Free Project-Invitro antimicrobial activity of aristolochia bracteolata on e_coli

  CHAPTER ONE

    INTRDODUCTION 

1.1MEDICINAL PLANT

Traditional medicine has always been an exemplary source of drugs and many of the currently available drugs have been derived directly or indirectly from them (Tenginas,1993). Traditional medicine has immense value in the health care systems of developing countries. The world health organization (WHO) estimates that more than 80% of health care needs in these countries are met through traditional health care practices (WHO,2002).The use of traditional medicines in West Africa is probably as old as the duration of human settlement in the region (Ahmad,2001). A medical plant provides an important sources of new chemical substances with potential therapeutic effects. These have been used in traditional medicine for the treatment of several diseases and aliments (Mukherjee et al., 1998). Plants are used medicinally in different countries and are a sources of many potent and powerful drugs (Srivastava et al., 1996). Folk healing, since ancient times has always included medicines of plant origin. Traditional healing practices and herbal remedies have stood the test of time because of their strong cultural ties, and easy accessibility (Dolin et al., 1994). Traditional medicines have and will continue to have positive roles in the lives of people throughout the world, traditional medicines have known to sustained mankind as a sources of medicines and are utilized in various ways for varied purpose (Tenginas, 1993).

Medicinal plants have been used for centuries to cure human disease (Roweha, 1994). According to WHO the increase of resistance to antibiotics by bacterial pathogens is a growing problem in both developing and developed countries. It has been proved that many of these plants exhibit general antifungal and antibacterial activities (Roweha, 1994). The systematic screening of antibacterial plant extract represents continuous efforts to finding newer compounds with the potential to act against drug resistant organisms (Roweha, 1994). Use of plant as a sources of medicine has been inherited and is an important aspect of healthcare system in Nigeria (Roweha, 1994).

1.2.1 Description and Occurance

Aristolochia Bracteolata is a herbaceous plant belonging to family aristochiaceae arisootlochia it is a large plant with over 500 species (Pushpangada, 1995). Aristolochia bracteolate. Lam called as worm killer (due to supposed antihelminthic activity) in English. Aristolochia species have been used as traditional medicines (Pushpangadan, 1995). They contain important natural toxins (Srivastava et al., 1996). Also Aristolochia bracteolate have been an integral part of life in various regional communities for food and drugs both. India has more than 3,000 years of medicinal heritage based on medicinal plants (Pushpangadan, 1995). Aistolochia Bracteolata are largely used by all divisions of the population either directly as folk medication or indirectly in the preparation of recepharmaceuticals (Pushpangadan, 1995). Numerous plants synthesize substances that are useful in the maintenance of health in humans and animals (Pushpangadan, 1995) Aristolochia Bracteolata are used medicinally in diffent countries and are a sources of many potent and powerful drugs (Srivastava et al., 1996).

The genus Aristolochia consist of about 400 species of herbaceous perennials, under shrubs or shrubs bearing essential oils and is widespread across tropical Asia, Africa and south America (Macmillan, 2008). In the indigenous system of medicine, the plant was used for the treatment of skin disease, inflammation and purgative (wealth of india 1982). Root extract was reported to have anti bacterial activity (Dirdiri et al., 1988) and also Toxicity of A, bracteolate was reported (Harborne et al., 1988). This species which had been shown to be nephrotoxic and carcinogenic due to the cytotoxicity of the Aristolochic acid constituents, the leaves of the plant which are used by native tribal and the villagers, it is commonly called as worm killer in English (Harborne et al., 2006). 

1.2.2 USES OF ARISTOLOCHA BRACTEOLATA

The use of Aristolocha Bracteolata to treat human disease has iits roots in pre historical times. Medicinal plants are used by 80% of the world populations as the only available medicines especially in developing countries (Hashim et al., 2010). The information of medicinal plants have been accumulated in the course of several centuries based on various medicinal systems such as AYURVADA, Homeopathy, Naturopathy, Amchi, Modern, Siddha and Unani, in india, it is declared that traditional healers use 2500 plants species 100 species of plant serve as natural principles of medicine (Seliya et al., 2010). With a view to increasing the wide range of medicinal usages, the present day entails new rugs with more potent and desired activity with less or no side effects against particular disease (Roy and Kumar, 2009). Therefore the use of leaf extract of Aristolochia bracteolate is used to cure human disease such as in diverse biological functions include hypertension relief, leukocyte ancement, rheumatism relief, edema therapy, as weel as analgestic and diuretic effects (Bensky et al,. 1993). Various Aristolocha species have been used in herbal medicines since antiquity in obstetrics and in treatment of snakebite (Meenatchi sundaram, 2009). Festering wound, and tumours and they remain in use particularly in chinese herbal medicine (Balachandran, 2005).

1.2.3 JUSTIFICATION OF THE RESEARCH

The use of herbal medicine is a rich tradition particularly among rural peoples because of its relative safety and affordability compared to the cost of modern medicine. Therefore, there is need to provide scientific basic for justification on the therapeutic uses of medicinal plants against infection disease. Some tradition medicines have potential and their effects have been tested and used in the production of modern medicines used in hospitals and medical centers 

1.2.4 AIM AND OBJECTIVES

The aim of this research is to evaluate in vitro and to correlate scientifically the phytochemical composition of Aristolochia Bracteolata with it is medicinal uses.

Objectives of the Research

  i.  To carry out qualitative phytochemical analysis of Aristolochia Bracteolata

  ii.  To quantify the phytochemicals present in Aristolochia Bracteolata

  iii.  To carry out the antibacterial activity of Aristolochia Bracteolata

CHAPTER TWO

2.0 LITERATURE REVIEW

Historically A. Bracteolata plants have provided a sources of inspiration for novel drug compounds. As plant derived medicines have made large contributions to human health and well being (Iwu et al., 1999). A Bracteolata constitutes an effective sources of both traditional and modern medicines (Iwu et al., 1999). A Bracteolata has been shown to have genuine utility and about 80% of rural population depends on it as primary health care (Farnsworth et al., 1985; Akinyemi et al., 2005). 

  Over the years, the World Health Organization advocated that countries should encourage traditional medicine with a view to identifying and exploiting aspects that provide safe and effective remedies for ailments of both microbial and non-microbial origins (WHO 1978). 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 (Doughari, 2006).

  Pharmaceutical screening of A. Bracteolata plans has revealed the presence of numerous chemical including alkaloids, tannins, flavonoids, steroids, glycosides, saponins etc (Iwu et al., 1999). Many plant extracts and phytochemicals show antioxidant/free radical scavenging properties (Larson 1988; Nair et al., 2007;Parekh and Chanda 2007a). Secondary metabolites of plants serve as defense mechanisms against predation by many microorganisms, insects and herbivores (Lutterodt et al., 1999; Marjorie 1999).

  Flavonoids are broad class of plant phenolics that are known to possess a well established protective ability against membrane lipoperoxidative damages (Sen et al., 2005) A. Bracteolata have been part of phytomedicines since time immemorial.

These can be derived from any part of the plant like bark, leaves, flowers, root, fruits, seeds etc (Gordon and David 2001) i.e any part of the plant may contain active component, knowledge of the chemical constituents of plants is desirable because such information will be of value for the synthesis of complex chemical substances. Such phytochemical screening of A. Braceolata is reported by many workers (Mojab et al., 2003; Parekh and Chanda 2007b; Parekh and Chanda 2008).

  The use of antimicrobial agents is critical to the successful treatment of infectious diseases. Although there are numerous classes of drugs that are routinely used to treat infections in humans, pathogenic microorganisms are constantly developing resistance to these drugs (Al-bari et al., 2006). Because of indiscriminate use of antibiotics (Gibbons 1992;Rahman et al., 2001).

The use of higher plants and preparations made from them to treat infections is a longstanding practice in a large part of the population, especially in the developing countries, where there is dependence on traditional medicine for a variety of ailments (Ahmad and Mohammad 1998). Interest in A. Bracteolata with antimicrobial properties increased because of current problems associated with the antibiotics (Emori and Gaynes 1993; Pannuti and Grinbaum 1995).

Recently, the antimicrobial effects of A. Bracteolata extracts against certain pathogens have been reported by a number of researchers (Ahmad and Beg 2001; Erasto et al., 2004; Nair et al., 2007b; Carneiro et al., 2008; Liasu and Ayandele 2008; Parekh and Chanda 2008; Chanda et al., 2009).

  Disc diffusion method is the most widely used procedure for testing antimicrobial susceptibility (Sambath kumar et al., 2006). The disc diffusion procedure (Kirby-Bauer method) has been accepted by the Food and Drug Administration (FDA) and as a standard by the national committee for clinical laboratory standards (Barry and Thornberry 1985;NCCLS 2003).

2.1 Phytochemistry

Phytochemistry are the byproduct of the primary metabolite functions of the plant, otherwise called the secondary metabolite (Richer et al., 1978). They are produced and used by the plants for protection and repair processes within the natural environment (Bako et al., 2005). Plant secondary products also have protective actions in relation to a biotic stress such those associated with the change in temperature, water, status, light level, Uv exposure, mineral and nutrient (Kaufman et al., 1999).

2.2 Tannins 

Tannins is a general descriptive name for group of polymeric/phenolic substances capable of tanning leather or precipitating gelatin from a solution, a property known as astringency (Harborne, 1973). They are divided in to two groups namely hydrolyzed and condensed tannins. Hydrolyzed tannins are based on garlic acid, usually as multiple esters with D-glucose, while the numerous condensed tannins (Often proanthocyanides are derived from flavonoid monomers (Harborne, 1973).

 2.3 Saponins

Saponins are glycosides of both triterpennes and steroids that are characterized by eithr bitter or astringent taste, foaming property, haemolytic effect on red blood cells and cholesterol binding properties (Okwu, 2005). Saponins have been shown to possess both beneficial (lowering cholesterol ) and deleterious (cytotoxic and permesbilization of intestinal epithelium) properties and to exhibit structure dependent biological activity (Okwu, 2005).

2.4 Flavonoids

Flavonoids are 15-carbon compounds generally distributed thoughout the plant kingdom (Hrborne, 1973). They are known to be synthensized by plants in response to microbial infection and have been found invitro to be effective against the wide arrays of microorganism (Harborne, 1973).

Terpenoids essential oil are the main compound found in the volatile steam distillation fraction responsible for the characteristic scent, odour or small found in many palnts(Harborne, 1973). They are commercially important as the basis of natural perfumes and also of spices and are used for flavouring purposes in the food industry (Harborne, 1973).

2.5 Cardiac Glyosides

This is another class of the plants secondary metabolites that are also produced by oxidation of the plants phenolic compound (Muell and Olugbade, 1996). It is often shown antimicrobials activity and considered to be associated with innate resistance of plants to disease (Muell and Olugbade, 1996).

2.6 Alkaloids

Alkaloids are group of naturally occurring chemical compounds that contain mostly basic Nitrogen atoms (Andreas and Luch, 2009). This group also includes some related compound that is the presence A reddish brown precipitate, (Andreas and Luch, 2009)

.

2.7 DESCRIPTION OF GENUS ARISTOLOCHI'A

Aristolchi'a is a genus of evergreen and deciduous lianas (woody vines) and herbaceous perennials . The smooth stem is erect or somewhat twining. The simple leaves are alternate and cordate, membranous, growing on leaf stalks.(Tomar A June 2017).There are no stipules . The flowers grow in the leaf axils . (Harborne, 1973). They are inflated and globose at the base, continuing as a long perianth tube, ending in a tongue-shaped, brightly colored lobe.(Bako et al., 2005). There is no corolla . The calyx is one to three whorled, and three to six toothed. The sepals are united (gamosepalous).(Gbadamosi IT,et., al. (2012).  There are six to 40 stamens in one whole. They are united with the style, forming a gynostemium. The ovary is inferior and is four to six locular. These flowers have a specialized pollination mechanism. The plants are aromatic and their strong scent attracts insects. (Sen et al., 2005). The inner part of the perianth tube is covered with hairs, acting as a fly-trap. These then wither to release the fly, covered with pollen.(Tomar A June 2017). The fruit is dehiscent capsule with many endospermic seeds. The common names Dutchman's pipe and pipevine (e.g. common pipevine, A. durior ) are an allusion to old-fashioned meerschaum pipes at one time common in the Netherlands and northern Germany. Birthwort (e.g. European birthwort A. clematitis ) refers to these species' flower shape, resembling a birth canal.

(Gbadamosi IT,et., al. (2012).

2.8 TAXONOMY OF THE GENUS ARISTOLOCHI'A

 Aristolochia:- is a large plant genus with over 500 species that is the type genus of the family Aristolochiaceae . Its members are commonly known as birthwort , pipevine or Dutchman's pipe and are widespread and occur in the most diverse climates. Some species, like A. utriformis and A. westlandii , are threatened with extinction.

2.9 PLANT TAXONOMY OF A.BRACTEOLATA

Kingdom: Plantae

Clade : Angiosperms

Clade : Magnoliids

Order: Piperales

Family: Aristolochiaceae

Genus: Aristolochia

Species: A. bracteolata

2.10 COMMON AND LOCAL NAME OF A.BRACTEOLATA

Aristolochia bracteolata also known as 'worm killer' in English due to its anthelminthic activity and trypanocidal effect, is a perennial herb growing from 1060 cm tall. The plant is important in traditional medicine in Africa , India and the Middle East . Madacin qasa in hausa language of Northen Nigeria.

2.1.1 ETHNOMEDICINAL USES OF A.BRACTEOLATA

Aristolochia bracteolata has been used in traditional medicine in Nigeria, India, and Ethiopia as an infusion of dried leaves to treat intestinal worms , skin itch, or insect bites. 

2.1.2 CULTIVATION OF A.BRACTEOLATA

Aristolochia bracteolata is usually gathered from the wild.

2.1.3 HERBALISM, TOXICITY AND CARCINOGENICITY

 The species Aristolochia clematitis was highly regarded as a medicinal plant since the ancient Egyptians, Greeks and Romans, and on to until the Early Modern era; it also plays a role in traditional Chinese medicine. (Harborne, 1973). Due to its resemblance to the uterus, the doctrine of signatures held that birthwort was useful in childbirth. A preparation was given to women upon delivery to expel the placenta, as noted by the herbalist Dioscurides in the 1st century AD. Despite its presence in ancient medicine, Aristolochia is known to contain the lethal toxin aristolochic acid.(Bako et al., 2005).

 The Bencao Gangmu, compiled by Li Shi-Zhen in the latter part of the sixteenth century, was based on the authors experience and on data obtained from earlier herbals; this Chinese herbal classic describes 1892 "drugs" (with 1110 drawings), including many species of Aristolochia.(Okwu, 2005). For 400 years, the Bencao Gangmu remained the principal source of information in traditional Chinese medicine and the work was translated into numerous languages, reflecting its influence in countries other than China. In the mid-twentieth century, the Bencao Gangmu was replaced by modern Materia Medica, the most comprehensive source being Zhong Hua Ben Cao (Encyclopedia of Chinese Materia Medica), published in 1999. (Sen et al., 2005). The Encyclopedia lists 23 species of Aristolochia, though with little mention of toxicity. The Chinese government currently lists the following Aristolochia herbs: A. manshuriensis (stems), A. fangchi (root), A. debilis (root and fruit), and A. contorta (fruit), two of which (madouling and qingmuxiang) appear in the 2005 Pharmacopoeia of the People's Republic of China.(Harborne, 2016).

 In traditional Chinese medicine Aristolochia species are used for certain forms of acute arthritis and edema.

 Despite the toxic properties of aristolochic acid, naturopaths claim that a decoction of birthwort stimulates the production and increases the activity of white blood cells, or that pipevines contain a disinfectant which assists in wound healing. Also, Aristolochia bracteolata is colloquially known as "worm killer" due to supposed antihelminthic activity.(Harborne, 1973). 

Aristolochia taxa have also been used as reptile repellents. A. serpentaria (Virginia snakeroot) is thus named because the root was used to treat snakebite, as "so offensive to these reptiles, that they not only avoid the places where it grows, but even flee from the traveler who carries a piece of it in his hand".A. pfeiferi, A. rugosa, and A. grill bats are also used in folk medicine to treat snakebites.(Harborne, 1973). 

2.1.4 TOXICITY AND CARCINOGENICITY

  In 1993, a series of end-stage renal disease cases was reported from Belgium associated with a weight loss treatment, where Stephania tetrandra in a herbal preparation was suspected of being substituted with Aristolochia fangchi. (Okwu, 2005). More than 105 patients were identified with nephropathy following the ingestion of this preparation from the same clinic from 1990 to 1992. Many required renal transplantation or dialysis.(Bako et al., 2005).

Aristolochia has been shown to be both a potent carcinogen and kidney toxin. Herbal compounds containing Aristolochia are classified as a Group 1 carcinogen by the International Agency for Research on Cancer. (Harborne, 1973). Epidemiological and laboratory studies have identified Aristolochia to be a dangerous kidney toxin; Aristolochia has been shown associated with more than 100 cases of kidney failure. Furthermore, it appears as if contamination of grain with European birthwort (A. clematitis) is a cause of Balkan nephropathy, a severe renal disease occurring in parts of southeast Europe.(Harborne, 1973). 

 Aristolochic acid was linked to aristolochic acid-associated urothelial cancer in a Taiwanese study in 2012. In 2013, two studies reported that aristolochic acid is a strong carcinogen. (Gupta, 2014). Whole-genome and exome analysis of individuals with a known exposure to aristolochic acid revealed a higher rate of somatic mutation in DNA. Metabolites of aristolochic acid enter the cell nucleus and form adducts on DNA. (Gupta, 2014). While adducts on the transcribed DNA strand within genes are detected and removed by transcription-coupled repair, the adducts on the non-transcribed strand remain and eventually cause DNA replication errors.(Devgan, 2013). These adducts have a preference for adenine bases, and cause A-to-T transversions. Furthermore, these metabolites appear to show a preference for CAG and TAG sequences.(Gupta, 2014).

2.1.5 ETIOLOGY OF BACTERIA

Bacteria are single celled microbes with a simple cell structure than that of other organisms as there is no nucleus or membrane bound organelles. Instead their control center containing the genetic information is contained in a single loop of DNA (Dyall et al., 2004).

Some bacteria have an extra circle of genetic material called a plasmid. The plasmid often contains genes that give the bacterium some advantage over other bacteria. For example it may contain a gene that makes the bacterium resistant to a certain antibiotic (Perilla, 2003). 

Bacteria are found in every habitat on earth: soil, oceans and even arctic snow. Some live in or on other organisms including plants and animals including humans (Schopf, 1995). There are approximately 10 times as many bacterial cells as human cells in the human body. A lot these bacterial cells are found lining in digestive system. Some bacterial live in soil or on dead plant matter where they play an important role in the cycling of nutrient. Some types couse food spoilage and crop damages but others are incredibly useful in the production of fermented food such as yoghurt and soy sauce. Relatively few bacteria are parasite or pathogens that cause disease in animal and plant. (Schopf, 1995). 

 Bacteria were also involve in second great evolutionary divergence,that of the archaca and the eukaryotes (Dyall et al.,2004).

 Here,eukaryotes resulted from the entering of ancient bacteria into endosymbiotic association with the ancestor of eukaryotic cells, which were themselves possibly related to the achaea (Poole and penny 2007). 

2.1.6 BACTERIA GROWTH

Bacteria growth follows four phases. When a population of bacteria enters a high nutrient environment that allows growth, the cells need to adapt to their new environment (Prats et al., 2006).  The first, Second, Third and fourth phase growth  are as follow respectively:

lag phase is a period of slow growth when cells are adapting to the high nutrient environment and preparing for fast growth (Prats et al., 2006). The lag phase has high  biosynthesis rate, as protein necessary for rapid growth are produced (Prats et al., 2006).

Log phase is also known as logarithmic or exponential phase is a period characterized by cell doubling (Skarstad et al., 1983). The number of new bacteria appearing per unit time is proportional to present population. If growth is not limited,  doubling will continue at a constant rate so both the number of cells and rate of population increase doubles with each consecutive time period  (Skarstad et al., 1983). For this type of exponential growth, plotting the natural logarithm of cell number against time produce a straight line.  The slope of this line is the specific growth rate of the organisms, which is a measure of the number of division per cell per unit time (Skarstad et al., 1983). The actual rate of this (i.e. the slope of the in the figure) depend upon the  growth conditions, which affect the  frequency of the cell division events and the  probability of the both daughter cell surviving. Under controlled conditions cyanobacteria can double their population four times a day (Marshall, 1989). Exponential growth can not continue independently, however, because the medium is soon depleted of nutrient and  enriched with waste (Skarstad et al., 1983).

Stationary phase is often due to growth liming factor such as the depletion of an essential nutrient and/or the formation of an inhibitory product such as an organic acid (Zwietering et al., 1990). Stationary phase result from a situation in which growth rate and death rate are equal. The number of new cell  created is  limited by the growth factor and as a result the rate of the cells growth marches the rate of cells death. The result is  a smooth horizontal linear part of  the curve during the stationary phase (Zwietering et al., 1990). 

Death phase (Decline phase) bacteria die, which could be due to lack of nutrient, a temperature which is too high or low, or the wrong living conditions (Novick, 1955).

2.1.7 CLASSIFICATION OF BACTERIA

Classification seek to describe the diversity of bacteria species by naming and grouping organisms base on similarities (Cavalier, 2002). Bacteria can classified on the basis of cell structure, cellular metabolism or on differences in cell components such as DNA, fatty acid, pigment, antigens and quinone (Thomson and Bertram, 2001). While these scheme allowed the identification and classification of bacterial strains, it was unclear whether these differences represent variation between distinct species or between strains of the same. This uncertainty was due to lack of distinctive structures in most bacteria, as well as lateral gene transfer between unrelated specie (Boucher et al.,2003).Due lateral gene transfer, some closely related bacteria can have different morphologies and metabolisms. to overcome  this uncertainty, modern bacterial classification emphasizes molecular systematic using genetic techniques such as guanine, cytosine ratio determination.

A definitive identification scheme for bacteria was first presented in 1984 in Bergey's manual of systematic bacteriology. In these scheme ,bacteria are classified on the basis of many characteristics (Gupta ,2000). Cell shape nature of multicell aggregate ,motality,formation of spore and reaction to the gram stain are important. These morphological features including the shape and colour of bacterial colonies are  not always constant and can be influenced by environmental conditions(Rappe et al; Dootittle 2005).Important in the identification of genus and species bacteria are biochemical test including the determination of the kinds of nutrient of a cell use,the product of its metabolism, the response to specific chemicals, and the present of particular characteristic enzymes. Other criteria used for the identification of some types of bacteria might be their antigenic composition, habitat, disease production,and requirement for specific nutrient (Who,1990). 

2.2.1 PATHOLOGY  OF ESCHERICHIA COLI AND SHIGELLA SPECIES INFECTION

2.2.2  ESCHERICHIA COLI  INFECTION

Although E. coli in human large intestine can assist with waste processing and food absorption, some strain of E. coli can cause severe infection in many animals, such as  humans, sheep , horses, dogs e.t.c. (Parry  and Sharon, 2002). The one that only  found in humans is called enteroaggregative  E. coli. Urinary tract infection, for example can be caused by ascending infections of urethra (Jennifer, 2005). Such infections can be found in both adult male and female, and some infant can be infected as well (Jennifer, 2005). 

E. coli is one of the most infective strains that can cause food poisoning (Atlanta, 2005). It belongs to enterohemorrhagic strain of the  E. coli and can lead to bloody diarrhoea and kidney failure when one get infected by contaminated  ground beef, unpasteurize milk or contaminated water (Atlanta, 2005).  the toxin that  E. coli 0157:H7 produces is a shiga-like toxin which is a regulated toxin that Catalytically inactive 60s ribosomal subunit of most Eukaryotic cells, blocking mRNA translation and thus causing cell death (Champman and Robinson, 2002). Some important symptoms are diarrhoea that is acute and severe, either bloody or not bloody, stomach  cramping, vomiting lost of appetite abdominal pain and fever. The causes can usually clear up on their own in 1-3 days with no treatment require. However patient should avoid dairy products because those products may induce temporary  intolerance, and therefore make the diarrhoea worst (Hagan and Mobley, 2007).

2.2.3 SHIGELLA  INFECTION

Shigella infection (shigellosis) is an intestinal disease caused by a family of bacteria known as shigella. The main sign of shigella infection is diarrhea, which often is bloody. Shigella can be passed through direct contact with the bacteria in the stool. (Hagan and mobley, 2007).For example, this can happen in a child care setting when staff members don't wash their hands well enough after changing diapers or helping toddlers with toilet training. Shigella bacteria also can be passed in contaminated food or by drinking or swimming in contaminated water.(Champman and Robinson,2002). Children under age 5 are most likely to get shigella infection, but it can occur at any age. A mild case usually clears up on its own within a week. When treatment is needed, doctors generally prescribe antibiotics.(Mobley, 2007).

Symptoms

Signs and symptoms of shigella infection usually begin a day or two after contact with shigella, but may take up to a week to develop. Signs and symptoms may include:

 Diarrhea (often containing blood or mucus)

 Abdominal pain or cramps

 Fever

Although some people have no symptoms after they've been infected with shigella, their feces may still be contagious up to a few weeks.

Causes

Infection occurs when you accidentally swallow shigella bacteria. This can happen when you:

 Touch your mouth. If you don't wash your hands well after changing the diaper of a child who has shigella infection, you may become infected yourself lf Direct person-to-person contact is the most common way the disease is spread.

 Eat contaminated food. Infected people who handle food can transmit the bacteria to people who eat the food. Food can also become contaminated if it grows in a field that contains sewage.

 Swallow contaminated water. Water may become contaminated either from sewage or from a person with shigella infection swimming in it.

2.3.1 TREATMENT AND CHEMOTHERAPY OF BACTERIAL INFECTION

2.3.2 TREATMENT OF  ESCHERICHIA INECTION

If E. coli bacteria escape the intestinal tract through a perforation (for example from an ulcer, ruptured appendix, or due to surgical error )  and enter the abdomen. They usually cause peritonitis that can be fatal without prompt treatment( Rolhion et al., 2017). However E. coli  are extremely sensitive to such antibiotics as streptomycin, gentamicin. Recent research  suggest treatment of entropathogenic  E. coli with antibiotic may not  improve the outcome of diseases, as it may significantly increase the chance of developing haemolytic-uremic syndrome ( Rolhion et al., 2017).

2.3.3 TREATMENT OF  SHIGELLA SPECIES INFECTION

Shigella infection usually runs its course in five to seven days. Replacing lost fluids from diarrhea may be all the treatment you need, particularly if your general health is good and your shigella infection is mild.(Rolhion et al., 2017). Avoid drugs intended to treat diarrhea, such as loperamide (Imodium) or atropine (Lomotil), because they can make your condition worse. (Rolhion et al., 2017).

Antibiotics

For severe shigella infection, antibiotics may shorten the duration of the illness. However, some shigella bacteria have become drug resistant. So it's better not to take

antibiotics unless your shigella infection is severe. Antibiotics may also be necessary for infants, older adults and people who have HIV infection, as well as in situations where there's a high risk of spreading the disease.

2.3.4 DIAGNOSIS OF BACTERIAL INFECTION

Diagnosis of infectious bacterial disease sometimes involves identifying the infectious agent either directly or indirectly (van den et al.,2004). practice most minor   infectious diseases such as warts, cutaneous abscesses, respiratory system infection and diarrheal diseases are diagnosed by their clinical representation (ryan,2004). Conclusions about the cause of disease are based upon the likelihood that a patient came in contact with a particular bacteria agent, the presence of a microbe in a community, and other epidemiological considerations. Given sufficient effort, all known infectious agents can be specifically identified. The benefit of identification, however, often greatly outweighed by the cost, as often there is no specific treatment,  the cause is obvious, or the outcome of an infection is benign (kayser et al., 2005).

diagnosis of infectious disease is nearly always initiated by medical history and physical examination (van den et al.,2010). More details identification techniques involve the culture of infectious agent isolated from a patient. Culture allows identification of infectious organism by examining their microscopic features, by detecting the presence of substances produced by pathogen, and by directly identify and organism by its genotype (Baron and Samuel ,1986). Other techniques (such  as X-rays,CAT scans, PET scans or MR) are used to produce images of internal abnormalities resulting from the growth of an infectious agent. The images are useful in detection of ,for example, a bone abscess or spongiform  encephalopathy produce by afrion(Robot ,2009).

CHAPTER THREE

3.0 STUDY AREA

Aliero is a town in northern Nigeria's Kebbi State. Located in the southeast of Kebbi State 12°16′42″N 4°27′6″E / 12.27833°N 4.45167°E, the name Aliero was originally from two prominent Fulani scholars Ali & Yero. The town is the headquarters of Aliero Local Government Area.

Most of the people in Aliero LGA are agrarian, with emphasis on vegetation, especially onion and paper. The town has the largest onion market in northwest Nigeria and is a major producer of onions in Nigeria. Aliero residents are known for bone setting across West and Central Africa. Aliero town is surrounded by mango trees.

3.1.0 Methods

3.1.1 Sample collection and preparation

The leaf extract of aristolochia bracteolata was collected from Aliero, Kebbi in Kebbi state, Nigeria and identified by Dr. D singh. A plant scientist in the Department biological science, Kebbi state university, Aliero, Kebbi state. Nigeria. The leaf extract of aristolochia bracteolata was droed for two weeks, and the dried plant material was ground into fine powder.

3.1.2 Test organisms

The test organisms used for the antimicrobial analysis include two bacteria. The bacteria include Escheria coli  and shigella species pure isolates of these organisms were obtained from Microbiology laboratory of the department of life science Kebbi state university of science and technology aliero.

3.1.3 Extraction

Two hundred grams (50g) of A.bracteolata sample was soaked in 500ml of acqeous and 500ml of methanol in a screw-cap bottle for 24 hours. The extract was filtered using a whatman No. 42 filter paper and concentrated with a rotary evaporator at 40°C. Further concentration was done on a water bath at 40°C after which extract was transferred into a sample bottle pending further analysis. The residue was dried and extracted with chloroform and then with methanol.

3.1.4 Qualitative phytochemical screening

Phytochemical screening was performed on the extract using standard procedure to identify chemical constituents as described by Trease and Evans (1989), Harbone (1973) and sofowara (1993).

3.1.4 Alkaloid (sofowara 1993)

1.0g of the extract was stirred in 5ml of 1%HCL  on a steam bath and filtered while hot. Distilled water was added to the residue and 1ml of the filtrate with a few drops of wagner's reagent. A reddish brown precipitate indicates the presence of alkaloids.

3.1.6 Flavonoids (Harbone 1973)

2ml of dilute sodium hydroxide was added to 2ml of extract. The appearance of a yellow colour indicates the presence of flavonoids.

3.1.7 Saponins (sofowora 1993)

1ml of distilled water was added to 1ml of the extract and shaken vigorously. A stable persistent froth indicated the presence of sapanins.

3.1.8 Tannins ( Trease and Evens 1989)

A portion of the extract was dissolved in wate, after which solution was clarified by filtration. 10% ferric chloride solution was the added to the resulting filtrate. The appearance of bluish black colour indicated the presence of tannins.

3.1.9 Anthraquinones (Harbone 1973)

0.5g of the extract was shaken with 10ml of benzene and filtered 10% of ammonia solution was added to filtrate and mixture was shaken. The formation of pink, red or violet colour on the ammonia cal phase indicated the presence of anthraquinones.

3.1.10 Cardiac glycosides (Harbone 1973)

0.5g of the extract was dissolved in 2ml glacial acetic acid containing 1  drop of ferric chloride solution. This was under layered with 2ml of concentrated sulphuric acid. A brown of deoxy sugar characteristics of cardiac glycosides.

3.2.0 Phlobatannins (sofowora 1993)

A few drops of 1%HCL was added to 1ml of extract and boiled. A red precipitation indicates the presence of phlobatannins.

3.2.1 Terpenoids (Salkowskl test) (Trease and Evens 1989)

5ml of each extract was mixed in 2ml of chloroform, and concentrated H2SO4 (3ml) was carefully added to form a layer. A reddish brown colouration of the inter face was formed to show positive results for the presence of terprnoids.

3.2.2 Cardenolides (Harbone 1973)

2ml of benzene was added to 1ml of these sample extract. The formation of a turbid brown colour is an indication of the presence of cardenolides.

3.2.3 Steroids ( Mbatchous and kosoono, 2012)

2ml of the sample was put in test tube; 6ml of chloroform was added. 4 ml of concentration H2SO4 was added by the slide of the test tube. The upper layer turned red.

3.2.4 Quantitative phytochemical analysis

Total alkaloid, flavonoids and saponins were determined using the method described by Krishnaiah et al, 2009.

3.2.5 Determination of alkaloids 

Five grams of fhe plant sample was placed in a 250ml beaker and 200ml of 10% CH3C02H in C2H50H was added. The mixture was covered and allowed to stand for 4 hourr. It was then filtered and the filtrate was concentrated on a water bath until it reaches a quarter of its original volume. Concentrated  NH40H was added until precipitation was complete. The mixture was allowed to settle and the precipitate collected on a weighed filter paper and washed with dilute NH4OH. The precipitate, alkaloid, was dried and wighed.

3.2.6 Determination of flavonoids

The grams of plant sample was repeatedly extracted with 100ml of 80% aqeous methanol at room temperature. The mixture was then filtered through a filter paper into a pre-weighed 250ml beaker. Tje filtrate was transferred into a water bath and allowed to evaporate to dryness and weighed. The percentage flavonoid was calculated by difference.

3.2.7 Determination of saponins

Twenty grams of plant sample was weighed into 250ml conical flask. 100ml of 20% C2H5OH was added. The mixture was heated over a hot water bath for 4 hours with continuous stirring at about 55° C. It was then filtered with a whatman No. 42 paper. The residue was re-extracted with another 200ml of 20% C2 H5OH.  The combined extract was reduced to 40ml over a water bath at about 90°C. The concentrated extract was then transferred into a 250ml seperator funnel and 20ml of (CH3CH2)02 was added to the extract and shaken vigorously. The aqeous layer was recovered while the (CH3CH2)2O layer was discarded. This purification process was repeated. 60ml of N-butanol was added and the combined N-butanol extract was washed twice with 10ml of 5% NaCL. The remaining solution was then heated on a water-bath In a pre-weighed 250ml beaker. After evaporation the residue was dried in Gallenkamp moisture extraction oven (size 1) to a constant weight. The % saponin was calculated by difference. Whereas, the sulphuric acid layer turned yellow with green fluorescence. This indicate the presence of steroids.

3.2.8 Determination of antimicrobial analysis

3.2.9 microbial screening of the crude extract 

The agar diffusion method was used as described by Dauda et al., (2011) and Jimoh et al., (2010). Sterile nutrient agar was prepared and placed in labeled Petri dishes and allowed to gel. Wells were bored into the nutrient agar using a 7mm sterile cork borer. Each crude extract was reconstituted by adding 2ml of its mother solvent. 0.2ml of the reconstituted extract was dispensed into each well and allowed to diffuse for 30minutes The test organisms were inoculated into the labeled Petri dishes with a swab stick before incubating at 37°C for 24hours.

                         CHAPTER FOUR

4.0 RESULT

Table 1. Qualitative phytochemical screening of A. bracteolata

Test

Aqeous

Methanol

Alkaloid

++

ND

Saponin

ND

ND

Tannins

+

+

Cardiac glycoside

+++

+

Steroid

+

+

Flavonoid

++

+

Anthraguinone

ND

ND

Terpenoid

ND

+

KEY

ND = not detected, + = Slightly present, ++ = Moderately present, +++ = Highly present.

Table 2. Quantitative screening of A.bracteolata leaf extract

Test

Value obtained

Alkaloids

5.3±0.3

Flavonoid

6.8±0.16

Tannins

5.1±0.1

Values are express as mean ± SD standard deviation in 3 replicate (n=3)

Table 3. Antimicrobial activity of A.bracteolata of aqeous extracts

Extract

Concentration in (u/ml)

zone of inhibition (mm)/ microorganism

E. Coli

zone of inhibition (mm)/ microorganism

Shigella

Aqeous

50

75

100

N.D

N.D

N.D

N.D

N.D

N.D

key:- N.D= no detected

Table 4. Antimicrobial activity of methanol extract

Extract

Concentration in (u/ml)

Zone of inhibition (mm)/ microorganism

E.coli

Zone of inhibition (mm)/ microorganism

Shigella

methanol

50

75

100

N.D

N.D

N.D

N.D

N.D

N.D

key:- no detected.

                             CHAPTER FIVE

5.0 DISCUSSION CONCLUSION AND RECOMMENDATION

5.1 DISCUSSION

Although result obtainable may vary depending on the method of extraction, type of A. Bracteolata, method and extent of drying and storage, e.t.c.

The result for phytochemical screening of the extracts of A. Bracteolata revealed the presence of cardiac glycoside, flavonoids and steroid in all extracts. Tannins, alkaloids and were not detected in the methanol extract. The tests also revealed that the aqueous extract have higher contents of the phytochemicals. Result for the quantitative analysis carried out on powdered sample of A. Bracteolata leaves as shown in table 1, revealed that A. Bracteolata leaves has flavonoids content of 6.9±0.02, Alkaloids content of 5.3±0.1 and Tannins content of 5.1±0.01  Ekwenye and Okorie, (2010) recorded 7.1±0.03, 6.8±0.4 and 6.1±0.06 of Flavonoid, alkaloids and Tannins respectively from tetrapleura tetraptera. In various Nigerian herbs. This showed that plants have different concentration of constituents and A.Bracteolata compares favorably.

The result obtained in these studies has shown that, the presence of some phytochemicals in A. Bracteolata leaf extracts could be responsible for the observed effect of this leaf in which tannins, flavonoids, terpenes, alkaloids, cardiac glycoside and steroid were found present in the leaf extracts. While saponin and Anthraquinones were found absent in leaf extract. Therefore, the medicinal values of this leaf may be related to their constituent phytochemicals. According to Varadarajan et al., (2008), reported that the secondary metabolites (phytochemicals) and other chemical constituents of medicinal leaf account for their medicinal value. For example saponins have hypotensive and cardiodepresant properties, flavonoids have anti-allergic, anti-inflammatory, anti-microbial, anti-cancerous and anti-diarrheal activities. Microorganisms are the concealed enemies to the mankind. They are small but cause a very profound damage in human body as well as other living organisms. The agent, which have the capacity to kill the microbes or arrest the multiplication, are called the antimicrobial agents or drugs. There are alot of antimicrobial drugs of which some are discovered or established and some are hidden in the nature. Hence, the last decade witnessed an increase in the investigation on plant as source of human disease management and more natural antimicrobials have driven scientists to investigate the effectiveness of inhibitory compounds such as extracts from plants. There are several reports of antibiotics resistance of human pathogens to available antibiotics. Bimolecules of plant origin appear to be one of the alternatives for the control of these antibiotic resistant human pathogens.

The sensitivity test showed that the extract were not active against E.coli and Shigella. Based on concentration of 50, 75 and 100u1.

5.2 CONCLUSION

In the presence study, the following conclusion were made; the phytochemical analysis present in the A. Bracteolata leave extracts are generally moderate. And therefore it has advantage on pharmacologically. It also concluded that the leave extract of A. Bracteolata has no antimicrobial activity based on concentration used for the study.

5.3 RECOMMENDATION

 Presently there is an increasing interest worldwide in herbal medicines accompanied by increased laboratory investigation into the pharmacological properties of the bioactive ingredients and their ability to treat various diseases.

 In future study the isolated principle needs to be evaluated in scientifically animal model and clinical trial to understand the molecular mechanism of action, in search of lead molecules from natural resources.

 As the global scenarios now changing towards the use of nontoxic plant product having traditional medicine use, development of modern drug from A.bracteolata should be emphasized for the control of various diseases.

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