plants, for their antimicrobial properties is gaining ground. Plants produce secondary metabolites (phytochemicals), which have demonstrated their potential as antibacterials when used alone and as synergists or potentiators of other antibacterial agents. Phytochemicals frequently act through different mechanisms than conventional antibiotics and could therefore be of use in the treatment of resistant bacteria (Abreu et al. 2012).
- 4 ACTIVE COMPONENTS OF PLANT EXTRACTS
The beneficial medicinal effects of plant materials typically result from the combination of secondary products present in plants. These compounds are mostly secondary metabolites such as alkaloids, steroids, tannins, and phenol compounds, which are synthesised and deposited in specific parts or in all parts of the plant (Joseph and Raj 2010). Generally, leaves are the favourable storage site for desiredcompounds. Fruits also contain a substantial amount of active ingredients, and thus are often consumed as juice via oral administration to obtain the desired compounds. Other parts of plants that can be extracted for therapeutic compounds are roots, aerial parts, flowers, seeds, stem barks, etc. (Chan et al. 2012).
Plant secondary metabolites are used as the basis for the production of valuable synthetic compounds such as pharmaceuticals, cosmetics, or more recently nutraceuticals (Bourgaudet al. 2001). These secondary metabolites are largely viewed as potential sources of new drugs, antibiotics, insecticides and herbicides (Crozieret al. 2006). This is because of their biological significance and potential health effects, such as antioxidant, anticancer, anti-aging, anti-atherosclerotic, antimicrobial and anti-inflammatory activities.
2.4 MECHANISM OF ACTION OF PLANT ACTIVE MEDICAL COMPOUNDS
Plant active compounds are usually classified according to their biosynthetic pathways. Three large molecular families are generally considered: phenolics, terpenes and steroids, and alkaloids. A good example of a widespread metabolite family is the phenolics, because these molecules are involved in lignin synthesis, they are common to all higher plants. Phenolic compounds are potent antioxidants and free radical scavengers which can act as hydrogen donors, reducing agents, metal chelators and singlet oxygen quenchers (Chew et al. 2009). Studies have shown that phenolic compounds such as catechin and quercetin are very efficient in stabilising phospholipid bilayers against peroxidation induced by reactive oxygen species (Gülçin 2010). Flavonoids, which are a subclass of phenolics, are known to be synthesised by plants in response to microbial infection and they have been found in vitro to be effective antimicrobial substances against awide array of microorganisms. Their activity is probably due to their ability to complex with extracellular and soluble proteins and to complex with bacterial cell walls (Cowan 1999). Tannins and flavonoids are thought to be responsible for antidiarrhoeal activity by increasing colonic water and electrolyte reabsorption (Palombo 2016).
Terpenoids are condensation products of C5 isoprene units which are important constituents of essential oils (Pichersky and Gershenzon 2012). They have been shown to be active against bacteria, fungi, viruses, and protozoa. The mechanism of action of terpenes is not fully understood but is speculated to involve membrane disruption by the lipophilic compounds (Cowan 1999).
Alkaloids are the best known nitrogen-containing metabolites of plants and are sparsely distributed in the plant kingdom, but much more specific to defined plant genera and species. This is probably due to the limited supply of nitrogen in plants (Harborne,2014). Alkaloids have been found to have antimicrobial properties with microbicide effects against Giardia and Entamoebaspecies as well as antidiarrhoeal effects, which are probably due to their effects on transit time in the small intestine (Cowan 2010).
In research carried out by Nitta et al. (2012), the active extracts obtained from the bark of Shoreahemsleyanaand roots of Cyphostemmabainessiwere separated into their components and these exhibited strong inhibitory activity against methicillin-resistant Staphylococcus aureus. These active compounds were identified as stilbene derivatives.
2.5 SIGNIFICANCE OF ANTIMICROBIAL SUSCEPTIBILITY TESTING
In screening new antimicrobials or antibiotics, evaluation of biological activity is essential for the assessment of susceptibility of pathogens to the antimicrobial agent. Antimicrobial susceptibility testing is used in pathology to determine the resistance of certain microbial strains to different antimicrobials and in pharmacology research it is used to determine the efficacy of novel antimicrobials from biological extracts against different microorganisms (Das et al. 2010). Microbial growth or its inhibition can be measured in a number of ways, e.g. viable counts, direct microscopic counts, turbidity measurement, bioluminescence and fluorimetry (Grareet al. 2008). Of the various antimicrobial susceptibility methods employed, the disk diffusion method and the broth microdilution method are commonly used to evaluate the effect of the plant extracts or any other antimicrobial on disease-causing pathogens.
The disk diffusion method is used in determining the zones of inhibition exhibited by the plant extracts, while the broth microdilution method, which has been recommended by the Clinical and Laboratory Standards Institute (2003), is used in determining the minimum inhibitory concentration (MIC) of plant extracts. This method is less cumbersome, less expensive and quite reproducible when compared with the disk diffusion method. The use of microplates allows large amounts of data to be generated quickly. Bacterial growth could be assessed either visually by grading turbidity or better spectrophotometrically by measuring optical density (Grare et al. 2008). The disadvantage of visual assessment of bacterial growth is that it lacks objectivity and precision; whereas the accuracy of spectrophotometric readings may be hampered by (i) additives or antibacterial compounds that affect the spectral characteristics of growth media, (ii) the aggregation of bacteria, or (iii) bacterial pigments (Eloff 1998). Colorimetric methods therefore could represent an alternativeapproach, using tetrazolium salts as indicators, since bacteria convert them to coloured formazan derivatives that can be quantified (Grareet al. 2008).
PREVIOUS STUDIES
Previous studies on the methanolic extract of A. digitateand Bridialmacranths have been reported that it has anti-trypanosomal activity against Trypanosomacongolenseand T. brucei(Atawodiet al. 2003). Stem and root barks of A. digitate and Bridal macranthaalso contain bioactive constituents such as tannins, phlobatannins, terpenoids, cardiac glycosides and saponins in the stem bark, as well as terpenoids in the aqueous extract of root bark, which are responsible for significant antibacterial activity of the crude extracts of this plant (Masolaet al. 2009).
Studies reported the usedofBridialmacrantha traditionally in treating diarrhoea; seeds, pulp and leaves of A. digitata, bark of G. livingstonei, and S. birrea(FAO 1993; De Caluweet al. 2009; Masolaet al. 2009; De Wet et al. 2010; Gouwakinnouet al. 2011), as seen in Table 1.0. Determination of antibacterial activity and the active components of these plants will provide baseline information on potential usage of extracts from these plants for the treatment of infectious diarrhoea caused by the following bacteria: Escherichia coli, Staphylococcus aureus, Klebsiellaoxytoca, Salmonella entericaand Shigellasonnei. The information obtained from this research could then be used further in developing drugs from these plants or synthesising drugs that mimic the active components in these plants.
