Table of Contents
CHAPTER ONE: Introduction
CHAPTER THREE: Material and Method
CHAPTER FOUR: Results
CHAPTER FIVE: Discussions and Conclusions
CHAPTER ONE: Introduction
1.1 Background of the Study
The increased usage of antibiotics has induced microorganisms to acquire resistance factors which have become a burning predicament (Abimbola et al., 2013). As a result there is an urgent need to find the alternative of chemotherapeutic drugs in diseases treatment particularly those of plants origin which are easily available and have considerably less side effects (Khulbe and Sati, 2015). The use of higher plants and their extracts for treating the infectious diseases has long been practiced in many parts of the world (Sofowora, 2014). The plant derived medicines may be used in many different forms including: powder, liquid or mixtures which could be raw or boiled such as, liniments, ointments and incisions. Ginger (Zingiber officinale) is a medicinal plant that has been widely used all over the world, since antiquity, for a wide array of unrelated ailments including arthritis, cramps, rheumatism, sprains, sore throats, muscular aches, pains, constipation, vomiting, hypertension, indigestion, dementia, fever and infectious diseases (Ali, 2018). Ginger has direct anti-microbial activity and thus can be used in treatment of bacterial infections (Tan and Vanitha, 2014). Ginger belongs to Zingiberaceae family (Sharma, 2010). The Zingiberaceous plants have strong aromatic and medicinal properties and are characterized by their tuberous or non-tuberous rhizomes (Chen, 2018). Ginger is relatively inexpensive due to their easy availability, universally acceptable and well tolerated by the most people. It has also “Generally Recognized as Safe” (GRAS) by the US FDA (ICMR Bulletin, http://icmr.nic.in/BUJUNE O3nwe.pdf). In many countries including Bangladesh, ginger is used in boiled food preparation.
1.2 Aim of the Study
The aim of this study is to determine the antimicrobial effects of Allium sativum (garlic) and Zinger officinale (ginger) on pathogenic bacteria.
1.3 Objectives of the Study
The specific objectives of the study are:
i. To determine the antimicrobial effects of Allium sativum (garlic) and Zinger officinale (ginger) on pathogenic bacteria Salmonella typhi and Staphylococcus aureus
ii. To determine the minimum inhibitory concentration of Allium sativum (garlic) and Zinger officinale (ginger) on pathogenic bacteria Salmonella typhi and Staphylococcus aureus .
iii. To determine the minimum bactericidal concentration of Allium sativum (garlic) and Zinger officinale (ginger) on pathogenic bacteria Salmonella typhi and Staphylococcus aureus .
CHAPTER TWO: Literature Review
2.1 History of Medicinal Plants
The use of plants as medicines could be dated back to the Middle Paleolithic Age, which is about 60,000 years ago, according to fossil records (Fabricant and Farnsworth, 2011). The first records written on clay tablets in cuneiform are from Mesopotamia and date from about 2, 600 BC. Some of the substances that were used were oils of Cedrus species (cedar) and Cupressus sempervirens (cypress), Glycyrrhiza glabra (licorice), Commiphora species (myrrh) and Papaver somniferum (poppy juice), most of which are still in use today for treating ailments ranging from coughs and colds to parasitic infections and inflammation (Gurib-Fakim, 2016).
Health care in ancient times included the use of leaves, flowers, stems, berries and roots of herbs for their therapeutic or medicinal value. These medicines initially took the form of tinctures, teas, poultices, powders, and other herbal formulations (Gurib-Fakim, 2016). Knowledge of the specific plants to be used and the methods of application for particular ailments were passed down through oral history and information regarding medicinal plants was eventually recorded (Balunasa and Kinghorn, 2015).
The ethnobotanical use of medicinal plants and/or their derivatives such as essential oil, resins and soluble extracts in Africa dates back to early civilization (Sofowora, 2010). This practice is globally perceived as comparatively cheaper and more widely accessible to most rural or less-privileged economies of the world than modern synthetic drugs (Lawal et al., 2012). The bulletin of the World Health Organization showed that close to 65% of the world population relied on medicinal plants for their primary healthcare drugs (Eddouks and Ghanimi, 2013). Consequently, it is estimated that about 39% of drugs developed since 1980 have being from natural plants, their derivatives or analogues (Verpoorte et al., 2010). In addition, Verpoorte et al. (2010) noted that approximately 25% of the currently used modern drugs are derived from plants, a number largely composed of analgesics (morphine), cardiotonics, chemotherapeutics and antimalarials (quinine and artemisinin).
There have been growing concerns on the rising cost of buying synthetic drugs, assessing their toxicological profile, and redressing their periodic side-effects and unstable efficacy (Gupta et al., 2016). These undermine their continuous use in modern healthcare delivery and cause a renaissance of herbal screening as well as the chronopharmacological process (Westh et al., 2014). The clinical and pharmacokinetics sustainability of the efficacy of many synthetic antibiotics, prophylactics and curative drugs is threatened by the growing emergence of multi-drug resistant pathogenic strains and cost of production (Bandow et al., 2013). These concerns caused researchers to shift focus and exploit more intensely natural plants and their allies such as ferns, fungi and algae for safer generic bioequivalents to synthetic drugs or substitutions with stable therapeutic values (Savoia, 2012).
Plant-derived medicine accounts for more than a quarter of today’s pharmacopoeia (Eddouks and Ghanimi, 2013). While the global inventory of ethnobotanicals is growing, the catalogue of their bioactive compounds that can improve human health is constantly being updated. Over 12,000 bioactive metabolites including primary and secondary metabolites and pigments of plant origin with a wide range of biological activities as well as therapeutic values were documented. Osemwegie et al. (2014) noted the inadequacy of current data in capturing the global totality of medicinal plants due to either neglect of plants in remote ecozones or bias against other related plant biota. While the prehistoric and historic knowledge of numerous health-beneficial plants in Africa seems threatened in recent times, their use in primary healthcare delivery predates modern medicine (Pasewu et al., 2008). This has facilitated the hybridization of both traditional and modern primary healthcare systems in some continents of the world (Eddouks and Ghanimi, 2013).
2.2 Extraction Techniques of Plant Extracts
Extraction is the crucial first step in the analysis of medicinal plants because it is necessary to extract the desired chemical components from the plant materials for further separation and characterization (Sasidharan et al., 2011). Different extraction techniques are available, but the most common ones used in plants extraction are the conventional techniques. In conventional extraction, the release of the desired compounds traditionally requires soaking and maceration in mild solvents (Chan et al., 2012).
Decoction in water is broadly employed in traditional Chinese medicinal practices and is an effective method that can be considered in cases where the presence of a chemical solvent is undesirable (Das et al., 2010). Other solvents that can be used in conventional extraction are acetone, petroleum ether, methanol and hexane. Liquid nitrogen has also been used as a form of extraction in some research work (Karuna et al., 2012). Techniques such as lyophilization (Chen et al., 2013) and sonification (Chukwujekwu et al., 2015) are further methods that can be employed other than solvent extraction. Non-conventional methods that can be used are the supercritical fluid extraction and microwave-assisted techniques. Supercritical fluid extraction was used to investigate the antioxidant activity of the extract of lotus gem (Li et al., 2015). Microwave-assisted extraction has also been used to investigate the bioactivity of tea flower polysaccharides (Wei et al., 2010).
2.3 Plant Secondary Metabolites
Natural products chemistry is the chemistry of metabolite products of plant, animals, insects, marine organisms and microorganisms. The metabolic products include alkaloids, flavonids, terpenoids, glycosids, amino acids, protein, and carbohydrates. The applications of natural products range from medicines, to sweeteners and pigments (Rensheng et al., 2010).
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 synthesized and deposited in specific parts or in all parts of the plant (Joseph and Raj, 2010). Generally, leaves are the favourable storage site for desired compounds.
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 and stem barks (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 (Chan et al., 2012). These secondary metabolites are largely viewed as potential sources of new drugs, antibiotics, insecticides and herbicides (Joseph and Raj, 2010). This is because of their biological significance and potential health effects, such as antioxidant, anticancer, anti-aging, anti-atherosclerotic, antimicrobial and anti-inflammatory activities.
Alkaloids are defined as natural plant compounds having a basic character and containing at least one nitrogen atom in a heterocyclic ring. The alkaloids are usually colorless, crystalline, non-volatile solids which are insoluble in water, but are soluble in ethanol, ether, chloroform. Some alkaloids are liquids which are soluble in water. Most alkaloids have a bitter taste and are optical active. They are generally tertiary nitrogen compounds and contain one or two nitrogen atoms usually in the tertiary state in a ring system, most of the alkaloids also contain oxygen (Saxena, 2016).
Flavonoids are plant secondary metabolites, aromatic and belong to the group of plant phenols. Concerning the plant phenol, and consequently the flavonoids, it may be considered as those compounds originating in the shikimate and phenyplpropanoid pathways. Notwithstanding, the flavonoids, as a differentiate subgroup inside the phenolic compound, show a characteristic metabolic intermediate, the naringeninchalcone, from which all the bioflavonoids originate. Exclusively from a chemical point of view the flavonoids are characterized by a skeleton of three units, C6 – C3 –C6, that forms a cyclic structure in most cases. In this skeleton tow aromatic ring, referred to as A and B, can be distinguished, and an additional third ring C, in the rest of the flavonoids.
Terpenoids are defined as materials with molecular structures containing carbon backbones made up of isoprene (2-methylbuta-1,3-diene) units. Isoprene contains five carbon atoms and therefore, the number of carbon atoms in any terpenoids is a multiple of five. Degradation products of terpenoids in which carbon atoms have been lost through chemical or biochemical processes may contain different number of carbon atoms, but their overall structure will indicate their terpenoid origin and they will still be considered as terpenoid. The generic name “terpene” was originally applied to the hydrocarbons found in turpentine, the suffix “ene” indicating the presence of olefinic bonds. Each of these materials containing 20 carbon atoms is named as diterpenes.
2.4 Mechanism of Action of Plant Metabolites
Plant secondary metabolites are usually classified according to their biosynthetic pathways. Three large molecular families are generally considered: phenolics, terpenes and steroids, and alkaloids (Chew et al., 2015). A good example of a widespread metabolite family is the phenolics, because these molecules are involved in lignin synthesis, and 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., 2015). 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 (Gulcin et al., 2010).
Tiwari and Rao (2002) reported that the different composition of the active components in plants give medicinal plants an edge as better therapeutic agents than chemotherapy in management of different ailments such as atherosclerosis, hypertension and diabetes.
Phytochemical are naturally occurring non-nutritive plant chemicals that are largely responsible for the protective health benefits of these plant-based foods beyond those conferred by their vitamins and minerals. They are not required by the human body for sustaining life, but recent research findings indicate that they can also protect humans against diseases (Saidu and Okorocha, 2013). Researchers have reported that various parts of Gongronema latifolium are rich in flavonoids, alkaloids, saponins, resins, cardiac glycosides, and β–sitosterol. These phytochemicals present in the plants have been linked to its ethno botanical use.
Flavonoids are known to inhibit formation of plagues in arteries and so prevent artherosclerosis, hypertension and other cardiovascular diseases. They are also very important antioxidants that mop up reactive oxygen radicals known to be involved in many conditions that cause cancers, diabetes, inflammatory diseases and neurodegenerative diseases. Saponin a very important phytoconstituent, lowers cholesterol and glucose level and are also involved in ulcer protection and certain antimicrobial activity (Ukwe et al., 2014). Alkaloids are involved in antimicrobial and hypoglycemic activities (Punitha et al., 2015). Resins and essential oils have also been involved in antimicrobial, anti-inflammatory and antioxidant properties (Lemos et al., 2016). Cardenolides and bufadienolide are useful for treatment of heart conditions (Enemor et al., 2014).
2.5 Antimicrobial Effects of Garlic
Garlic has been used since ancient times for its health beneficial properties and modern research has provided a scientific basis for this practice . Garlic compounds have been shown to decrease cholesterol and fatty acid levels in the blood and lower blood pressure ; thus, garlic consumption can contribute to the prevention of cardiovascular diseases . Anti-tumour activities of garlic compounds have been demonstrated, providing for a potential use in cancer-therapy and prevention . Another very important garlic property is the antimicrobial activity observed in raw garlic extract. The main anti-bacterial compound of fresh garlic is allicin, a thiosulfinate with two allyl groups as carbon chains (diallylthiosulfinate) . Besides bacteria, the effects of allicin have been investigated against fungi, protozoa and viruses. Methicillin-resistant Staphylococcus aureus (MRSA) isolates were also shown to be susceptible to allicin . Allicin is produced from the non-protein amino acid alliin (S-allylcysteine sulfoxide) upon tissue damage in a reaction that is catalyzed by the enzyme alliinase. Structurally analogous thiosulfinates are produced in nature by other Allium and Petiveria spp., and M antimicrobial activity has been reported for this group of compounds. Unlike conventional antibiotics, allicin is volatile and can kill bacteria via the gas phase. This is particularly interesting since many lung-pathogenic bacteria are susceptible to allicin. Although allicin is also toxic to human cells, the successful treatment of tuberculosis by breathing in the vapour from crushed garlic preparations was reported in the pre-antibiotic era.
2.6 Antimicrobial Effects of Ginger
Ginger (Zingiber officinale Roscoe), which belongs to the Zingiberaceae family and the Zingiber genus, has been commonly consumed as a spice and an herbal medicine for a long time (Viega etal.,2006). Ginger root is used to attenuate and treat several common diseases, such as headaches, colds, nausea, and emesis. Many bioactive compounds in ginger have been identified, such as phenolic and terpene compounds. The phenolic compounds are mainly gingerols, shogaols, and paradols, which account for M the various bioactivities of ginger . In recent years, ginger has been found to possess biological activities, such as antioxidant, anti-inflammatory, antimicrobial (Tijjani et al., 2009) and anticancer activities. In addition, accumulating studies have demonstrated that ginger possesses the potential to prevent and manage several diseases, such as neurodegenerative diseases cardiovascular diseases,obesity,diabetes mellitus, chemotherapy-induced nausea and emesis (Keneman etal.,1997) and respiratory disorders In a review, focus on the bioactive compounds and bioactivities of ginger, and with special attention to its mechanisms of action.
In recent years, ginger has been reported to show antibacterial, antifungal, and antiviral activities (Moon et al., 2018). Biofilm formation is an important part of infection and antimicrobial resistance. One result found that ginger inhibited the growth of a multidrug-resistant strain of Pseudomonas aeruginosa by affecting membrane integrity and inhibiting biofilm formation (Chakatiga etal.,2017). In addition, treatment with ginger extract blocked biofilm formation via a reduction in the level of bis-(3-5)- cyclic dimeric guanosine monophosphate (c- di-GMP) in Pseudomonas aeruginosa PA14 (Kin etal.,2013). Moreover, a crude extract and methanolic fraction of ginger inhibited biofilm formation, glucan synthesis, and the adherence of Streptococcus mutans by downregulating virulence genes. Consistent with the in vitro study, a reduction in caries development caused by Streptococcus mutans was found in a treated group of rats (Hosa etal.,2018). Furthermore, an in vitro study revealed that gingerenone-A and 6-shogaol exhibited an inhibitory effect on Staphylococcus aureus by inhibiting the activity of 6-hydroxymethyl-7, 8- dihydropterin pyrophosphokinase in the pathogen (Rampogu etal.,2018). The compounds in ginger essential oil possess lipophilic properties, making the cell wall as well as the cytoplasmic membrane more permeable and inducing a loss of membrane integrity in fungi (Nerilo.,etal.,2016). An in vitro study revealed that ginger essential oil effectively inhibited the growth of Fusarium verticillioides by reducing ergosterol biosynthesis and affecting membrane integrity. It could also decrease the production of fumonisin B and fumonisin B (Gracia.,203). In addition, ginger essential oil had efficacy in suppressing the growth of Aspergillus flavus as well as aflatoxin and ergosterol production . Moreover, the γ-terpinene and citral in ginger essential oil showed potent antifungal properties against Aspergillus flavus and reduced the expression of some genes related to aflatoxin biosynthesis (Moon etal.,2018).