CHAPTER ONE

1.0 INTRODUCTION
Plants are living things that grows in the ground, usually has leaves or flowers and needs sun and

water to survive. They are mainly, predominantly photosynthetic eukaryotes of the kingdom

plantae.

Medicinal plants also called medicinal herbs have been discovered and used in traditional

medicine practices since prehistoric times. Plants synthesis hundreds of chemical compounds for

functions including defence against insects, fungi, diseases and herbivorous mammals.

History of medicinal plant use

The use of medicinal plants for their therapeutic capabilities is an old age tradition which

continues to develop gradually with scientific research and new discoveries (Eddouks et al.,

2012). Generally, there is a strong association between medicinal plants and the local area within

which they exist and this has resulted in several plant-based medicinal systems such as the

Ayurvedic and Unani from India, the Chinese and Tibetan originating from other regions of

Asia, the native American and the Amazonian systems from North and South America,

respectively; as well as others from Africa and the Caribbean (Mamedov, 2012).

According to the WHO, about 70 percent of the global population is dependent on plants to

supply their primary health care needs. Approximately 35,000 to 70,000 (14 to 28%) of the

250,000 plant species have been manipulated for their healing properties and to date [10-13],

only about fifty major drugs have been developed from tropical plants (De Padulaet al., 1999).

This could possibly be attributed to the fact that of the 250,000 species of higher plants

worldwide, only about 17% have been scientifically researched for their medical efficacy.

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1.1 PETIVERIA ALLIACEA

Petiveria alliacea is a common medicinal plant that thrives naturally in tropical climates and has

been used in traditional medicine for the treatment of cancer, diabetes, muscular pain, skin

diseases, various central nervous system disorders, respiratory and pulmonary infections, malaria

among other ailments. Petiveria alliacea is a plant from the family Phytolaccaceae, known by

different names in different countries of Central and South America, the Caribbean and Africa.

The genus name of this herb is derived from Jacob Petiver, who dedicated his work to medicinal

plant study, while the epithet is related to the pungent smell of garlic released after tissue

disruption. For hundreds of years it has been used for pain relief, and as an anti-influenza, anti-

inflammatory, anti-tumor, anti-bacterial, anti-fungal, anti-hyperlipidemia, and anti-diabetic drug

(Tropical Plant Database-Anam, 2011). This plant also grows in Indonesia, but it has not been

used extensively. It is traditionally used in Indonesia as an analgesic, anti-inflammatory and for

treatment of hemoptysis (Mulyani et al., 2012).

Widowati, (2007) reported that P. Alliacea can reduce the length of therapy with standard drugs

in tuberculosis patients. Several volatile compound like; benzyl-2-hydroxyethyl trisulfide, cis-

3,5-diphenyl-1,2,4-trithiolan (trithiolaniacin), benzaldehyde, benzoic acid, elemental sulfur, and

trans-stilbene, dibenzyltrisulfide and benzaldehyde, benzyl alcohol, cis- and trans-stilbenes,

benzyl benzoate, dibenzyldisulfide, and dibenzyltrisulfide. Other compounds such as flavonoids,

terpenoids, and benzenoids are commonly identified in P. alliacea extracts. In the 17th century,

African slaves used to use preparations obtained from this plant to make their masters lethargic,

and for this reason, P. alliacea is widely known in Brazil as the herb to “tame the master.The

plant is also called mucuracaá, tipi, guiné, pipi, apacin, herbe aux Poules, anamu, and

embayayendo. Nowadays, herbal medicines derived from P. alliacea are available on the market

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in Paraguay, Cuba, and Japan. P. alliacea has been used in traditional medicine with different

purposes in many countries, such as antirheumatic, analgesic, and to treat respiratory conditions.

Pharmacological investigations have shown the therapeutic potential of P.alliacea as an

immunomodulator, analgesic, antimicrobial, and anticancer.

1.2 BOTANICAL CONSIDERATIONS

P. alliacea is a perennial subshrub, sub Woody, erect, and branched with long branches, which

are delicate and ascending, measuring up to 1 m in height. The leaves are 5–10 cm long and 2–

6 cm in width, discolor, oblong lanceolate, acuminate, with a cuneiform base and short petioles,

its texture ranges from membranaceous to herbaceous, with prominent midrib in the abaxial face

and secondary arqued veins. P. alliacea roots are pivoting type and may reach 30 cm in length

and 1 cm in diameter in the base; it has a yellowish–brown surface, tortuous, pale externally, and

bright whiteness internally, with an acre flavor and a garlic like odor (Rocha et al., 2006).

1.3 SIGNIFICANCE OF THE STUDY

The significance of this study is to determine the antimicrobial and phytochemical potential of

methanolic extract of plant of Petiveria alliacea and to collate the scientific evidence related to

the traditional and medicinal uses of Petiveria alliacea in the treatment of chronic diseases.

1.4 AIM AND OBJECTIVES

The aim of this study is to determine the antimicrobial and petrochemical constituents of

methanolic extracts of petiveria alliacea against some human pathogenic bacteria.

The objectives of this study are as follows:

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1. Determination of Antimicrobial analysis of Petiviera alliacea leaves against some human

pathogenic bacteria

2. Determination of Phytochemical (qualitative and quantitative) constituent of Petiviera alliacea

leaves against some human pathogenic bacteria

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 CHAPTER TWO

2.0 LITERATURE REVIEW

Potential of herbs and other plant-based formulations have been increasingly recognized in

prevention and treatment of human diseases. The discipline of ethno-pharmacology, the study of

biologically active agents traditionally employed or observed by man, has in recent years

received increased attention, and there is presently a widespread interest in medicinal plants used

by different cultures (IJAR, 2016).

Extracts from plants have been found to contain minerals and primary metabolites but not only

these; they have also been found to contain a diverse array of secondary metabolites with

antioxidant potential and these have made the medicinal value of plants to assume a more

important dimension in the past few decades (Akinmoladun et al., 2007). Antioxidant substances

act as protective shield for our bodies against certain diseases like cardiac disease,

atherosclerosis, cancer and in the aging (Marthur et al., 2011). This they do by removing the

harmful effect of free radicals within our body. They slow or delay the organic matter oxidation

promoted by these free radicals by removing the excess free radical intermediates and inhibit

other oxidation reactions by going through oxidation themselves (Fasola et al., 2011). Free

radicals are involved in the pathogenesis of a large number of diseases thus a potent scavenger of

free radicals may serve as a possible preventive intervention for diseases (Charturvedi et al.,

2015).

It is commonly perceived that infection plays a major role in many cancers. P. alliacea is widely

used in folk medicine for treating infections. Many clinical reports and studies document that the

plant shows broad-spectrum antimicrobial properties against numerous strains of bacteria,

viruses, fungi, and yeast. (Ruffa et al., 2002) revealed that extracts of P. alliacea inhibited the

5
replication of the bovine diarrhoea virus; (test model for hepatitis C virus). The antimicrobial

properties of the plant have been documented by Cuban scientists who conducted in vitro studies

of extracts against numerous pathogens, including Escherichia coli, Staphylococcus,

Pseudomonas and Shigella (Pacheco, 2013). Crude water extracts of the plant, outperformed the

alcohol extracts. The antimicrobial activity was attributed to the presence of phenolic (Scalona,

2005) (Oguleye and Ibioteye, 2003), and sulphur compounds (Kim et al., 2006), which were

previously found to display antimicrobial action. A German group documented good activity

against several bacteria, Mycobacteriunm tuberculosis, several strains of fungi

and Candida. Anamu’s antifungal properties were documented by one research group in 1991,

and again by a separate research group in 2001. Its antimicrobial activity was further

demonstrated by researchers from Guatemala and Austria who, in separate studies in 1998,

confirmed its activity in vitro and in vivo studies against several strains of protozoa, bacteria, and

fungi.

Ayodele (2015), revealed that leaves of P. alliacea are rich in phytochemicals (Saponinis,

Alkaloids, Flavonoids, Phenolics and Terpenoids among others) which could be the basis for it

being used for medicinal purposes. However, caution should be taken in the intake and

administration of the leaf extracts because of the saponin and alkaloid content which may induce

some side effect

Joãoet al.( 2018) reported that P.alliaceais a cosmopolitan plant that provides easy access to

consumption by population. Till date, data have revealed that P.alliacea preparations present in

its constitution many biologically active compounds. In this sense, several sulfur containing

compounds (i.e., polysulfides and thiosulfinates) have revealed higher antimicrobial activity

against pathogenic bacteria and fungi at low concentrations.

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Guedes et al.,(2009)evaluated the antimicrobial activity by broth micro dilution method of

diffferent extracts obtained from P.alliacea leaves. In this regard, the hexane extract was more

active to inhibit Staphylococcus aureus than the polar extract (ethanol 70%), with minimum

inhibitory concentrations (MICs) values of 240¼g/ml and 3960¼g/ ml respectively .On

the other hand, the methanol extract presented activity against Enterococcus faecalis

(MIC'f 240¼ g/ mL) .

Kim et al. (2006) also evaluated the antimicrobial activity of different polysulfides isolated

from P . alliacea .In this sense, thiosulfinates and their degradation products inhibited at low

concentrations (MICs values d"6 4¼ g / m L) the bacteria Bacillus cereus, Mycobacterium

smegmatis, Micrococcus luteus, S.agalactiae, Staphylococcus aureus, Escherichia coli,

Stenotrophomonasmaltophila, Klebsiellapneumonie.

2.1 ORIGIN OF PETIVERIA ALLIACEA

Petiveria alliacea L. belongs to the Phytolaccaceae family which is considered to be the most

primitive family of the caryophyllales. There are about 17 genera and 120 pan-tropical species in

this group which are often found throughout North and South America. The family is comprised

mainly of shrubs and herbs and very few trees. Alliacea is the sole species within the Petiveria

genus. The plant is native to Florida and the Lower Rio Grande Valley of Texas in the United

States (USDA, 2008), tropical areas of Central and South America and the Caribbean. Introduced

populations exist in Benin and Nigeria.

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2.2 DESCRIPTION OF PETIVERIA ALLIACEA

Petiveria alliacea is a species of flowering plant in the pokeweed, family, phytolaccaceae

(Wikipedia, 2015). It is a native to Florida and the lower Rio Grande valley of Texas in the

United State, Mexico, Central America. It is commonly called Guinea Hen Weed, the Yorubas

call it Ewesoro.

The plant has several applications both in medicine and industry. It is used in teas, extracts and

capsules. It has been used to reduce inflammation and pain. It has antibacterial ability, antifungi

and antiviral effects (Schmelzer and Gurib-Fakim, 2008).

Schmelzer and Gurib-Fakim (2008) have revealed that the plant has tendency to reduce blood

sugar level and also destroy cancer cells. The plant is also used as bait and insect repellant

(Wikipedia, 2015).

Extract of this plant have been used for cancer therapy, this was due to the immune stimulatory

and cytotoxic properties of the plant (Eggenschwier et al., 2007). It has been used as anticancer,

antidiabetic, antihypertensive and as all purpose herb. It is used in Ondo State as topical

antibiotic in form of pastes for the treatment of wounds and other skin infections (Yusuf et al.,

2013).

Synthetic antibacterial used nowadays are associated with different complications leading to

different diseases like blood cancer and upper gastrointestinal complications. Not only that, they

are expensive and are not within the reach of the poor masses, there is therefore need for cost

effective and natural antibiotic. Some of the underutilized plants grown in Nigeria have these

properties.

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2.2.1 TAXONOMY OF PETIVERIA ALLIACEA

KINGDOM Plantae

CLASS Angiosperm

ORDER Caryophyllales

FAMILY Petiveriaceae

GENUS Petiveria L.

SPECIE P.alliacea

2.3 ANTIMICROBIAL ACTIVITY OF PETIVERIA ALLIACEA

Antimicrobial is an agent that kills microorganisms or stop their growth. It is used to describe

substances which demonstrate the ability to reduce the presence of microbes such as bacteria and

mould.

2.4 ANTIBACTERIAL ACTIVITY OF PETIVERIA ALLIACEA

ORGANISM’S PROFILE

2.4.1Bacillus subtilis is a ubiquitous bacterium commonly recovered from water, soil, air, and

decomposing plant residue. The bacterium produces an endospore that allows it to endure

extreme conditions of heat and desiccation in the environment. B. subtilis produces a variety of

proteases and other enzymes that enable it to degrade a variety of natural substrates and

contribute to nutrient cycling. However, under most conditions the organism is not biologically

active but exists in the spore form. B. subtilis is considered a benign organism as it does not

possess traits that cause disease. It is not considered pathogenic or toxigenic to humans, animals,

or plants. The potential risk associated with the use of this bacterium in fermentation facilities is

low. Bacillus subtilis is not a frank human pathogen, but has on several occasions been isolated

9
from human infections. Infections attributed to B. subtilis include bacteremia, endocarditis,

pneumonia, and septicemia.

2.4.2 Pseudomonas aeruginosa is a gram negative bacillus that commonly colonizes hospital

environment. It contaminates water and hospital devices and is known as aetiological agent of

nosocomial infection. Community-acquired infection of this organism had been reported (Hadi et

al., 2007). The prevalence of P. aeruginosa bacteremia varies from one to other institutions and

the sources of bacteremia were identified in 66 % of cases (Vitkauskien et al., 2010). Altogether

21% of these bacterial infections were classified as community acquired infection (Parkins et al.,

2010). Pseudomonas aeruginosa is a leading cause of nosocomial infection and associated with

high mortality rate (Fazlul et al., 2011). Pneumonia and sepsis are the most common infections

associated with these bacteria (Tam et al., 2009). This is a case of Pseudomonas aeruginosa

bacteremia, which we believe might have acquired from community setting. Pseudomonas

aeruginosa is an uncommon cause of community-acquired bacteremia. Cases of community-

acquired P. aeruginosa bacteremia represent about 21 % of all cases of P. aeruginosa bacteremia

(Parkins et al., 2010). Community-acquired P. aeruginosa infections occurred in those with

specific predisposing factors such as neutropenic and chronic structural lung diseases such as

cystic fibrosis and bronchitis (Schechner et al., 2009). In our case, we could not find any

significant risk factors that are associated with community-acquired P. aeruginosa infection.

2.4.3 Esherichia coli are gram-negative bacilli of the family Enterobacteriaceae. They are

facultative anaerobes and nonsporulating. E.coli strains with the K1capsular polysaccharide

antigen cause approximately 40% of cases of septicemia and 80% of cases of meningitis.

Different strains of E.coli are associated with a number of distinctive diarrheal illnesses. Among

these are the enterotoxigenic E.coli (ETEC), enteroinvasive E coli (EIEC), and Shiga toxin–

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producing E.coli (STEC). Of the STEC, E.coliO157:H7is the prototypic strain. Each class of E.

coli has distinct somatic(O) and flagellar (H) antigens and specific virulence characteristics.

2.4.4 Staphylococcus aureus is a Gram-positive, round-shaped bacterium that is a member of the

Firmicutes, and it is a usual member of the microbiota of the body, frequently found in the upper

respiratory tract and on the skin . It is often positive for catalase and nitrate reduction and is a

facultative anaerobe that can grow without the need for oxygen.

2.4.5 Klebsiellaoxytoca is a Gram-negative, rod-shaped bacterium that is closely related to K.

pneumoniae, from which it is distinguished by being indole-positive; it also has slightly different

growth characteristics in that it is able to grow on melezitose, but not 3-hydroxybutyrate.

2.5 PHYTOCHEMICAL ACTIVITY OF PETIVERIA ALLIACEA

Phytochemicals are naturally occurring chemicals produced by plants, they are found in fruits,

vegetables, legumes, grains, plant leaves, and so on. They give plants its colour, flavor, smell and

play a role in plant growth or defense against competitors, pathogens or predators. (Onyeka and

Nwabueke, 2007). Eating lots of plant foods rich in phytochemicals may help to prevent at least

one in every five cases of cancer, as well as other serious ailments such as heart disease. Plants

produced these chemicals to protect themselves, but recent researches demonstrate that they can

also protect humans against diseases (Liu, 2004) and (Anderson, 2004). There are about 6000

known phytochemicals in natural products and they have been isolated and characterized from

fruits, vegetables, spices, beverages and many other sources (Doughari and Obidah, 2008).

The antimicrobial properties exhibited by the extracts may be associated with the presence of

tannins, saponins and alkaloids found in the plant extracts. A large number of flavonoids have

been reported to possess antimicrobial properties (Boris, 1996), (Olowusulu and Ibrahim, 2006),

(Akinjobiet al., 2006).

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Dharmananda (2003), reported that herbs that have tannins as their component are astringent in

nature and are used for the treatment of gastrointestinal disorders such as diarrhea and dysentery.

2.5.1 ALKALOIDS

Alkaloids are natural products that contain heterocyclic nitrogen atoms and are always basic in

character. The name of alkaloids derives from the ‘alkaline’ nature and it was used to describe

any nitrogen-containing base. Almost all the alkaloids have a bitter taste. The alkaloid quinine,

for example, is one of the bitter tasting substances known and is significantly bitter (1x10-5) at a

molar concentration. Alkaloids are so numerous and involve such a variety of molecular

structure that their rational classification is difficult. However, the best approach is to group them

into families, depending on the type of heterocyclic ring system present in the molecule.

Alkaloids are significant for the survival of plant because they ensure their protection against

micro-organisms (antibacterial and antifungal activities), insects and herbivores (feeding

deterrents) and also against other plants by means of allelopathy. The use of alkaloids containing

plants as dyes, spices, drugs or poisons can be traced back almost to the beginning of

civilization. Alkaloids have many pharmacological activities including anti-hypertensive effects

(many indole alkaloids), anti-arrhythmic effect (quini-dine, spareien), anti-malarial activity

(quinine), and anti-cancer actions (dimericindoles, vincristine, vinblastine). These are just a few

examples illustrating the great economic importance of this group of plant constituents. Some

alkaloids have stimulant property as caffeine and nicotine, morphine are used as the analgesic

and quinine as the antimalarial drug.

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2.5.2 STEROIDS

Steroidis a biologically active compound which comprises of four rigs arranged in s specific

molecular configuration. Steroids have two principal biological functions as important

component of cell membrane which alter membrane fluidity and as signaling molecule

2.5.3 FLAVONOIDS

Flavonoids are important group of polyphenols widely distributed among the plant flora. They

are the most diverse group of phytochemicals. Flavonoids are a class of non-nitrogenous

biological pigments (biochromes) that includes the anthocyanins and the anthoxanthins. Though

extensively represented in plants, the flavonoids are of relatively minor and limited occurrence in

animals, which derive the pigments from plants.

Flavonoids are a group of plant that provide health benefits through cell signalling pathways and

antioxidant effects (Kar, 2007). They have also been shown to possess anti-inflamtory, anti-

carcinogenic, antithrombotic, anti-allergic and hepato-protective capabilities (Tapas, 2008)

2.5.4 SAPONINS

The term saponin is derived from Saponaria vaccaria , a plant, which abounds in saponins and

was once used as soap. Saponins therefore possess ‘soaplike’ behaviour in water, i.e. they

produce foam. On hydrolysis, an aglycone is produced, which is called sapogenin. There are two

types of sapogenin: steroidal and triterpenoidal. Saponins are extremely poisonous as they cause

haemolysis of blood (Kar, 2007). However, they are shown to have hypolipidermic and

anticancer activity (Sarker and Nahar, 2007).

2.5.5. TERPENOIDS

Terpennoids are flammable unsaturated hydrocarbon of plant origin of general formula (C5H8)

in existing in liquid form commonly found in essential oils, resins or oleoresins. Terpenoids are

13
classified according to the number of isoprene units in their structures. The diterpenes, C20 (4

isoprene units) are used as anti-cancer agents, the triterpenes C30, (6 isoprene units) show anti-

inflammatory, sedative and insecticidal activies (Martinez et al., 2008).

2.5.6. PHENOLIC

Phenolics are chemical components that occur ubiquitously as natural color pigments responsible

for the color of fruits of plants. Phenolics in plants are mostly synthesized from phenylalanine

via the action of phenylalanine ammonia lyase (PAL). They are very important to plants and

have multiple functions. The most important role may be in plant defence against pathogens and

herbivore predators, and thus are applied in the control of human pathogenic infections (Galm

and Shen, 2007). They are classified into (i) phenolic acids and (ii) flavonoid polyphenolics

(flavonones, flavones, xanthones and catechins) and (iii) non-flavonoid polyphenolies. Phenolics

essentially represent a host of natural antioxidants, used as nutraceuticals, and found in apples,

green-tea, and red-wine for their enormous ability to combat cancer and are also thought to

prevent heart ailments to an appreciable degree and sometimes are anti-inflammatory agents.

2.5.7 CARDIAC GYLCOSIDE

Cardiac glycosides are a class of organic compounds that increase the output force of the heart

and increase its rate of contractions by acting on the cellular sodium-potassium AT Pose pump.

Their beneficial medical uses are as treatments for congestive heart failure and cardiac

arrhythmias: however, their relative toxicity prevents them from being widely used. Most

commonly found as secondary metabolites in several plants such as foxglove plants, these

compounds nevertheless have a diverse range of biochemical effects regarding cardiac cell

function and have also been suggested for use in cancer treatment.

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2.6 FUNCTIONS OF PHYTOCHEMICALS IN THE BODY

• To stimulate the immune system in the body and body’s defense against bacteria, viruses and

other disease-causing agents.

• Phytochemical block the potential carcinogens i.e cancer causing substances to be formed in

the body from substances we eat, drink and absorb from the environment.

• It slows the growth rate of cancer cells.

• It helps to reduce inflammation that provides a setting favorable for cancer growth.

• To prevent DNA damage and help with DNA repair and mechanisms.

• Phytochemical reduce oxidation, the damage to cells that occurs with aging and exposure to

pollution.

• It regulates the hormones such as estrogen and insulin.

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 CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 COLLECTION OF PLANT

The leaves of Petiveria alliacea were collected from a wild along Abule Oko, Ijoko, Ogun State,

Nigeria and the plants were washed after collection to remove dust particles.

Figure 1: The extracts of Petiveria alliacea, field picture, 2019.

3.2 DRYING OF PLANT MATERIAL

Leaves of Petiveria alliacea were shade dried for 21days.

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3.3 GRINDING OF PLANT MATERIAL

The leaves were pulverized andpacked in airtight bottle for the preparation of extracts.

3.4 EXTRACTION OF PLANT MATERIAL

300g of grinded Petiveria alliacea leaves was weighed and soaked in 1200ml of methanol for

7days was sieved using muslin cloth and then re-sieved with Whattman’s No 1 filter paper and

stored in a bottle with cap.

3.5 RECONSTITUTION OF PLANT MATERIALS

The content was poured into evaporating dish and was evaporated in the hot air oven at 60oC.

The extract after drying remained green. It was stored in universal bottles and labeled.

3.6 SAMPLE PREPARATION FOR THE EXTRACTS

The working samples for the extract were of three values, 600g/ml , 300mg/ml, and 150mg/ml

which were achieved by weighing 6g of extract and dissolving in the appropriate solvent and

then double diluting to have other working values.

6g of extract in 10ml of preparation, concentration = 600mg/ml

2ml of A + 2ml of sterile distilled (diluent), concentration = 300mg/ml

2ml of B + 2ml of sterile distilled water (diluent), concentration =150mg/ml

Contents of bottles A,B, and C were the working samples.

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3.6.1 STANDARD PREPARATION

CIPROFLOXACIN STANDARD CONCENTRATIONS

Similarly, working standards were of four values, 40µg/ml,20µg/ml,10µg/ml and 5µg/ml

solutions of the sample were achieved by double diluting the sample as follows;

A. 2000µg/ml of the proprietary standard stock solution in normal saline solution.

B.0.2ml of A + 9.8ml of sterile diluent , concentration = 40 µg/ml

C.2ml of B + 2ml of sterile diluent, concentration = 20 µg/ml

D.2ml of C + 2ml of sterile diluent , concentration = 10 µg/ml

E.2ml of D+ 2ml of sterile diluent , concentration = 5 µg/ml

Contents of bottles B, C, D and E were the working standards of neomycin.

3.6.2 MEDIA PREPARATION

3.6.2.1 MUELLER HINTON AGAR ( MHA )

The media were prepared following the inscriptions by the manufacturers. They were weighed

and dispersed in the specified volume of distilled water. They were heated to melt in the water

bath at 1000C. The molten agar gels so formed were dispensed in 25ml portions into sample

bottles and autoclaved at 1210C for15 minutes. The 25ml portion of the agar gel in each of the

sample bottles was the working volume of the agar.

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3.6.2.2NUTRIENT AGAR (NA)

This medium was used for sub-culturing and recovery of the organisms from the stock cultures.

It was prepared according to the instructions on the manufacturer’s label. A quantity weighing

28g was dispersed in 1 liter of distilled water. It was heated to melt in water bath and then

homogenized. The nutrient agar gel was autoclaved at 1210c for 15 minutes. After sterilization,

the sterile molten agar was pour plated under the lamina flow system. They were dried under the

same condition before streaking the surface with test organisms.

3.6.3 STERILIZATION OF GLASSWARES

Glassware were washed and rinsed with tap water. They were further rinsed with distilled water

and dried in a hot air oven at 1000Cuntil they were dry. They were then sterilized at 1600C for

one hour in the hot air oven and allowed to cool to the Laboratory ambient temperature before

use.

The assay organisms which were bacterial group include Escherichia coli, Pseudomonas

aeruginosa, Bacillus subtilis and Staphylococcus aureus and Klebsiella oxytoca. They were

primarily isolated on various diagnostic media accordingly and then subcultured to further

purify. They were then subcultured onto Nutrient Agar to remove the effects of indicators and

suppressive chemical agents in primary isolation media. They were then subcultured into sterile

nutrient broth for optical density calibration. Incubation periods were 24 hours for all the bacteria

at 370c .

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3.6.5 CALIBRATION OF ASSAY ORGANISMS

The biological load of the bacterial assay organisms were adjusted using certain criteria. All the

bacterial suspensions were adjusted using sterile normal saline. The bacterial liquid cultures were

added dropwise to the normal saline until the turbidity matched that of the densometer, 0.5

McFarland turbidity standard. The adjusted suspension was used for the assay. All molds were

calibrated using their spores in 0.05 tween 80 in normal saline. The spore load was adjusted to

10spore forming unit per millilitre , SFU/ml, using serial dilution and plating out technique.

3.6.6 SEEDING OF ASSAY ORGANISMS

The assay medium which was prepared and measured was maintained at 450C so as to make it

remain molten. 1ml of calibrated organisms was seeded into the warm agar and was mixed

thoroughly using the roll-palm method before pour-plating. After solidifying under sterile

condition in a biological safety cabinet, they were prepared for cork boring.

3.6.7 CORK BORING AND DISPENSING OF STANDARDS AND SAMPLES

After allowing all the seeded agars to set, a cork borer, size 10mm cross-section was used for

boring the wells. It was flamed and allowed to cool before using it to gently punch a hole in each

of the sectors of the Petri dishes. All the cut portions were thrown into a dish of disinfectant. 150

µl of various working concentrations was dispensed into the wells and allowed to stand for four

hours before incubation.

3.6.8INCUBATION OF PLATES AND READINGS

All the Petri dishes were incubated lid-up position. This was so in order to avoid spillage. After

four hours on the Laboratory bench for the samples and standard concentration to diffuse, the

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plates for antibacterial studies were incubated at 370C and observed after 24 hours and observed

for zones of inhibition as a result of growth of the bacteria. Several readings were taken with

zone reader and average zone values were determined and recorded.

3.7 PHYTOCHEMICAL SCREENING USING (Edeoga et al)

Chemical tests were carried out on the aqueous extract and on thepowdered specimens using

standard procedures to identify theconstituents

Test for tannins: About 0.5 g of the dried powdered samples wasboiled in 20 ml of water in a

test tube and then filtered. A few dropsof 0.1% ferric chloride was added and observed for

brownish green or a blue-black colouration.

Test for phlobatannins: Deposition of a red precipitate when anaqueous extract of each plant

sample was boiled with 1% aqueoushydrochloric acid was taken as evidence for the presence of

phlobatannins.

Test for saponin: About 2 g of the powdered sample was boiled in20 ml of distilled water in a

water bath and filtered. 10ml of thefiltrate was mixed with 5 ml of distilled water and shaken

vigorouslyfor a stable persistent froth. The frothing was mixed with 3 drops ofolive oil and

shaken vigorously, then observed for the formation ofemulsion.

Test for flavonoids: Three methods were used to determine thepresence of flavonoids in the

plant sample. 5 ml of dilute ammonia solution were added to aportion of the aqueous filtrate of

each plant extract followed byaddition of concentrated H2S04. A yellow colouration observed in

each extract indicated the presence of flavonoids. The yellow colouration disappeared on

standing. Few drops of 1% aluminium solution were added to a portion ofeach filtrate. A yellow

colouration was observed indicating thepresence of flavonoids.A portion of the powdered plant

sample was in each case heatedwith 10 ml of ethyl acetate over a steam bath for 3 min. The
21
mixturewas filtered and 4 ml of the filtrate was shaken with 1 ml of diluteammonia solution. A

yellow colouration was observed indicating apositive test for flavonoids.

Test for steroids: Two ml of acetic anhydride was added to 0.5 gethanolic extract of each

sample with 2 ml H2S04. The colour changed from violet to blue or green in some samples

indicatingthe presence of steroids.

Test for terpenoids (Salkowski test): Five ml of each extract wasmixed in 2 ml of chloroform,

and concentrated H2S04 (3 ml) wascarefully added to form a layer. A reddish brown colouration

of the inter face was formed to show positive results for the presence of terpenoids.

Test for cardiac glycosides (Keller-Killani test): Five ml of eachextracts was treated with 2 ml

of glacial acetic acid containing onedrop of ferric chloride solution. This was underlaid with 1 ml

of concentrated sulphuric acid. A brown ring of the interface indicates a deoxysugar

characteristic of cardenolides. A violet ring mayappear below the brown ring, while in the acetic

acid layer, agreenish ring may form just gradually throughout thin layer.

QUANTITATIVE DETERMINATION OF THE CHEMICAL CONSTITUENTS

Preparation of fat free sample: 2 g of the sample were defattedwith 100 ml of diethyl ether

using a soxhlet apparatus for 2 h.

Determination of total phenols by spectrophotometric method:The fat free sample was

boiled with 50 ml of ether for the extractionof the phenolic component for 15 min. 5 ml of the

extract waspipetted into a 50 ml flask, then 10 ml of distilled water was added.2 ml of

ammonium hydroxide solution and 5 ml of concentrated amylalcohol were also added. The

samples were made up to markand left to react for 30 min for colour development. This was

measured at 505 nm.

22
Alkaloid determination using Harborne (1973) method: 5 g ofthe sample was weighed into a

250 ml beaker and 200 ml of 10%acetic acid in ethanol was added and covered and allowed to

standfor 4 h. This was filtered and the extract was concentrated on a waterbath to one-quarter of

the original volume. Concentratedammonium hydroxide was added drop wise to the extract until

theprecipitation was complete. The whole solution was allowed to settle and the precipitated was

collected and washed with diluteammonium hydroxide and then filtered. The residue is the

alkaloid,which was dried and weighed.

Tannin determination by Van-Burden and Robinson (1981)method: 500 mg of the sample

was weighed into a 50 ml plasticbottle. 50 ml of distilled water was added and shaken for 1 h in

amechanical shaker. This was filtered into a 50 ml volumetric flaskand made up to the mark.

Then 5 ml of the filtered was pipetted outinto a test tube and mixed with 2 ml of 0.1 M FeCl3 in

0.I N HCl and0.008 M potassium ferrocyanide. The absorbance was measuredat 120 nm within

10 min.

Saponin determination: The samples were ground and 20 g of eachwere put into a conical flask

and 100 cm3 of 20% aqueous ethanolwere added. The samples were heated over a hot water bath

for 4hours with continuous stirring at about 55°C. The mixture was filteredand the residue re-

extracted with another 200 ml 20% ethanol. Thecombined extracts were reduced to 40 ml over

water bath at about90°C. The concentrate was transferred into a 250 ml separatory funnel and 20

ml of diethyl ether was added and shaken vigorously.The aqueous layer was recovered while the

ether layer wasdiscarded. The purification process was repeated.60 ml of n-butanol was added.

The combined n-butanol extracts were washed twice with 10 ml of 5% aqueous sodium chloride.

The remaining solution was heated in a waterbath. After evaporation the samples were dried in

the oven to a constant weight; the saponin content was calculated as percentage.

23
Flavonoid determination by the method of Bohm and Kocipai-Abyazan (1994): 10 g of the

plant sample was extractedrepeatedly with 100 ml of 80% aqueous methanol at

roomtemperature. The whole solution was filtered through whatman filter paper No 42 (125

mm). The filtrate was later transferred into acrucible and evaporated into dryness over a water

bath and weighed to a constant weight.

24
 CHAPTER FOUR

4.0 RESULTS

AVERAGE INHIBITION ZONE DIAMETER (MM) OF SAMPLE PETIVERIA

ALLIACEA EXTRACT ON BACTERIA

The methanolic extract of Petiveria alliacea at concentration 600mg/ml was carried out on the

antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus

aureus, Bacillus subtilis, and Klebsiella oxytoca and this result were compared to that of the

standard antibiotic drug called Ciprofloxacin. The Standard antibiotic drug has higher

antimicrobial activity than that of the methanolic extract of Petiveria alliacea. The methanolic

extract of Petiveria alliacea were susceptible to both Gram positive and some Gram negative

bacteria except Klebsiella oxytoca.

25
Table 4.1 showed the antibacterial effect of the methanolic extract of Petiveria alliacea leaves at

various concentrations. At 600mg/ml: showed the highest susceptibility Klebsiella oxytoca

(10.85) followed by Bacillus subtilis (10.20), Pseudomonas aeruginosa (8.50),

Stapylococcusaureus (7.90), Escherichia coli (5.50).

26
TABLE 4.1 AVERAGE INHIBITION ZONE DIAMETER (MM) OF SAMPLE PA
EXTRACT ON BACTERIA

ASSAY ORGANISM 600MG/ML

Escherichia coli 5.50

Pseudomonas 8.50
auregenosa

Staphylococcus 7.90
aureus

Bacillus subtilis 10.20

Klebsiella oxytoca 10.85

27
INHIBITION ZONE DIAMETER (MM) OF CIPROFLOXACIN CONTROL

STANDARD ON BACTERIA

Table 4.2 showed the antibacterial effect of the Standard drug Ciprofloxacin at various

concentrations. At 40µg/ml: Pseudomonas aeruginosa shows the highest susceptibility

(30.40±0.37) followed by Bacillus subtitis (25.40±0.37), Staphylococcus aureaus (24.75±0.25),

Escherichia coli (21.60±0.20), and Klebsiella oxytoca (0.00). At 20µg/ml: Pseudomonas

aeruginosa shows the highest susceptibility (27.30±0.25) followed by Bacillus subtitis

(22.60±0.20), Staphylococcus aureaus (20.60±0.20)g, Escherichia coli (19.30±0.25)and

Klebsiella oxytoca (0.00). At 10µg/ml: Pseudomonas aeruginosa shows the highest

susceptibility (24.30±0.25) followed by Bacillus subtitis (21.20±0.25), Escherichia coli

(17.70±0.40), Staphylococcus aureaus (17.30±0.25) and Klebsiella oxytoca (0.00). At 5 µg/ml

Bacillus subtilis shows the highest susceptibility (18.20±0.25) followed by Escherichia coli

(15.20±0.25), Staphylococcus aureaus (14.50±0.320), Pseudomonas aeruginosa (0.00) and

Klebsiella oxytoca (0.00).

28
TABLE 4.2 INHIBITION ZONE DIAMETER (MM) OF CIPROFLOXACIN STANDARD
ON BACTERIA

ASSAY ORGANISM 40µg/ml 20µg/ml 10µg/ml 5 µg/ml

Escherichia coli 21.60±0.20 19.30±0.25 17.70±0.40 15.20±0.25

Pseudomonas 30.40±0.37 27.30±0.25 24.30±0.25 0.00

Staphylococcus 24.75±0.25 20.60±0.20 17.30±0.25 14.50±0.32

Bacillus subtilis 25.40±0.37 22.60±0.20 21.20±0.25 18.20±0.25

Klebsiella oxytoca 0.00 0.00 0.00 0.00

29
PHYTOCHEMICAL (QUALITATIVE) ANALYSIS OF PETIVERIA ALLIACEA

Table 4.3 showed the qualitative analysis of the methanolic extract of Petiveria alliacea leaves.

Alkaloid(+), saponin(+), phlobatanin(+), Terpenoid(+), flavonoid(+), Cardiac glycoside(+)were

shown to be presentwhile Steroid(-) was absent.

30
Table 4.3 SHOWING RESULTS OF PHYTOCHEMICAL (QUALITATIVE ANALYSIS)

OF PETIVERIA ALLIACEA

Compound Methanol extract

Alkaloid +

Saponin +

Steroid -

Phlobatanin +

Terpenoid +

Flavonoid +

Cardiac glycoside +

+=Presence of constituent

- = Absence of constituent

31
QUANTITATIVE ANALYSIS OF PETIVERIA ALLIACEA mg/100ml

Table 4.4 showed the quantitative analysis of the methanolic extract of Petiveria alliacea leave

at mg/100ml. Alkaloid (22.50), Saponin (12.30), Steroid (04.40), Phlobatanin (22.40), Terpenoid

(12.40), Flavonoid (30.20), Cardiac glycoside (22.20) and Phenol (10.00).Alkaloid has the

highest value and steroid has lowest value.

32
Table 4.4 QUANTITATIVE ANALYSIS OF PETIVERIA

ALLIACEAmg/100ml

Compound Methanolic extract

Alkaloid 22.50

Saponin 12.30

Steroid 04.40

Phlobatanin 22.40

Terpenoid 12.40

Flavonoid 30.20

Cardiac glycoside 22.20

Phenol 10.40

33
 CHAPTER FIVE

5.0 DISCUSSION, CONCLUSION AND RECOMMENDATION

5.1 DISCUSSION

According to Wasimet al., (2013), showed the phytochemical screening of methanolic extracts of

Petiveria alliacea, the results confirmed the presence of glycosides, phytosterol, steroids,

saponins, tannins and flavonoids in extracts of the plant. These phytochemical constituents are

good source of antimicrobial and antioxidant activity Phytochemical analysis results of Petiveria

alliacea leaves were reported as glycosides(+), phytosterol(+), steroids(+), saponins(-),

tannins(+) and flavonoids(+). In the present study glycosides, saponins, flavonoids were present

while steroids absent.

According to Sathiyabalan et al., (2017), showed the phytochemical of the methanolic and

ethanolic extracts of whole plant of P. alliacea shows the presence of alkaloid, anthraquinone,

catechin, flavonoid, phenol, quinone, saponin, steroid, tannin, sugar, glycoside and xanthoprotein

but only methanolic extracts was used in the present study in which steroids was absent.

According to Adesipo et al., (2007), showed that the phytochemical of the methanolic extracts

of whole plant of P. alliacea showed the presence of alkaloid, anthraquinone, catechin,

flavonoid, phenol, quinone, saponin, steroid, xanthoprotein, tannin, sugar but the absence of

cardiac glycoside while in the present study, cardiac glycoside was present while steroids was

absent.

According to Olufunmilayo et al., (2015), confirmed that the phytochemical screening of Petiveria

alliacea can be attributed to the presence of phytochemicals such as flavonoids, saponins,

alkaloids, antioxidants, phenolics and terpenoids in the plants extract while in the present, study

34
there was presence of flavonoids, tannins, alkaloids, steroids, phenols, cardiac glycosides and

terpenoids in the plants extract while in the present study there was absence of steroids.

According to Tavs and Doris. (2012), confirmed that the antifungal activities of the

phytochemical screening of Petiveria alliacea can be attributed to the presence of

phytochemicals such as flavonoids, tannins, alkaloids, steroids, coumarines, cardiac glycosides,

quinines and terpenoids in the plants extract while in the present study there was absence of

steroids.

According to Seokwonet al., (2006), showed that the phytochemical screening of the methanolic

extract of Petiveria alliacea, in that tannins and phenols were present while in the present study,

cardiac glycosides, saponins, terponoids, phenols, flavonoids were present while steroids absent.

According to (Adzu et al., 2005), they confirmed that antifungal activities of Petiveria alliacea

can be attributed to the presence of phytochemicals such as flavonoids, tannins, alkaloids,

steroids, coumarines, cardiac glycosides, quinines and terpenoids in the plants extract while in

the present study there was absence of steroids.

5.2 CONCLUSION

The result obtained from this study has shown that phytochemical screening of the methanolic

extract of Petiveria alliacea revealed the presence of alkaloids, Flavonoids, saponins, tannins,

anthraquinones, terpenoids but absence and Steroids. The extracts also demonstrated significant

antimicrobial and antifungal activities against the tested organisms which are responsible for

various biological activities. This study revealed that the leaves of Petiveria alliacea exhibit

maximum antifungal activity against some fungi such as Aspergillus niger. The methanolic leaf

extract showed a stronger antimicrobial activity against Aspergillus flavus and Penicillium sp.

This plant can be used as alternative medicine for treating many diseases cancer, diabetes,

35
muscular pain skin diseases, various central nervous system disorders, pain relief, and as an anti-

influenza, anti-inflammatory, anti-tumor, anti-bacterial, anti-fungal, anti-hyperlipidemia, and

anti-diabetic drug, respiratory and pulmonary infections, malaria among other ailments.

5.3 RECOMMENDATION

I recommend that the use of Petiveria alliacea should be rational as it can be used in traditional

medicine for the treatment of cancer, diabetes, muscular pain skin diseases, various central

nervous system disorders, pain relief, and as an anti-influenza, anti-inflammatory, anti-tumor,

anti-bacterial, anti-fungal, anti-hyperlipidemia, and anti-diabetic drug, respiratory and pulmonary

infections, malaria among other ailments and that pregnant women should stay away from it

because of its presence of skunk which may leads to miscarriage in them.

36
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41
 APPENDIX

CHART SHOWING ANTIMICROBIAL ANALYSIS OF PETIVERIA ALLIACEA

600MG/ML
12

10

6
600MG/ML
4

0
EC PA SA BS KO

Key: EC = Escherichia coli, PA = Pseudomonas auregenosa, SA = Staphylococcus aureus, BS =

Bacillus subtilis, and KO = Klebsiella oxytoca

Figure 2: Bar chart showing Antimicrobial analysis of Petiveria alliacea plant extract.

42
CHART SHOWING THE PHYTOCHEMICAL (QUANTITATIVE) ANALYSIS OF

PETIVERIA ALLIACEA

Methanolic extract
35
30
25
20
15
10
Methanolic extract
5
0

Figure 3: Bar chart showing the phytochemical (quantitative) screening of Petiveria alliacea

43