CHAPTER ONE

INTRODUCTION

The term of medicinal plants include a various types of plants used in herbalism and some of these plants have

a medicinal activities. These medicinal plants consider as a rich resources of ingredients which can be used in

drug development and synthesis. Besides that these plants

play a critical role in the development of human cultures around the whole world.

Moreover, some plants consider as important source of nutrition and as a result of that these plants

recommended for their therapeutic values. These plants include ginger, green tea, walnuts and some others

plants. Other plants their derivatives consider as important source for active ingredients which are used in

aspirin and toothpaste.

Alternative Medicine

These days the term “Alternative Medicine” became very common in western culture, it focus on the idea of

using the plants for medicinal purpose. But the current belief that medicines which come in capsules or pills are

the only medicines that we can trust and use. Even so most of these pills and capsules we take and use during

our daily life came from plants. Medicinal plants frequently used as raw materials for extraction of active

ingredients which used in the synthesis of different drugs. Like in case of laxatives, blood thinners, antibiotics

and antimalaria medications, contain ingredients from plants. Moreover the active ingredients of Taxol,

vincristine, and morphine isolated from fox glove, periwinkle, yew and opium poppy respectively.

Future of Medicinal Plants

Medicinal plants have a promising future because there are about half million plants around the world, and most

of them their medical activities have not investigate yet, and their medical activities could be decisive in the

treatment of present or future studies.

Characteristics of Medicinal Plants

Medicinal plants have many characteristics when used as a treatment, as follow:

damage others or neutralize their possible negative effects.

• Support of official medicine- In the treatment of complex cases like cancer diseases the components of the

plants proved to be very effective.

• Preventive medicine- It has been proven that the component of the plants also characterize by their ability to

prevent the appearance of some diseases. This will help to reduce the use of the chemical remedies which will

be used when the disease is already present i.e., reduce the side effect of synthetic treatment.

The use of plant and its products has a long history that began with folk medicine and through the years has

been incorporated into traditional and allopathic medicine 1. Since antiquity, many plants species reported to

have pharmacological properties as they are known to posses various secondary metabolites like glycosides,

saponins, flavonoids, steroids, tannins, alkaloids, tirpenes which is therefore, should be utilized to combat the

disease causing pathogens.

With the advancement in Science and Technology, remarkable progress has been made in the field of medicine

with the discoveries of many natural and synthetic drugs. Antibiotics are undeniably one of the most important

therapeutic discoveries of the 20th century that had effectiveness against serious bacterial infections. However,

only one third of the infectious diseases known have been treated from these synthetic products. This is because

of the emergence of resistant pathogens that is beyond doubt the consequence of years of widespread

indiscriminate use, incessant and misuse of antibiotics. Antibiotic resistance has increased substantially in the

recent years and is posing an ever increasing therapeutic problem. One of the methods to reduce the resistance

to antibiotics is by using antibiotic resistance inhibitors from plants. Plants are known to produce a variety of

compounds to protect themselves against a variety of pathogens. It is expected that plant extracts showing target

sites other than those used by antibiotics will be active against drug resistant pathogens. Medicinal plants have

been used as traditional treatments for numerous human diseases for thousands of years and in many parts of the

world. Hence, researchers have recently paid attention to safer phytomedicines and biologically active

compounds isolated from plant species used in herbal medicines with acceptable therapeutic index for the

development of novel drugs.

LITERATURE REVIEW

Tetrapleura tetrapleura, is traditionally been used as anti ulcerative, anti hypertensive, anti inflammatory

and anti conversant.

After scrutiny of published literature showing its medicinal importance, the present protocol has been outlined

regarding the antimicrobial activity on these selected plant using ethanoic extract. It is in view of this, that the

present research was set up to evaluate the antimicrobial activity of Tetrapleura tetrapleura, using its

extractions against some pathogenic gram negative bacteria.

It was discovered long ago that some plant materials exhibit antibacterial properties. The use of

these plant materials and as preservative and as a means of preventing microorganism

development in foods has become the subject of extensive studies (Gould, 1996). Importantly,

the inhibitory effects of Tetrapleura tetraptera extracts against some human pathogens have

been reported. Currently, there is a growing

demand worldwide of consumers for minimizing chemical preservation that can be detrimental

to human health consequently, spices, herbs and naturally occurring phenolics from various

plants sources are being studied in detail in response to consumer requirements for fresher and

more natural additive-free products

(Nychas,1995).

Tetrapleura tetraptera is one of the medicinal plants in Nigeria. The documented biological or

pharmacological activities are found to be molluscicidal, cardiovascular, neuromuscular,

hypotensive, anti-conversant, anti ulcerative, anti-inflammatory and anti-microbial.

post parturition and as a lactation aid (Enwere, 1998).The antibacteial activity of this plant has

been formulated into soap bases using palm kernel oil. At the same time, most of the folkloric

chains agree in the traditional use of the fruit for management of convulsion, leprosy,

inflammation and rheumatoid pains (Dalziel,1948).

Alcoholic and water extracts of Tetrapleura tetraptera inhibited the growth of

Staphylococcus aureus (Salako,et al.,1990).The antibacterial activities of these plants has

been exploited in the formulation of the dried powdered fruit of the plant. Thus, dried

powdered herbs have been formulated into soap bases using palm kernel oil, shea butter and

mixture of bases. The formulated soaps were evaluated for organoleptic properties and foaming

ability. Soaps with mixture of these two bases were of better qualities than those with the

individual base. Incorporation of powdered plant materials influenced both the foaming

property and the hardness of the soaps. Except for the Tetrapleura tetraptera fruit powder

which improved the foaming ability of these soaps all other herbs including Acalypha

wilkensia,Harugana madagascariensis and Ficus exasperate depressed the foaming

ability of the soaps.The extract from Tetrapleura tetraptera exhibited anti-convulsant activity,

which could be linked to their ability to depress the central nervous system(Akah and

Nwambie,1993). The ethanol extract and saponins from stem-bark of Tetrapleura tetraptera

exerted an inhibitory effect on luteinizing hormone released by pituitary cells (El Izziet et

al.,1990) suggesting its use as a contraceptive agent. The nutritive quality of the dry fruit of

Tetraptera tetraptera used as spice was assessed. The fruit shell, fruit pulp and seed
contained varying amount of nutrients such as proteins, lipids and minerals which were

comparable and some were higher than popular spices such as red pepper, onion curry and

ginger (Essien et al.,1994).

Previous researchers repeated the presence of glycosides and tannins in water and ethanolic

extracts of Tetrapleura tetraptera and observed that such phytochemical metabolites were

effective inhibitors of growth of bacterial. Apart from food qualities, vegetables have been

found t contain phytochemical with demonstrated medicinal usefulness (Ames,1983).

Oguntana(1988) attributed the protection offered by some fruits and vegetables against diseases

such as cancer and heart diseases to the presence of some phytochemical.

The molluscicidal activity of the extracts from the leaf, leaf-stalk, stem-bark and root-bark have

been exploited for long, but studies on the antibacterial effects of the essential oil from its fruit

are scarce. The purpose of this study was to examine the antibacterial effects of the essential oil

of the pod of Tetrapleura tetraptera extracted using ethanoic solvent to identify the chemical

components of the extract and to determine at which concentration they were inhibitory to some

human pathogens like Escherichia coli, Klebsiella specie , Pseudomonas aeruginosa

and Salmonella typhi. Also, to provide a guide or direction on the concentration of the fruit

extracts populace who use them for the treatment of diseases and to prevent side effects

associated with the diseases.

BACTERIA

Bacteria are microscopic organims, typically a few micometers in length. They have wide range of

shapes,ranging from spheres to rod and spirals.They are present in most habitats on earth and grow in soil,
water, organic matter, and also in plants and animals. The Gram staining method is widely used to different

between bacteria species into Gram negative and Gram positive.

The Danish bacteriologist J.M.C Gram (1853-1938) devised a method of staining bacteria using a dye called

crystal violet. Gram negative bacteria are bacteria that do not retain the crystal violet stain during gram staining.

Klebsiella

Klebsiella is a Gram-negative bacteria (bacillus) that can cause different types of healthcare-associated

infections, including pneumonia, bloodstream infections, wound or surgical site infections (SSI), and

meningitis. This organism is normally found (normal flora) in the human intestines and is usually excreted

in human stool. In healthcare settings, Klebsiella infections commonly occur among sick patients who are

receiving treatment for other conditions. Patients whose care requires devices like ventilators or

intravenous catheters, and patients who are taking long courses of certain antibiotics are most at risk for

Klebsiella infections. Healthy people usually do not get Klebsiella infections.

TRANSMISSION

In healthcare settings, Klebsiella bacteria can be spread through person-to-person contact. This may

occur via the contaminated hands of healthcare personnel, or by contamination of the environment or

equipment. The bacteria are not spread through the air. Patients may be exposed to Klebsiella when they

are on ventilators, or have intravenous catheters or wounds that are caused by either injury or due to

surgical procedures. Because these medical devices and conditions are associated with breaks in the skin

or other mucous membranes, this may allow Klebsiella to enter the body and cause infection.

Prevention

To prevent spreading Klebsiella infections between patients, healthcare personnel must follow

specific infection control precautions. These precautions may include strict adherence to hand

hygiene and wearing gowns and gloves when they enter rooms where patients with Klebsiella–related

infection are staying. Healthcare facilities must also follow proper cleaning procedures to prevent the

spread of Klebsiella. Certain protocols should be followed to prevent device-associated infections,

other healthcare pathogens.

Drug-resistant Klebsiella

Some Klebsiella strains have become highly resistant to antibiotics. This occurs when bacteria such as

Klebsiella pneumoniae produce an enzyme known as a carbapenemase (referred to as KPC-producing

organisms). Klebsiella species belongs to the family Enterobacteriaceae, a normal flora of the

gastrointestinal tract that can become carbapenem-resistant (CRE stands for carbapenem-resistant

Enterobacteriaceae).Unfortunately; carbapenems are often the last

Treatment

Klebsiella infections that are not drug-resistant can be treated with antibiotics. Infections caused by

KPC-producing bacteria can be difficult to treat because fewer antibiotics are effective against them.

In such cases, a microbiology laboratory must run tests to determine which antibiotics will treat the

infection.

Guidelines and Recommendations

There are no guidelines specific for this organism. Healthcare facilities must follow the general

guidelines for hand hygiene, environmental hygiene, and guidelines for the prevention of device

associated infections an SSI.

Pseudomonas

Pseudomonas is a Gram-negative bacterium (bacillus) that can cause different types of healthcare-

associated infections. The most common species, Pseudomonas aeruginosa, is commonly found in

soil and ground water. It rarely affects healthy people and most community acquired infections are

associated with prolonged contact with contaminated water. P. aeruginosa is increasingly important

clinically as it is a major cause of both healthcare-associated infections and chronic lung infections in

people with cystic fibrosis. Although P. aeruginosa is an opportunistic pathogen (i.e. more likely to
infect those patients who are already very sick as opposed to healthy patients), it can cause a wide

range of infections, particularly among immune-compromised people (HIV or cancer patients) and

persons with severe burns, diabetes mellitus or cystic fibrosis. In hospitals the organism contaminates

moist/wet reservoirs such as respiratory equipment and indwelling catheters and infections can occur

in almost every body site but are particularly serious in the bloodstream (bacteraemia).

Transmission

In healthcare settings, Pseudomonas may spread through person-to-person contact. This may occur

via the contaminated hands of healthcare personnel, or through direct contact with contaminated

environmental surfaces or equipment. Patients may be exposed to Pseudomonas when they are on

ventilators, or have intravenous catheters, urinary catheters, or wounds that are caused by either

injury or due to surgical procedures. Because these medical devices and conditions are associated

with breaks in the skin or other mucous membranes, this may allow Pseudomonas to enter the body

and cause infection. The effects of P. aeruginosa on people vary widely, ranging from no symptoms at

all to serious respiratory infections, especially in patients with cystic fibrosis.

Prevention

To prevent spreading Pseudomonas infections between patients, healthcare personnel must follow

specific infection control precautions. These precautions may include strict adherence to hand

hygiene and wearing gowns and gloves when they enter rooms where patients infected with

Pseudomonas are staying. Healthcare facilities must also follow proper cleaning procedures to

prevent the spread of Pseudomonas. Certain protocols should be followed to prevent device-

associated infections, wound infections and SSIs. A breakdown in these protocols may lead to

infections with Pseudomonas or other healthcare pathogens.

Drug-resistant Pseudomonas

Most infections are susceptible to third generation cephalosporins. P. aeruginosa is one of the more

common causes of healthcare-associated infections and is increasingly resistant to many antibiotics.

P. aeruginosa is the second most common cause of pneumonia, the third most common cause of

urinary tract infections and the eighth most frequently isolated pathogen from the bloodstream. Rates
of resistance to antibiotics are on the rise. Multidrug-resistant (MDR) Pseudomonas is defined as

resistance to at least three of four antibiotic classes.

Treatment

Pseudomonas infections that are not drug-resistant can be treated with antibiotics. Infections caused

by MDR Pseudomonas can be very challenging. Therapeutic options are limited which may lead to

prolonged hospitalization and even death.

Guidelines and Recommendations

There are no specific guidelines for this organism. Healthcare facilities must follow the general

guidelines for hand hygiene, environmental hygiene, and guidelines for the prevention of device-

associated infections and SSIs.

Salmonella

Salmonella spp. are a group of bacteria which reside in the intestinal tract of human

beings and warm blooded animals and are capable of causing disease. They are the

second most common cause of bacterial foodborne illness in Ireland (Campylobacter spp.

is the most frequent cause). They are facultative anaerobic Gramnegative rods.

Salmonella spp. are members of the Enterobacteriaceae group. The genus Salmonella

contains 2 species:

• Salmonella enterica

• Salmonella bongori

Salmonella enterica is an important agent of foodborne illness. This species is sub-

classified into 6 subspecies of which S. enterica subspecies enterica is the most

important for human health.

The genus Salmonella can be subdivided into more than 2,400 serotypes. Salmonella

enterica subsp. Enteric serotype Typhimurium (S. Typhimurium) and Salmonella enterica

subsp. enterica serotype Enteritidis (S.Enteritidis) are the most frequently isolated

serotypes in humans in Ireland. Serotypes are further subdivided by their resistance to

biochemical characteristics (biovars or biotypes) or their sensitivity to or production of

bacteriocins.

TRANSMISSION

Salmonella spp. reside in the intestinal tract of humans and warm blooded animals. They

are shed in the faeces.

Poor hygiene practices can result in the spread of this pathogen to the hands of humans

while on the farm. Feet, hair and skin of animals can become contaminated as they walk,

sit or lie in faecally contaminated ground or litter.

The spread of Salmonella spp. to carcasses may occur in the abattoir environment during

slaughter.

Furthermore, spread of Salmonella spp. to agricultural commodities may occur through

faecal contamination of streams, rivers and coastal waters and the use of improperly

treated sewage for agricultural purposes. In the food processing environment, spread of

this pathogen can occur through cross contamination from raw food or infected food

handlers. The human infection caused by Salmonella spp. is referred to as salmonellosis.

Although salmonellosis can arise from contact with infected animals, consumption of

contaminated food is the most usual cause.

CONTROL

Since low numbers of Salmonella spp. can cause illness, it is important that control

measures are taken at all stages in the food chain. These are essential to protect the

health of consumers and the integrity of the business. Examples of control measures

include:

• Implementation of Good Hygiene Practices (GHP) and Good Manufacturing Practices

(GMP) at all stages in the food chain, i.e. at farm level, slaughterhouse, manufacturing,
processing, catering, retail etc. Particular attention should be paid to the prevention of

cross contamination between raw and ready-to-eat foods

• Implementation of a food safety management system based on the principles of

HACCP. This includes good process control, e.g. temperature control during cooking,

storage

• Testing against microbiological criteria, as appropriate, when validating and verifying

the correct functioning of their HACCP based procedures and other hygiene control

measures.

CHAPTER THREE

MATERIALS AND METHODS

Renewed interest in the therapeutic potential of medicinal plants means that researchers are

concerned not only with validating ethnopharmacological usage of plant, but also with identifying,

isolating and characterizing the active components (Fennell eta/., 2004). However, the presence of

numerous inactive components makes the screening and isolation of the target component(s)

extremely cumbersome (Sticher, 2008). In choosing medicinal plants for scientific evaluation of their

biological activities and validation of ethnopharmacological usage, some criteria such as

• Evidence of ethnopharmacological usage by the native population.

• The ailment(s) which the plant(s) is used to cure.

• The availability of the plant in its natural habitat.

• The sustainable use of the part(s) of the plant (root, leaves, stem, bark or whole plant) (Baker et a/.,

1995; Vander Watt and Pretorious, 2001 ).

• Mode of preparation and administration by traditional healers must also be considered.

Plant quality and pre-treatment are also important determinant of the phytochemical constituents and

invariably the biological activities of an extract. These factors depend on plant parts used, genetic

variation, geographical location, climatic conditions, collection period, drying methods, and storage
conditions. Due to these possible variations, plant material from recognized botanical gardens or

herbaria is usually recommended because they are protected, correctly identified and serve as

reliable sources for subsequent collections. Preparation of voucher specimens is also an important

aspect of medicinal plant research. Standard procedures for pretreatment of plant materials have

been developed (Eioff et a/., 2008). The basic steps include pre-washing if necessary, air drying

under shade at room temperature, grinding into powder and storage in an air tight container at

appropriate temperature (room or refrigerated).

MATERIALS

The materials used for this research include four gram negative organisms, pods of

Tetrapleura tetraptera, concentrated ethanol, soxhlet apparatus, agar, autoclave, bunsen

burner, incubator, Petri dish, inoculating loop, cotton wool, beaker, weighing balance,

incubator, fridge, normal saline,

Collection and Identification

The test spice sample, Tetrapleura tetraptera was obtained from Central Market and was identified by

Mr. Olaiwola of the Department of Biological Sciences, Ramon Adedoyin college of Natural and

Applied Sciences of Oduduwa university .

Sample Preparation

The test pods of Tetrapleura tetraptera was first air dried for some weeks before it was ground and

sieved to obtain a ground (powdery) processed sample which was used for the extraction.

Extraction method
The general techniques of medicinal plant extraction include maceration, infusion percolation,

digestion, decoction, hot continuous extraction (soxhlet), aqueous alcoholic extraction by

fermentation, counter current extraction, ultrasound extraction, supercritical fluid. Etc.

For the purpose of this research we made use of maceration and soxhlet apparatus for extraction.

Maceration

Maceration is generally, to soften by soaking in a liquid. In biology it is used to describe multiple

actions.

50gram of grounded pod was put into a conical flask and weighed using a weighing balance.

500ml of concentrated ethanol was then measured and the measured sample was poured into the

ethanol.

The procedure was repeated with same sample and same measurements and left to stand for

48hours and 72 hours respectively.

The sample was sieved after the above period of time and was extracted using the soxhlet apparatus.

Soxhlet Extraction
The standard method for the determination of free fat in foodstuffs is the

Soxhlet extraction.

behr equipment for the extraction fulfils the most varied requirements of

daily laboratory use.

• Extractor sizes from 30 to 250 ml.

• Kompakt units for single sample processing.

• Heating block units with especially exact temperature control

forsimultaneously processing up to6 samples.

• Extraction systems for four or six samples with linear configuration of

individual extrators.

• Extractors with a specially developed siphon tube guarantee uniformity

of extraction in all sample positions.

• Extractors provided with spigots for draining solvent obviate need to

distill solvent before reuse.

• No rotary evaporator is required.

The test human bacteria pathogens Escherichia coli , Salmonella typhi, Klebsiella

specie and Pseudomonas aeruginosa were sourced from the microbiology Laboratory

of Ramon Adedoyin College of Natural and Applied Sciences Oduduwa university

Ipetumodu, Osun state.The test organisms were further identified and the biochemical

and morphological characteristics were confirmed by standard methods .

PHYTOCHEMICAL SCREENING

Qualitative Analysis

The extract was subjected to screening to establish the presence or absence of some

specific active principles.

Test for Tannins

The ferric chloride test described by Harborn (1973) was used. An aqueous extract of

the sample was obtained by shaking 10g of thepowdered sample in 100ml of distilled

water for 30minutes.After filtration, the extract was used for the test as described below.

2mls of theaqueous extract mixed with equal volume of distilled water in a test tube and

drops of diluted ferric chloride solution. The presence of dark precipitate gave an

indication of the presence of tannin in the extract.

The combined froth and emulsion test was used to test for the presence of saponin in

the sample.2mls of the aqueous extract was mixed with 5mls of distilled water in a test

tube. The mixture was shaken well and observed. The formation of a stable froth (foam)

gave a positive result. However, this was confirmed by the addition of few drops of olive

oil and shaken again. The formation of an emulsion confirmed the presence of saponin

in the test sample.

Test for Flavonoid

The alkaline acid test (Harborn, 1973)was used. A drop pf bench ammonia solution was

added to 3mls of the aqueous extract of the sample in a test tube. The formation of

yellow coloration which clears on the addition of concentrated acid solution was taken

as a positive result for the test.

Test for Cyanogenic glycoside

An alkaline picrate colorimetric method was dispersed in 150mls of distilled water in

conical flask. An alkaline picrate paper was suspended over the mixture and held in

place by the rubber bung (stopper).Care was taken to avoid the paper touching the

surface of the mixture. The arrangement was allowed to stand for 18hours (over-night)

at room temperature. The picrate paper was examined for colour change from yellow to

orange as a positive test for the presence of cyanogenic glycoside(HCN).

The Mayors and Haglens test (Harborn,1973) was used to test for alkaloid in the

sample.2mls of an ethanolic extract was mixed with equal volume of Mayors and

Haglens reagents in separate test tubes. The formulation orangebrown precipitate

indicated the presence of alkaloid.

Test for Steroids

The Dragendert’s test was used. A portion of the ethanolic extract was mixed with acetyl

anhydride in a test tube. The presence of a brown colouration at the interface between

the two layers indicated a positive result for steroid.

Preparation of media

The media used for this work; Nutrient agar (NA), MacConkey agar and Salmonella-

Shigella(SSA) were prepared according to manufacturer’s instructions.