INTRODUCTION

1.1                                                  Background of the Study

Medicinal plants have been used for centuries as remedies for human diseases because they contain chemical components of therapeutic value (Nostro et al., 2000). Of the 252 drugs considered essential by the World Health Organization, 11% were derived from flowering plants (Rates, 2001). More than 80% of the world's population relies on traditional medicine for their primary healthcare needs (Alagesaboopathi, 2011). Nigeria is covered with a large number of plant species, some of which have been used for centuries in folkloric medicines to diagnose, prevent and treat various ailments (El-Mahmood et al., 2010). Among the diseases that have been managed successfully by traditional (herbal) medicine include malaria, epilepsy, infertility, convulsion, diarrhoea, dysentery, gonorrhoea, flatulence, tonsillitis, bacterial and fungal infections, mental illness and worm infections (Sofowora, 1996). 

Plants generally produce many secondary metabolites which constitute an important source of microbicides, pesticides and many pharmaceutical drugs. Ogundipe et al. (1998) asserted that plant products still remain the principal source of pharmaceutical drugs and agents used in traditional medicine. Studies have been conducted on the antimicrobial potentials of crude extract of leaves, bark, bulbs, stems and roots of various plants (Atata and Sani, 2003; Olafimihan, 2004). A number of phytotherapy manuals have stated various medicinal plants for treating infectious diseases owing to their availability and low mammalian toxicity (Lee et al., 2007). The effects of plant extracts on bacteria have been studied widely from different parts of the world (Ateb and Erdourul, 2003).

New antioxidants such as plant phenolic compounds are sought for general health maintenance (Wah Chan et al., 2008). The phenolic compounds cover a very large and diverse group of chemical compounds, including flavonoids, lignins, tannins, phenolic acids, coumarins, phenols, phenylpropanoids, quinines, stilbenoids and xanthones. (Vermerris and Nicholson, 2006). Flavonoids are a large group of polyphenolic compounds, subdivided into anthocyanins, flavanols including proanthocyanidins, flavonols, dihydroflavonols, flavones, isoflavonoids, flavonones, chalcones and dihydrochalcones (Li and Beta, 2013). Flavonoid-based herbal medicines are available in different countries as antiinflammatory, antispasmodic, antiallergic, antibacterial and antifungal remedies (Rice-Euans and Parker, 2003).

Garlic (Allium sativum Linn.) is one of those plants that had been heavily investigated over a period of time (Cavallito and Bailey, 1994; Jabar and Al-Mossawi, 2007) and one that has already reached the market together with ginger (Zingiber officinale). It has been used for centuries by Egyptians to fight infections (Onyeagba et al., 2006; Abubakar, 2009). According to Abubakar (2009), Jabar and Al-Mossawi (2007), the early Egyptians used it to treat diarrhoea, the ancient Greeks used it to treat intestinal and extra-intestinal diseases, while the ancient Japanese and Chinese used it to treat headache, flu, sore throat and fever. In Africa, particularly in Nigeria, garlic has been reported to be used to treat abdominal discomfort, diarrhea, otitis media and respiratory tract infections (Ankri and Mirelman, 1999; Jabar and Al-Mossawi, 2007). The phytochemical constituents of garlic have already been established by several workers (Farbman et al., 1993; Cavallito and Bailey, 1994; Ankri and Mirelman, 1999; Prodos-Rosales et al., 2003). The antimicrobial properties of garlic were first described by Pasteur in 1858, and since then, research had demonstrated its effectiveness against bacteria, protozoa, fungi and some viruses (Jabar and Al Mossawi, 2007; Abubakar (2009). Allium ascalonicum L. (Liliaceae) is widely used along with other Allium as spice for food. It contains factors like potassium, calcium, and vitamin C among others (Antia, 1976). There are reports showing that ingestion of Allium lowers blood cholesterol and reduce the risk for coronary artery disease (Antia, 1976; Bordia et al., 1996). 

Aloe vera (L.) Burm.Fil (Synonym A. brobadensis Miller) is a cactus-like plant of the family Liliaceae. It is believed to be effective in treating constipation, stomach ailments, gastrointestinal problems, skin diseases and radiation injury because of its antiinflammatory effect. It is also used for wound healing and burns, diabetes and also as an anti-ulcer (Arunkumar and Muthuselvam, 2009). It has been reported to contain tannins, saponins, flavonoids and terpenoids (Arunkumar and Muthuselvam, 2009). Aloe vera gel and leaf extracts have been shown to suppress the growth of Streptococcus pyogenes, Shigella flexneri, Pseudomonas aeroginosa, Staphylococcus aureus and Escherichia coli (Ferro et al, 2003; Arunkumar and Muthuselvam, 2009; Johnson et al., 2011; Thiruppathi et al., 2012).

Citrus aurantifolia Linn popularly known as lime has been used to treat ailments such as common cold, depressive illness and alcoholism. Several researchers have demonstrated the antimicrobial potentials of lime (Oboh et al., 1992, Oboh and Abulu, 1997; Onyeagba et al., 2006). It has been acclaimed to possess anti-inflammatory, anti-rheumatic, antiscorbutic, anti-coagulant and anti-spasmodic properties (Taiwo et al., 2007). 

The ginger plant, Zingiber officinale (Zingiberaceae), has a biennial or perennial creeping rhizome, locally called Citta (Hausa) and Ginger (English). Ginger is an essential ingredient in many traditional Chinese medicines and has been used since the 4th Century BC. The Chinese take ginger for a wide variety of medical problems such as stomachache, diarrhoea, nausea, cholera, asthma, heart conditions, respiratory disorders, toothache and rheumatic complaints (Wagner and Hikino, 1965). Africans and West Indians also use ginger medically and the Greeks and Romans use it as a spice (Melvin et al., 2009).

Anogeissus leiocarpus (DC) Guill and Perr family Combretaceae (Common name: Axlewood tree) has many applications in Nigeria. It is used medically as an ascaricide and for the treatment of gonorrhoea, general body pain, blood clots, asthma, cough and tuberculosis (Mann et al., 2003). Information obtained from the Yorubas and South-Eastern people of Nigeria showed that the plant is also used as an antimicrobial agent against bacterial infections (Dweek, 1996). The leaves of the plant are used externally as a decoction in the eastern part of Nigeria for the treatment of skin diseases and the itch of psoriasis. The powdered bark is applied to wounds, sores, boils, cysts and diabetic ulcers with good results. The powdered bark has also been mixed with „green clay‟ and applied as an unusual face mask for serious blackheads (Dweek, 1996). The infusion and decoctions are used as cough medicine, the pulped roots are applied to wounds and ulcers, the powdered bark is also rubbed to reduce toothache on gums, it is also used as vermifuges and the leaves decoction is used for washing and fumigation (Ibrahim et al., 1997). Anogeissus leiocarpus is traditionally acclaimed to be effective in treating infectious wounds in man and animals (Dweek, 1996).

Traditionally, the leaves, flowers, fruit follicles and bark of Cola acuminata Schott and Endl (Sterculiaceae) have been used to prepare a tonic as a remedy for dysentery, cough, diarrhea, and vomiting (Ayensu, 1978). They also ease hunger and thirst, eliminate fatigue, provide energy by stimulating the muscles and nerves and enhance intellectual activity (Sundstrom, 1966; Nickalls, 1986). The Cola acuminata nut's primary social and economic significance lies in its being a very concentrated source of Central Nervous System (CNS) stimulants, caffeine which may help in relieving migraine (Kiple and Ornelas 2000), theobromine which act as cerebral vasodilator and is thought to relieve pain and neuralgia (Hirt and M'pia, 2001) and proanthocyanidin which is used as an antitrypanosoma compound effective against Trypanosoma brucei (Kubata et al., 2005).

Cyperus rotundus Linn. (Family Cyperaceae) is a pestiferous perennial weed with dark green glabrous culms, arising from underground tubers (Nadkarni and Nadkarni, 1996; Kirtikar and Basu, 2001). The rhizome part of Cyperus rotundus is one of the oldest known medicinal plants used for treatment of dysmenorrheal and menstrual irregularities (Bhattarai, 1993).  A number of pharmacological and biological activities including antidiabetic, antidiarrhoeal, cytoprotective, antimutagenic, antioxidant, antimalarial, antiinflammatory, antipyretic and analgesic activities have been reported for this plant (Raut and Gaikwad, 2006).

Costus afer Ker Gawl (Costaceae) is a tall terrestrial perennial herb which finds use in folkloric medicine as a remedy for cough, rheumatic pains, sleepiness and as a cardiotonic (Agoha, 1974; Iwu, 1982). It has also been shown to exhibit anti-arthritic activities (Iwu and Anyanwu, 1982). The phytochemical screening of C. afer revealed the presence of alkaloids, flavonoids, saponins, tannins, steroids and cardiac glycosides (Odoemena et al., 2008; Akpan et al., 2012).

Escherichia coli is a Gram-negative, toxin producing bacterium that causes serious food poisoning in humans (Vogt and Dippold, 2005). It is a model organism for bacteria (Peter et al., 1998) and extremely sensitive to antibiotics such as streptomycin or gentamycin but rapidly changing and acquiring drug resistance (Chapman et al., 2002) due to overuse of antibiotics in humans (Johnson et al., 2006). 

Salmonella typhi is a facultatively anaerobic Gram-negative rod-shaped bacterium (Krieg and Holt, 1984) and is one of the most important food borne pathogens. If present in food, the bacteria do not affect the taste, smell or appearance of the food. Frequent hand washing, throwing out expired food, avoiding eating raw or undercooked eggs, meats, seafood or poultry are the key to preventing Salmonella food poisoning. Antibiotics (such as ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline) may be prescribed for moderate to severe cases of Salmonella food poisoning or when it occurs in a person who is at risk for complications. However, probably as a consequence of the extensive use of antibiotics, the incidence and severity of human diseases related to Salmonella caused by antibacterial resistant Salmonella is rising in many countries (Breuil et al., 2000). Presence of the bacterium Salmonella in food and the disease Salmonella food poisoning and typhoid fever continue to be a major public health problem worldwide. Millions of human cases are reported worldwide every year and the disease results in thousands of deaths. The increasingly resistance to antibiotics of food borne Salmonella

(Breuil et al., 2000) drive much of the current interest on plant antibacterial molecules.

Staphylococcus aureus is an enteric Gram-positive bacterium, capable of causing diarrhoea. It is also responsible for skin diseases (Murray et al., 1998; Lansing et al., 1999). Foodborne diseases are of major concern worldwide and S. aureus is a leading cause of gastroenteritis resulting from the consumption of contaminated food (Le Loir et al., 2003).

1.2                               Statement of Research Problem

The wide acceptance of traditional medicine as an alternative form of healthcare and the alarming increase in the incidence of new and re-emerging infectious diseases bring about the necessity to investigate these medicinal plants. There is a continuous and urgent need to discover plants with antimicrobial activities. Moreover, since many plants are unexamined, therapeutic results have been mixed resulting to poisoning (Nostro et al., 2000). Only about 20% of the world medicinal plants have been screened for pharmacological and biological activities (Reynold and Lawson, 1978; Ndukwe et al., 2005, 2007). Another concern is the development of resistance of disease pathogens to the antibiotics in current clinical use (Ertürk et al., 2006).

The high cost of important conventional drugs for treating diseases and/or inaccessibility to modern health care facilities has led to reliance on traditional medicine since it is affordable and available to rural people (Kitonde et al., 2014). Scientific investigations and information on the therapeutic potentials of medicinal plants are limited. This lack of scientific knowledge has restricted the use of traditional herbs as remedies to be used in conjunction with or as an alternative to orthodox medical treatment (El-Mahmood et al., 2010). 

1.3                                                       Justification

The ever increasing demand for safer and cheaper herbal recipes in the developed countries has led to the extraction and development of several drugs and chemotherapeutic agents from plants as well as from traditional herbal remedies (Falodun et al., 2006). There are considerable economic benefits in the development of indigenous medicines and in the use of medicinal spices for the treatment of various diseases (Azaizeh et al., 2003). The scientific literature is full of reports describing plants as the sleeping giant of the pharmaceutical industry (Smith, 1991; Michael, 2002), which when fully exploited will provide novel compounds to fight infectious diseases (Onyeagba et al., 2006; El-Mahmood and Ameh, 2007; Jabar and Al-Mossawi, 2007).

Even though pharmacological industries have produced a number of new antibiotics in the last three decades, resistance to these drugs by microorganisms has increased (Vital and Rivera, 2009). In general, bacteria have the genetic ability to transmit and acquire resistance to drugs, which are utilized as therapeutic agents (Nascimento et al., 2000) and the increase in number of drug resistant bacteria is no longer matched by discoveries of new drugs to treat variant infections (Whitman, 2008). Therefore, there is a need to source for alternative antimicrobial agents. 

Also the claims of effective therapy for the treatment of various diseases by traditional herbalists worldwide have prompted interest in specific investigations of such herbal medication.

This study will however provide scientific evidence on the use of the selected plants combination which is being utilized as herbal therapy.

1.4                                            Aim and Objectives

The aim of this study is to screen for the secondary metabolites and antibacterial activity of a combination of medicinal plants extracts on Escherichia coli, Salmonella typhi and Staphylococcus aureus.

The specific objectives of this study are to:

1.      determine the phytochemical secondary metabolites of the combination of

selected plants.

2.      determine the antibacterial susceptibility of Escherichia coli, Salmonella typhi and Staphylococcus aureus to the different concentrations of the selected plants combination extracts.

3.      determine the minimum inhibitory concentrations (MICs) of the selected plants combination extracts on Escherichia coli, Salmonella typhi and Staphylococcus aureus.

4.      determine the minimum bactericidal concentrations (MBCs) of the selected plants combination extracts on Escherichia coli, Salmonella typhi and Staphylococcus aureus.

1.5                                                       Research Questions

i.               What are the secondary metabolites of the selected plants combination extracts?

ii.            Are the test organisms, Escherichia coli, Salmonella typhi and Staphylococcus aureus susceptible to the selected plants combination extracts?

iii.          Do the different concentrations of extracts have bacteriostatic effects on Escherichia coli, Salmonella typhi and Staphylococcus aureus? iv. Do the different concentrations of extracts have bactericidal effects on Escherichia coli, Salmonella typhi and Staphylococcus aureus?

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