THE PHYTOCHEMICAL ANALYSIS AND ANTIBACTERIAL ACTIVITY OF PARKIA BIGLOBOSA (AFRICAN LOCUST BEAN) leaf plant, commonly found in Aliero town, Kebbi State.

LIST OF FIGURES

Figure 1.1 Parkia biglobosa tree plant 7

Figure 1.2 Structure of alkaloids 10

Figure 1.3 structure of saponin 11

Figure 1.4 structure of flavone 12

Figure 1.5 structure of terpenoids 13

Figure 1.6 structure of tanins 14

Figure 2.1 antibacterial activity of bacteria against control 25

Figure 2.2 antibacterial activities of salmonella against plant extract 26

Figure 2.3 antibacterial activities of E-coli against plant extract 27

LIST OF TABLES

Table 1.1 list of chemicals & reagents 16

Table 1.2 list of apparatus & equipment 18

Table 2.1 phytochemical constituents of parkia biglobosa leaf extract 23

Table 2.2 antibacterial activities of parkia biglobosa leaf extract 24

ABSTRACT

The phytochemical content of parkia biglobosa leaf plant extract and its antibacterial activity against pathogenic bacteria was investigated. The phytochemical analysis revealed the presence of tanins, flavonoids, glycosides, phenols, sterols, alkaloids, terpenoids, saponins and the absence of Anthraquinones in the leaf extract. The methanol extracts of the investigated leaf extract inhibited the growth of some pathogenic bacteria (salmonella & E-coli).

 CHAPTER ONE

1.0 INTRODUCTION AND LITERATURE REVIEW

1.1 INTRODUCTION

Dependence on herbs as medicine in the treatment of disease is common among a large proportion of the rural populace because of its availability and affordability (Sani et al., 2009). Due to the increasing awareness of the importance of traditional medicine in human and animal

Health care, researches into the efficacy of some of the herbs used in the treatment of some illness would be worthwhile. This is not only to supplement modern drugs with effective local herbs for economic reasons but also to fill the gap created by recurring shortage of conventional drugs (Sofowora, 1982). WHO (1993) support the use of effective and safe remedies and accept traditional medicine as a valuable and readily available resources for primary health care. The use of traditional medicine and medicinal plants in most developing countries, as a normative basis for the maintenance of good health, has been widely observed. Hyperglycemia is a condition characterized by a high amount of serum glucose causing damage to small vessels, hence increase the risk of late stage diabetes complications such as neuropathy, Heart attack, hypertension, stroke,

A medicinal plant is any plant which is one or more of its organ, contains substance that can be used for the therapeutic purpose or which are precursors for the synthesis of useful drug. (sofowora, 2008),An herbal remedy is one in which  he main therapeutic activity depends upon the plant or fungal metabolites which it contain some plant are purely dietary and are necessary for health(fresh, vegetable, carrots, fruit ,which we now known provided essential vitamins) many plant products are consumed in reasonable quantity as food butknown to have medicinal plant, few apparently quite safe and other more potent( e.g  containing cardioactive  glycosides) which can only be consumed is small quantities but which as such dosage are suitable for the treatments of disease. The medicinal value of plants lies in some chemical substances that produce a definite physiological action on the human body. The most important of these bioactive constituent of plants are alkaloids, tannins, flavonoids and phenolic compounds.

The uses of plants as medicines predate written human history and forms the origin of modern medicine .Ethno botany (the study of traditional human uses of plants) is recognized as an effective way to discover future medicines. Many synthetic drugs originated from plant sources. In Nigeria, various plant parts are used for curing different ailments with remarkable success. Among the enormous number of these medicinal plants are members of the genus parkia bigdlobosa (African locus beans)

1.2 Justification

parkia biglobosa leaf has been  believed to be rich in chemical constituent by traditionalists without proper research of the plant, due to this  chemical richness, the leaves are used in traditional medicines for various purposes. 

In spite of the ever increasing efforts by researchers to discover medicinal potential of plants, the potential of many higher plants as source for new drugs is still largely unexplored. Antibacterial infection can be treated with a wide range of antibiotics. Most of these antibiotics are either too expensive or not readily accessible by many peoples particularly those living in rural areas. Further to this, the development of antibacterial resistance to many to many existing antibiotics is a major concern. These concerns justify the urgent need to discover novel drugs for treatment of antibacterial infections.

1.3 Aim and Objectives

1.3.1 Aim: To determine the phytochemicals and antibacterial activity of parkia biglobosa methanolic leaf plant extract. 

1.3.2 Objectives:

To evaluate the presence of phytochemical composition of parkia biglobosa leaf plant. 

To evaluate the antibacterial activity of methanolic leaf extract of parkia biglobosa.

1.4 Literature Review

Antibacterial activity is the ability of a substance to inhibit or kill bacterial cells. Different types of antibiotics and chemotherapeutic agents are being used in the treatment of one form of disease or the other. Most of these antibiotics were originally derived from micro-organisms while the chemotherapeutic agents are from plants. However, nowadays these antibiotics and chemotherapeutic agents are obtained by various synthetic processes. Most countries in West African especially Nigerian are richly blessed with forests containing arrays of different herbs, shrubs and trees.

Antibacterial properties of plant extracts have been a very hot topic for the researchers of this field. Everyone is concerned with the high and growing number of diseases associated with microorganism especially bacteria and fungi. Bacteria also have become far more resistant o many antibacterial agents. For instance, of the million people who acquired bacterial infection in United States of American (USA) hospitals annually, 70% of the cases involved the strains that are resistant to at least one antibacterial agent (cushine and lamb, 2005). The emergence of antibiotic-resistant microorganism had swiftly reversed the advances of previous fifty years of research on antibiotics. And also the side effect associated with the available antibiotics has been alarming too. Therefore, it has been a challenge for the researchers to overcome these problems. Over the past two-three decades, researchers have turned their eyes towards the traditional folk medicines or natural products to uncover the scientific basis of remedial effects such as antibacterial agents. Beside plants fruits also have been studied by the researchers for the presence of bioactive compounds close related with herbs, commonly referred to as phytochemicals such as tannins, carotenoids, and polyphenols and anthocyanin’s.

1.4.1 Description of the Plants

African locust bean is a medium-sized legume tree that reaches 20-30 m high. It has a dense, widely spreading umbrella-shaped crown and a cylindrical trunk can branch low. The bark is longitudinally fissured, scaly between the fissures, thick, ash-grey to greyish-brown in colour. It exudes an amber gum when cut. Parkia biglobosa is a dicotyledonous angiosperm belonging the family Fabaceae – Mimosoideae. It is categorized under spermatophytes, vascular plants.It is a deciduous perennial that grows to between 7 and 20 metres high, in some cases up to 30 metres. The tree is a fire-resistant helophyte characterized by a thick dark gray-brown bark. The pods of the tree, commonly referred to as locust beans, are pink in the beginning and turn dark brown when fully mature. They are 30-40 centimetres long on average, with some reaching lengths of about 45 centimetres. Each pod can contain up to 30 seeds. The African locust bean tree, Parkia biglobosa (Jacq) Benth is a perennial tree legume which belongs to the subfamily Mimosideae and family Leguminosae. The fermented seeds of P. biglobosa are used in all parts of Nigeria for seasoning traditional soups. Parkia has found use as foods, medicinal agents and are of high commercial value (Ajayeoba, 2002).

1.4.2 Taxonomy

African locust bean is a medium-sized legume tree that reaches 20-30 m high. It has a dense, widely spreading umbrella-shaped crown and a cylindrical trunk that can reach 130 cm in diameter, often branching low. The bark is longitudinally fissured, scaly between the fissures, thick, ash-grey to greyish-brown in colour. It exudes an amber gum when cut. The leaves are alternate and bipinnately compound, 30-40 cm long, bearing up to 17 pairs of pinnae. Its beignets are numerous (13-60/pinna), sub opposite, 8-30 mm long x 1.5-10 mm wide, rounded or obtuse at apex, glabrous but slightly ciliate near apex. The inflorescence is held on a long (10-35 cm) drooping peduncle. It is biglobose, showy, red in colour, and it looks like an electric bulb. The flower head is 4.5-7 cm long x 3.5-6 cm broad and it has a strong pungent smell. The many flowers are bisexual, sterile or nectar-bearing. Bisexual flowers are pentamerous, 1-1.4 cm long, and corolla lobes are fused at their base. Sterile flowers are shorter and are borne near the peduncle, in the upper part of the inflorescence, and their nectar is attractive to bats that pollinate the flowers. The flowers begin to open at dusk, close and wilt at dawn, lasting only a single night. The fruit is a linear, glabrous and smooth, indehiscent pod that becomes brown at maturity. It is 12-30 (-35) cm long x 1.5-2.5 cm wide and contains up to 23 seeds embedded in a yellowish mealy pulp. The seeds are globose-ovoid, 5-15 mm, smooth and glossy dark in colour. There are about 2800-6700 seeds/kg. The seeds are hardcoated and can remain viable up to 8 years (Orwa et al., 2009; NRC, 2006; Sina et al., 2002; Hopkins, 1983). African locust bean is native to tropical Africa. It occurs in open savannah woodlands, in bush fallow and wooded farmland where cultivation is semi-permanent, in areas ranging from tropical forests with high and well-distributed rainfall, to arid zones (Orwa et al., 2009; Sina et al., 2002). It was introduced as a food plant into the West Indies and is now naturalized in Haiti. It has also been introduced into Sao Tomé by the Portuguese (Hopkins, 1983).  The leaves are traditionally used, and whole branches are lopped for fodder (NRC, 2006). They can be used with success as forage to feed goats and large livestock, generally together with a concentrate (Sabiiti et al., 1992; Okagbare et al., 2004). The potential degradability of African locust bean fodder in cattle, sheep and goats was shown to be among the lowest compared with other typical browse species of Nigeria (Larbi et al., 1997). In particular, Parkia biglobosa leaves had lower in vitro DM digestibility than Gliricidia sepium, which could be attributed to a higher NDF, and probably tannin, contents (Sabiiti et al., 1992). The in vivo OM digestibility of Parkia leaves hay measured in goats was low (32%) (Adeloye et al., 1993).

African locust bean leaves included at up to 15% in the diet of Yankasa rams receiving Pennisetum pedicellatum as roughage and a concentrate had no ill-effect on blood parameters of the rams (Wada et al., 2014). African locust bean leaves given as sole forage did not result in live weight gain in West African dwarf female goats, suggesting that this forage would require a supplementation (Adeloye, 1994). Parkia filicoidea hay included as a supplement in a cassava-peel diet and compared to a control diet of Andropogon gayanus, maize and soybean meal, resulted in better DM intake than that obtained with the cassava peels-only supplement or with the control diet. Acceptability’s were enhanced at 25:75 and 50:50 cassava peel: Parkia hay mixtures. Nutrient digestibility’s were higher with the 50% replacement diet, except for crude fiber and total ash. The availability of the cassava peel and Parkia hay at little or no cost and the good digestibility of the 50:50 combination would make the 50:50 combination of the plant products an acceptable dry season feed and a suitably cheap feed in subsistence goat-production (Adeloye et al., 1993)

Figure 1.1: parkia biglobosa tree

1.4.3 Scientific Classification

Kingdom: Plantae

Phylum: angiosperm

Class: Eudiocots

Sub-class: Rosids

Order: Fabales

Family: Fabaceae

Genus: Parkia

Specie: P.biglobosa

1.4.4 Uses of plant

African locust bean, commonly known as ‘iru’ by Yoruba’, ‘ogiri’ by Igbo’s and ‘dawa-dawa’ by Hausa’s is a local seasoning or condiment used in stews. A very popular soup ingredient, it is found in a wide range of environments in Africa and is primarily grown for its pods that contain both pulp and valuable seeds.

The yellow pulp, which contains the seeds, is naturally sweet and is processed into a valuable carbohydrate food known as sikomu and daddawa among the Yoruba and Hausa people respectively. The most valuable part of the locust bean are high in lipid (29%), protein (35%), carbohydrate(16%) it is also a good source of fat and calcium for rural dwellers. Some traditional uses of locust bean are: It is used in controlling diabetes and cholesterol level, It helps to promote good sight and aids digestion, It is used for treating stroke and hypertension, The water and alcoholic extracts of fermented locust bean is used to reduce blood sugar, It is used in the management of bacterial infections, It is also used in the treatment of diarrhea, It is a potential benefit for enhancing weight loss, The bark of the tree is employed in wound healing and serves as one of the ingredients in treating leprosy.

Researchers in Dakar Senegal, tried out locust beans on rats to find out whether it actually has any impact in controlling blood pressure and the result obtained showed that adequate doses of locust beans helped to decrease blood pressure.(naij.et.al.,2015)

1.4.5 Phytochemicals

Phytochemicals are naturally occurring, biologically active chemical compound in plants. The presence of certain types of phytochemicals in some plants acts as a natural defense system providing protection against the insect that attack and grazing animals. Other plants produce phytochemicals that provide colour, aroma and flavour, thus inviting attention from potential consumers (Wada and Ou, 2002). More than 4,000 of these compound have been discovered, and it is expected that scientist will discover many more (Wada and Ou, 2002). Phytochemicals are a large group of plant-derived compounds hypothesized to be responsible for much of the disease protection conferred from diets high fruits, vegetables, beans, cereals, and plant-based beverages such as tea and wine (Arts and Hollman, 2000).

 

1.4.5.1 Alkaloids

Alkaloids are a group of naturally occurring chemical compounds that contain mostly basic nitrogen atom. This group also includes some related compounds with neutral and even weakly acidic properties (Robert, 1998). Some synthetic compounds of similar structure are also attributed to alkaloids (Robert, 1998). They often have pharmacological effects and are used as medication as recreational drugs, examples are the local anesthetic and stimulant cocaine, the psychedelic psilocin, the stimulant caffeine and nicotine (Robert, 1998).

Figure 1.2: Structure of Alkaloid.

1.4.5.2 Saponins

Saponins are a class of chemical compounds, one of main secondary metabolites found in natural sources, with saponins found in particular abundance in various plant species. More specifically, they are ampiphatic glycoside grouped, in terms of phenomenology, by the soap-like foaming they produce when shaken in aqueous solutions, and in terms of structure, by their composition of one or more hydrophilic glycoside moieties combined with lipophilic triterpene derivatives (Hostettmann and Martson, 1995). Saponins cause a depletion of body cholesterol by preventing its reabsorption, this increasing its excretion, in much the same way as other cholesterol lowering drugs, such as cholestylamine (Hostettmann and Martson, 1995). Saponins have been found to be useful in the treatment for hypercholesterolemia. Saponins bind with cholesterol so it cannot be reabsorbed into the system and is excreted from the body (Hostettmann and Martson, 1995).

Figure 1.3: Structure of Saponin.

1.4.5.3 Flavonoids

This large group of phytochemicals includes more than 1500 separated compounds with varied functions. Flavonoids are found in almost all plant based food and beverages, but levels vary depending on the degree of ripeness, variety and processing. Flavanoids enhance the effect of vitamin C and function as antioxidants. They are also known to be biologically active liver toxins, tumors, viruses and other microbes, allergies and inflammation (; De Soussa, 2007; Cushnie and Lamb, 2011). They protect blood vessels especially the tiny capillaries that carry oxygen and nutrients to our cell and are believed to slow down the development of cataracts in person who have diabetes (McGee and Harold, 2004). Flavanoids are the most important plant pigment for flower coloration producing yellow or red/blue pigmentation in petals designed to attract pollinator animals.

Figure 1.4: Structure of Flavone.

1.4.5.4 Terpenoids

Terpenoids also called “isprenoids” constitute one of the largest families of natural products encounting for more than 40,000 individual compounds of both primary and secondary metabolism. Most of them are of plant origin, and hundreds of new structures are reported every year (Sacchettini and Poulter, 1997). All organisms naturally produce some terpenoids as part of primary metabolism, but many produce terpenoids via secondary metabolism. In particular, terpenoids are contained in many herbal plants and several terpenoids have been shown to be available for pharmaceutical applications, for example, artemisinin and taxol as malaria and cancer medicine (Withers and Keasling, 2007).

Figure 1.5: Structure of Terpenoids.

1.4.5.5 Tannins

Tannins are naturally occurring plant polyphenols. The main characteristic is that they bind and precipitate proteins. They have a large influence on the nutritive value of many foods eaten by humans and feedstuff eaten by animals. Tanins are common in fruit grapes, persimmon, blueberry, in tea, in chocolate, in legume forages, in legume trees (Acacia spp., Sesbania spp., etc.), in grasses (sorghum, corn, tannin, also called tannic acid, any of a group of pale-yellow to light-brown amorphous substances in the form of powder, flakes, or a spongy mass, widely distributed in plants and used chiefly in tannin leather dyeing fabric, making ink and in various medical applications) (Souza et al., 2006). Tannin solutions are acid and have an anstrigent taste. Tannin is responsible for the astringency, colour and some of the flavour in tea. Tannins occur normally in the root, wood, bark, leaves and fruit of many plants, particularly in the bark of oak species and in sumac and myrobalan. They also occur in galls, pathological growths resulting from insect attacks (Souza et al., 2006).

Figure 1.6: Structure of tannin.

1.4.5.6 Glycosides

Glycosides are organic compounds containing glycosides (sugar) that on the contractile force of the cardiac muscle. Because of their potency in disrupting the function of the heart, most are extremely toxic. These glycosides are found as secondary metabolites in several plants, but also in some insects, such as the milkweed butterflies (singh,B.and Rastogi, R.P.1970). From ancient times humans have used glycoside-containing plants and their crude extract as arrow coating, homicidal or suicidal aids, and rat poisons heart tonics diuretics and emetics.

1.4.6 Description of the Test Organisms

1.4.6.1 Salmonella typhi

Salmonella typhi is a subspecies of Salmonella enterica, the rod shaped, flagellated, aerobic, Gram-negative bacterium. It is a member of the genus salmonella (Giannella, 1996). Many of the pathogenic serovars of the S. enterica species are in this species. Salmonella typhi causes typhoid fever,paratyphoid fever is caused by S. Paratyphi, S .schottmuelleri, and S. hirschfeldii, which are considered variant of S. enteritidis. Electrolytes may be replenishing with oral rehydration supplements (typically containing salts sodium chloride and potassium chloride). Appropriate antibiotics, such as ceftriaxone, are given to kill the bacterian. Azithromycin has been suggested to be better at treating typhoid in resistant population than both fluoroquinolone drugs and ceftriaxone (murray.et al.s, 2009).

1.4.6.2 Escherichia coli

Escherichia coli a gram negative, rod shaped bacterium that is commonly found in lower intestine of warm blooded organism (vogt, 2005). The harmless strains are part of normal flora of the gut and can benefit their host by producing vitamin K2 and by preventing the establishment of pathogenic bacteria within the intestine. E.coli and related bacteria constitute about 0.1% of gut flora, and fecal oral transmission is the major route through which pathogenic strains of the bacterium causes disease. Cells are able to survive outside the body for a limited amount of time, which makes them ideal indicator organisms to test environmental samples for fecal contamination. There is however, a growing body research that has examined environmentally persistent E.coli which can survive for extended period of time of the host.

                               

CHAPTER TWO

2.0 MATERIALS AND METHODS

2.1 Materials

2.1.1 Chemical and Reagents

Table 1.1 list of chemical & reagents

Chemicals and Reagents

Grade

%purity

Manufacturer

Hydrochloric acid

Analar

96

BDH Chemical Ltd, England

Acetic acid

Analar

BDH Chemical Ltd, England

Sulphuric acid

Analar

BDH Chemical Ltd, England

Ferric chloride

LR

98

BDH Chemical Ltd, England

Copper acetate

BDH Chemical Ltd, England

Distilled water

LR

Laboratory

Petroleum ether

Analar

BDH Chemical Ltd, England

Meyer’s reagent

BDH

BDH Chemical Ltd, England

NA2HPO4 salt

Methanol

Glucose

Haemoglobin

DNS

LR

BDH Chemical Ltd, England

2.1.2 Apparatus and Equipments

Table 1.2 list of Apparatus and Equipment

Apparatus

Volume(ml)

Types

Company

Beaker

250

Glass

Pyrex, England

Pipette

50

Glass

Pyrex, England

Measuring cylinder

500

Glass

Pyrex, England

Volumetric flask

500

Glass

Pyrex, England

Funnel

Glass

Pyrex, England

Test tubes

Glass

Pyrex, England

Muslin cloth

WRC, Chaina

Cotton wool

US,pharmacopeia America

Petri dishes

Water bath

HH-S stainless steel

England

Spectrophotometer

SMS7525

Spectrum lab 23A guflex, medical

Weighing balance 

Suntex, kontes

USA

Incubation 

DHG9101

Boekel scientific, England

Centrifuge

Refrigerator 

Haier-thermocool, Nigeria

2.2 Methods

2.2.1 Collection and Identification of Plant

A part of the parkia biglobosa leaves were obtained around the Aliero metropolis, Kebbi state. It was it was collected and authenticated by a botanist in the department of Biological Sciences, Kebbi State University of Science and Technology, Aleiro.

2.2.2 Preparation of Plant Material

The leaves of parkia biglobosa was dried at room temperature and grounded using pestle and mortar. It was kept in an air tight container for use.

2.2.3 Extraction of Plant Material

The plant extract was extracted with methanol. 100g of the grounded parkia biglobosa leaf was added to 800ml 0f methanol in a conical flask, the conical flask is then closed using foil paper  and cotton wool to avoid evaporation its content is then shaken and kept in room temperature for 72 hours. The macerate was filtered through double layer muslin cloth and the extract obtained was left to evaporate in the oven, with the marc left to dry. The dried extract were weighed and stored for future use.

2.2.4 Qualitative Phytochemical Screening

The parkia biglobosa leaf extracts were analyzed for glycosides, alkaloids, saponins, tannins, flavonoids, steroids, phenols, terpenoid and Anthraquinones using standard procedures.

2.2.4.1 Test for Glycosides: 2ml of acetic acid was added to 2ml of the extract. The mixture was cooled in cold water bath. 2ml of concentrated H2S04 was then added, colour development from blue to bluish green indicates the presence of glycosides (Valsala and Karpagaganapathy, 2002).

2.2.4.2 Test for Alkaloids: 1.27g of iodine and 2g of potassium iodide dissolved in distilled water (Wagner’s reagent), this was added to the plant extract. Appearance of reddish brown colour confirmed the presence of alkaloids (Harbone, 1998).

2.2.4.3 Test for saponins: To 2ml of the extract, 2ml of distilled water was added and agitated in a test tube for 5minutes. The formation of foams indicates the presence of saponin (Valsala and Karpagaganapathy, 2002).

2.2.4.4 Test for tannins: 5 drops of 0.1% ferric chloride was added to 2ml of extract, a brownish green or blue black coloration indicates the presence of tannins (Valsala and Karpagaganapathy, 2002)

2.2.4.5 Test for Flavonoid: 2ml of 10% Sodium hydroxide was added to 2ml of the extract in a test tube. An intense yellow colour was formed which turned colorless upon addition of 2ml of dilute hydrochloric acid indicating the presence of flavonoid (Valsala and Karpagaganapathy, 2002).

2.2.4.6 Test for Steroids: To 1ml of the extract, 0.5ml of acetic anhydride and 0.5ml chloroform were added and concentrated sulphuric acid later added. Formation of a brownish green ring at the contact of the two liquids indicates the presence of steroids (Harbone, 1998).

2.2.4.7 Test for Phenols: 2ml of the extract was mixed was mixed with ferric chloride solution. Green or dirty green precipitates indicate the presence of phenolic compounds (Elmahmood and Doughari, 2008).

2.2.4.8 Test for Terpenoids: 5ml of the extract was added to 2ml chloroform.3ml of conc.H2SO4 was carefully added to form a layer. Reddish brown coloration of the interface indicates the presence of terpenoids.

2.2.4.9 Test for Anthraquinones: 2ml of the extract were boiled with 5ml of 10% hydrochloric acid for 3minutes. 5ml of chloroform was added. 5 drops of 10% ammonia was added. A rose pink coloration indicates the presence of Antraquinones (Harbone, 1998).

2.2.3 Test for Bacteria

The tests for bacteria that are used in this study are isolates of Escherichia coli and salmonella typhi. The clinical isolate were obtained from department of microbiology, Kebbi State University of Science and Technology Aliero Kebbi state, the bacteria isolates were subculture as needed.

2.2.4 Media Preparation

The media used were Muller Hinton broth and Muller Hinton agar for antibacterial sensitivity testing. These were all prepared according to laboratory standard procedures as provided in the container label.

2.2.5 Preparation of Muller Hinton Agar

Muller Hinton agar was prepared by weighing 7.6gram of the powder on a weighing balance and transferred into 200ml on distilled water contained in a conical flask. The mixture was heated on a hot plate to dissolve the powder completely followed by autoclaving at 121C for 15mininutes. It was then allowed to cool to about 47C and poured into petri dishes for solidification. (Wolfgang and Hilda 1976).

2.2.6 Antibacterial Activity Studies

The Kirby bauser disc diffusion method was used to analyze the antibacterial effect of the leaf of parkia biglobosa.

2.2.7 Preparation of Culture and Inoculation

Pure culture of Escherichia coli and Salmonella typhi were obtained from the sample gotten from microbiology department Kebbi State University of Science and Technology Aliero Kebbi state. Muller Hinton agar, a portion of the pure culture of the test organism was picked using a sterilized wire loop and streaked in a zigzag manner on the surface of the media until all the surface were covered.

2.2.8 Agar Well Diffusion method

For Agar well diffusion method. A sterile punch was used to cut a series of wells on the surface of media in preparation arrange pattern on the agar plate, Using a sterile cork borer. The bacteria broth suspension was streaked on the medium with sterile wire loop and various and extract was put into well with 2% plane agar for solidification, the plate were incubated at 37C overnight. After 24 hours of incubation the inhibition zone diameter were determined. The concentrations used were 30, 60,90mg\ml.

CHAPTER THREE

3.0 RESULTS

The results for the phytochemical screening are presented in in Table 2.1 while those of the antibacterial activity are present in Table 2.2

Table 2.1:  Phytochemicals Constituents of parkia biglobosa Methanolic leaf Extract

Phytochemical

Observation

  Glycoside

                  + + +

Alkaloid

                    +

Flavonoid

                  + + +

Terpenoid

                  + + 

Steroids

                  + +

Phenols

                    +

Anthraquinones

                     _

Tannin

                   + + +

Saponin

                   + + 

Key: (+): present, (-): Not detected, (+ +): moderately present, (+ + +): abundantly present

Table 2.2: Antibacterial Activity of parkia biglobosa Methanolic leaf Extract

Test organisms

                         Diameter      of  Zone of inhibition (mm)

Control (0.05mg)

30mg/ml (extract)

60mg/ml (extract)

90mg/ml (extract)

Salmonella

 Typhi

22.00mm

12.00mm

16.00mm

17.00mm

Escherichia coli

25.00mm

12.00mm

13.00mm

16.00mm

NOTE: diameter of cork borer used =12mm. Positive control drug used for bacteria; ciprpfloxin

.

KEY: mm >12 = inhibition (positive)

          Mm<12 = no inhibition (negative

Figure 2.1: antibacterial activity of bacteria against control (ciprofloxacin)

Figure 2.2: antibacterial activity of salmonella with extract

Fgue 2.3: antibacterial activity of E coli with extract                           

CHAPTER FOUR

4.0 DISCUSSION, CONCLUSION AND RECOMMENDATION

4.1 DISCUSSION

In table 3.1, the phytochemical screening of parkia biglobosa methalonic leaf extract showed that the plant contains good amount of glycoside, flavonoid, terpenoid, steroids, tannin, and saponin. Alkaloid and phenols were fairly presence in the extracts. Anthraquinones however were not detected. The presence of alkaloid and flavonoid in methanolic leaf extracts may be used to explain the traditional use of this plant. Alkaloids acts as plant stimulants or regulators in activities like growth, metabolism and reproduction. The presence of flavonoid proves that parkia biglobosa is a good medicinal plant.

Table 3.2, shows the result of zone inhibition of methanolic leaf extract against the test organisms. The leaf of parkia biglobosa had zones of inhibition against E.coli and , Salmonella typhi at concentration of 30, 60 and 90mg/ml when compared with the control drug. Though with the results the concentration of the extract can’t be compared to the control drug but if the concentration is increased the zone of inhibition may increase. Parkia biglobosa is widely used in the treatment of leprosy, high blood pressure, arterial blood pressure, wound healing, indigestion might possibly contain anti-oxidant compound as additional ingredient. Thus further investigation is recommended to ascertain the traditional use of this plant.

 In literature, it has been indicated that the antibacterial activity is due to different chemical agents in the extract; including flavonoid, terpenoid and other compounds of phenolic nature or free hydroxyl group, which are classified as active antimicrobial compounds (Rojas et al 1992). These antibacterial effects of the plants were attributable to the presence of the active phytochemicals like tannins, saponin alkaloid and glycosides in the extracts (Enweren, et al., 2001, 2003). (Dapar, et al.,2007) reported that tannins form complexes with protein through hydrogen and covalent bonds as well as other hydrophobic effects which inhibit cell protein synthesis. The presence of these phytochemicals therefore has been adduced for the significant antibacterial activity of the extracts

4.2 CONCLUSION 

The phytochemical screening of methanolic stem extract of parkia biglobosa  reveal the presence of alkaloid, flavonoid, tannin, phenol, terpenoid, steroid and cardiac glycoside .Zone of inhibition observed when testing the plant extracts against E. coli, and Salmonella typhi. The results of this research is promising since the result gotten from this research proves that parkia biglobosa may be a good source of antibacterial drugs or starting material for the production of the drug.

4.3 RECOMMENDATION

The findings in the work have confirmed the activity of the plant against the tested bacteria. Therefore, to take the work to a beneficial level, the following recommendations need to be made. Further studies should be carried out to isolate, identify and purify the bioactive components of this plant. And also quantitative phytochemical analysis should be carried out on this plant.

REFERENCES

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