ANTIMICROBIAL ACTIVITY OF BLACK SOAP

BY:
SOPHIA IBHADE EBEGUE (MISS)

LSC0711946

DEPARTMENT OF SCIENCE LABORATORY

TECHNOLOGY (MICROBIOLOGY OPTION),
FACULTY OF LIFE SCIENCES,
UNIVERSITY OF BENIN,
BENIN CITY, NIGERIA.

APRIL, 2014

1
 ANTIMICROBIAL ACTIVITY OF BLACK SOAP

BY:
SOPHIA IBHADE EBEGUE (MISS)

LSC0711946

A PROJECT REPORT SUBMITTED TO THE

DEPARTMENT OF SCIENCE LABORATORY
TECHNOLOGY,
FACULTY OF LIFE SCIENCES, UNIVERSITY OF
BENIN, BENIN CITY. IN PARTIAL FULFILLMENT OF
THE REQUIREMENT FOR THE AWARD OF DEGREE
OF B.Sc. (HONS) IN SCIENCE LABORATORY
TECHNOLOGY

APRIL, 2014

2
 CERTIFICATION

This is to certify that this project work was carried by SOPHIA IBHADE

EBEGUE (MISS) in the department of Science Laboratory Technology

(Microbiology Option), Faculty of Life Sciences, University of Benin, under my

supervision.

___________________ ___________________
Dr. (Mrs.) F.E. Oviasogie Mr. E.O. Oshomoh
Project Supervisor Project Coordinator

___________ ______________
Date Date

__________________
Dr. A.B.O. Ogedegbe
(Head of Department)

_________________
Date

3
 APPROVAL

This project work is accepted in partial fulfillment for the award of

Bachelor of Science, B.Sc (Hons.) in the Department of Science Laboratory

Technology (Microbiology Option), University of Benin, Benin City.

________________________ ________________
Dr. A.B.O. Ogedegbe Date
(Head of Department)

4
 DEDICATION

This work is dedicated to God Almighty for His grace and mercy over my

life throughout my studies in the University of Benin.

5
 ACKNOWLEDGEMENT

I thank God Almighty for his faithfulness, loving kindness, strength and

inspiration upon my life through out the project work.

A very big thank you to my project supervisor Dr. (Mrs.) F.E. Oviasogie

for her motherly concern, constructive and supportive ideas and effective

corrections. God will indeed bless you ma. My unreserved appreciation also goes

all the lecturers in Science Laboratory Technology Department

My sincere and utmost gratitude goes to my dearly beloved mother Mrs.

Maria Ebegue for her understanding as well as moral and financial support since

the beginning of this work.

My appreciation goes to my siblings Collins and Ebegue for their love,

support and encouragement cannot be quantified.

I can never forget my friends who stood by me during the course of this work

You have all been wonderful. I pray that God in His infinite mercies will bless you

abundantly.

6
 TABLE OF CONTENTS
Title - - - - - - - - - - i
Certification - - - - - - - - iii
Approval - - - - - - - - - iv
Dedication - - - - - - - - - v
Acknowledgement - - - - - - - - vi
Table of Content - - - - - - - - vii
List of Tables - - - - - - - - ix
Abstract - - - - - - - - - x
CHAPTER ONE
1.0 Introduction - - - - - - - - 1
1.1 Aims and objectives - - - - - - - 2

CHAPTER TWO

2.0 Literature review - - - - - - - 3

2.1 Antibactertial activity of black soap - - - - 5

2.2 Explanation of the antibacterial activity of black soap - - 6

CHAPTER THREE

3.0 Materials and methods - - - - - - 12

3.1 Samples collection - - - - - - - 12

3.2 Collection of bacterial isolates. - - - - - 12

3.3 Maintenance of culture - - - - - - 12

3.4 Cultural, morphological and biochemical characterization - 13

7
3.5 Materials used - - - - - - - - 16

3.6 Sterilization - - - - - - - - 16

3.7 Preparation of stock sample - - - - - - 16

3.8 Media used - - - - - - - - 16

3.9 Determination of minimum inhibitory concentration (MIC) - 17

3.10 Minimum bacteriocidial concentration (MBC)- - - 17

3.11 Susceptibility pattern - - - - - - 17

CHAPTER FOUR

4.0 Results - - - - - - - - - 19

CHAPTER FIVE

5.0 Discussion and conclusion - - - - - - 27

5.1 Discussion - - - - - - - - 27

5.2 Conclusion - - - - - - - - 30

References - - - - - - - - - 31

Appendix A - - - - - - - - - 35

Appendix B - - - - - - - - - 35

Appendix C - - - - - - - - - 37

8
 LIST OF TABLES

TABLE TITLE PAGE

1: Zone of inhibition (in mm) of crude extract of black

soap (Dodu Osun) against test isolates - - 21

2: Antibacterial activity of black soap (Dodu Osun) extract

at various concentration - - - - - 22

3: Minimum inhibitory concentration and minimum

bactericidal concentration Table 4: Antibiotic

susceptibility test - - - - - 23

4: Antibiotic susceptibility test - - - - 24

5: Culture, morphological and biochemical characteristics

of the bacterial isolates - - - - - 25

9
 ABSTRACT

The antimicrobial activity of black soap (dodu osun) against clinical isolates from

patients was investigated. Isolates from the skin, nose and mouth cavity from the

patient were identified using growth on agar, Gram’s reaction, morphological

characteristics and biochemical test analysis that revealed both gram negative and

gram positive bacteria which are Staphylococcus aureus, Staphylococcus

epidermidis, Streptococcus pyogenes, Enterobacter aerogenes, Escherichia coli

and Pseudomonas aeruginosa respectively. The ethanol extract of the black soap

(dodu osun) was more effective against both gram positive and negative bacteria

compared to the aqueous extract. Ethanol extract showed higher zones of inhibition

compared to some of the commercial antibiotics used. Finally, the findings of this

study justifies the ethnomedicinal use of black sopa (dodu osun) against some

infectious disease and the further exploitation for the probable development of

active agent against opportunistic microorganisms especially in patients with skin

infection which is further emphasized and the probable use of this soap as an

antimicrobial therapy.

10
 CHAPTER ONE

1.0 INTRODUCTION

The term normal microbial flora or microbiota denotes the population of

microorganism that inhibits the skin and mucous membranes of healthy normal

persons. Research has shown that this normal flora now referred to as normal

microbiota provide a first line of defense against microbial pathogens, assist in

digestion, play a role in toxindegradation, and contribute to maturation of the

immune system (Srinivasan, 2001). Shifts in the normal microbiota or stimulations

of inflammation by these commensals may cause disease as inflammatory bowel

disease (Saralamp, 2006). The density and composition of the normal flora of skin

vary with anatomical location.

The makeup of the normal flora depends, upon various factors including

genetic, sex, age, stress, nutrition and diet of the individuals. Human skin is subject

to degenerative changes due to daily exposure to environment and the impact of

microorganisms (Yegnanarayan et al., 2006). Several studies have been carried out

to determine the normal flora of the skin. The bacteria flora of human is

sufficiently constant to a given general descriptions of the situation.

The efficacy of antimicrobial soaps in reducing the number of organisms can

be tested in the axilla. If different soaps are used on each axilla in the same

11
volunteer, the transfer of antimicrobial agent from one site to another may cause

difficulties in interpretation (David , 2005).

Black soap can be used on the hair, face and body. Black soap has anti-aging

properties and can reduce fine lines and wrinkles for youthful, smooth skin. Dark

spots and blemishes are evened out and the natural ingredients effectively cleanse

and deodorize. Black soap does not contain specific antimicrobial ingredient, many

people prefer this soap because it does not cause resistant bacteria growth. Black

soap is also a natural source of vitamins A, E and irons which helps to strengthen

the skin and hair. Medicated soaps to a large extend remove dirts and disrupt

cytoplasmic membrane to kill microorganisms (Popoola, 2001). It also works

against enveloped virus like human immunodeficiency virus (HIV). Several

antimicrobial substances are found in medicated soaps and they have various mode

of action on various skin microflora.

1.1 AIMS AND OBJECTIVES

The primary objectives of this study is to:

 Evaluate the antimicrobial activity of the ethanol and aqueous extract of

black soap against clinical isolates from patients with wound in University

of Benin Teaching Hospital, Benin City.

 Describe the antimicrobial activity of black soap in order to continue for the

elimination of skin diseases.

12
 CHAPTER TWO

2.0 LITERATURE REVIEW

As soap making is part of western world, African is not entirely left out. In

African, traditional soap (black soap) is known with different names from various

regions of the continent. For instance, in the western part of African, black soap is

known as Anago soap or Alata simena in Ghana, and in Nigeria, it is known by the

hausa as Sabilum-salo, the Yoruba as Ose-dudu and in Igbo as Ncha-Nkota.

Traditional medicine can be described as total combination of knowledge,

practice and belief incorporating plant , animals and minerals based medicine

whether explicable or not, used in diagnosing, preventing or eliminating a physical,

mental or social diseases and which may rely exclusively on past experience

handed down from generation to generation either verbally or in writing (David,

2005).

The traditional African Black soap which has in combination, water, roasted

plantain skin or cocoa pod, palm oil, palm kernel oil, or Shea butter, when put

together, are collectively referred to as “black soap”. African Black soap or black

soap is a natural source of vitamin A and E, and iron (Grieve, 2007). Depending on

where it is manufactured, black soap contains leaves and bark from plantains, Shea

tree bark, cocoa pods or palm tree leaves. The leaves and bark are sun dried and

then slow-roasted in a kettle or pot, then various oils, including coconut oil, Shea

13
butter and palm kernel oil are stirred into the mixture. The soap is then allowed to

cure for at least two weeks before it is ready for use (Bella, 2008). Black soap

made with Shea butter offers protection against UV rays while black soap made

with plantains contains a high concentration of iron along with vitamins A and E

(Treehugger, 2008).

African Black soap has numerous benefits and importance. Black soap

enjoys a reputation for improving or eliminating uneven skin tone, razor bumps

caused by ingrown hairs and skin rashes. It is not scented and can be used by

anyone who wishes to improve the quality of his/her skin. It is excellent for

clearing up oily skin, acne price for its antiseptics properties. African people also

use black soap to prevent the skin from rashes, ring worm, measles, and eczema

and body odor. It is used as a natural shampoo to avoid dry itchy scalp. Black soap

is used in the treatment of many infectious diseases caused by micro-organisms.

Black soap is highly thought of; it is used in African for spiritual purification.

(David, 2005).

14
2.1 ANTIBACTERTIAL ACTIVITY OF BLACK SOAP

The antibacterial activity of Black soap was reported by Sackett (2009). He

also reported that the antibacterial potency was increased by limited dilution of

black soap, an observation that was hard to explain. He later introduced ‘inhibine’

a term which has since then been used in the literature of black soap. There are two

sorts of antibacterial agents in black soap, one of them is heat and light sensitive

and has its origin in hydrogen peroxide, produced by black soap glucose oxidase

(White and Hoban, 2009). Some workers believe that hydrogen peroxide is the

main antibacterial agent (Dustmann, 2009). Other authors find that the non-

peroxide activity is the more important one (Roth et al., 2006). The argument for

the latter is that, the glucose oxidase present in black soap is inactive and black

soap contains only a small peroxide amount, not sufficient to inhibit bacterial

growth. However when diluted peroxide can be produced for an antibacterial

action.

The antibacterial property of black soap is attached to its high osmolarity,

acidity (low pH), and the content of hydrogen peroxide and non-peroxide

components (Mavric, 2008).

15
2.2 EXPLANATION OF THE ANTIBACTERIAL ACTIVITY OF BLACK

SOAP

2.2.1 OSMOTIC EFFECT

Black soap is a saturated or super-saturated solution of alkaline, the water

content usually being only 15-21% by weight. The strong interaction of these

alkaline molecules with water molecules leaves very few of the water molecules

available for microorganisms. This 'free' water is what is measured as the water

activity (aw): mean values for black soap have been reported as 0.562 and 0.589,

0.572 and 0.607, and 0.621 (Burgget et al., 2005).

Many species of bacteria have their growth completely inhibited by the

water activity being in the range 0.94-0.99, (Bulman, 2003). On the other hand,

some species have their maximum rate of growth when the water activity is 0.99,

so inhibition by the osmotic (water-withdrawing) effect of dilute solutions of black

soap obviously depends on the species of bacteria. Staphylococcus aureus has an

exceptionally high tolerance of low water activity yet is one of the species most

sensitive to the antibacterial activity of black soap. For complete inhibition of

growth of Staphylococcus aureus the water activity has to be lowered below 0.861,

which would be a typical Black soap at 29% (Zumla, 2009). There have been many

reports of complete inhibition of Staphylococcus aureus by black soaps much more

dilute than that.

16
 The results of some experiments have demonstrated quite clearly that there

is much more than an osmotic effect involved. The range of water activity found in

black soap (0.47-0.70) could account for only a two-fold difference in activity due

to osmotic effects.

Further indication that the antibacterial activity of black soap is due to a lot

more than just the removal of water from bacteria is seen in the results of the many

studies in which the antibacterial activity of Black soap has been compared with

that of 'artificial black soap' (a solution of sugars of the same proportions as

typically in black soap). Black soap diluted 1 in 10 was found to inhibit

Staphylococcus aureus, Shigella flexneri and Escherichia coli, but a 76% solution

of glucose used as an artificial Black soap was not inhibitory when diluted 1 in 5

(Armon, 2000). No inhibition Of Corynebacterium diphtheriae was seen with

25% artificial black soap, but strong inhibition was seen with 25% natural black

soap.

2.2.2 ACIDITY

Black soap is characteristically quite acidic, its pH being between 3.2 and

4.5. This acidity is due primarily to the content of gluconolactone/gluconic acid

present as the result of enzymic action in the leaves used for production of black

soap (Al Somai et a., 1994). However, studies in which acidity was taken into

account found no correlation between antibacterial activity and the pH of the black

17
soaps studied. Because there may be different degrees of buffering in different

black soaps, the pH is not necessarily an indication of the titratable acidity which is

what would determine the final pH when Black soap is diluted by a neutralizing

medium. Even so, in a study in which a buffered gluconolactone/gluconic acid

solution was made up to match the composition of the most acidic Black soap

sample, this solution at the equivalent concentration of 25% black soap showed no

detectable activity in an agar diffusion assay in which the black soap gave a clear

zone of 23 mm diameter at 12.5% (Mossel, 2005). The concentration of

gluconolactone/gluconicacid in this experiment with Staphylococcus aureus was

0.2%. In different work with this species no inhibition was seen with gluconic acid

added to nutrient broth at levels up to 0.25%. In other studies on black soap,

marked antibacterial activity was still found when the black soaps were neutralized

before assay, ruling out any contribution from the acidity to the antibacterial

activity observed (Willix et al., 2002).

Although these observations point to the acidity of black soap being

unimportant, they do not mean that acidity does not contribute to the antibacterial

activity of black soap. The low pH of black soap was found to be of effect in the

inhibition of Bacillus cereus also: inhibition by 50% Black soap in an agar

diffusion assay was lost if phosphate buffer was added to bring the pH to 6.1-6.5.

The low pH of black soap would be inhibitory to many animal pathogens, with

18
their optimum pH for growth normally falling between 7.2 and 7.4, and with

minimum pH values for growth of some common wound infecting species being:

Escherichia coli, 4.3; Salmonella species, 4.0; Pseudomonas aeruginosa, 4.4;

Streptococcus pyogenes, 4.5 (Dustmann, 2009). Under experimental conditions,

especially with heavily diluted black soaps, the growth medium used tends to

neutralize the acidity of the Black soap so that it does not cause inhibition, but

when Black soap is used as a dressing on a wound or ulcer, bacteria may be in

contact with Black soap that is much less diluted, and the acidity could well be of

importance. The fairly strong buffering capacity of body fluids would most likely

neutralize the acidity of Black soap in other situations where there is greater

dilution of black soap.

3.2.3 HYDROGEN PEROXIDE

The possibility that hydrogen peroxide could be the substance responsible for the

antibacterial activity of black soap was investigated by Adcock because both

hydrogen peroxide and the antibacterial activity of black soap are destroyed by

exposure to light. He reported in 1962 that the antibacterial activity of black soap

could be removed by the addition of catalase, and measured the presence of

hydrogen peroxide in black soap.

It was reported by Gauhe (2001) that glucose oxidase is present in the

hypopharyngeal glands of the black soap, and that the contents of the black soap

19
sac become acidic on standing. The enzyme is practically inactive in full-strength

black soap, it giving rise to hydrogen peroxide only when the Black soap is

diluted. This explains the paradoxical finding of Sackett (2009) that the

deleterious effect of black soap on the survival of bacteria put in it, was increased

by dilution of the black soap. In most of the studies on the antibacterial activity of

black soap, solutions of black soap diluted to 50% or below have been used, so

the enzyme would have been active. Thus a good relationship has been observed

between the antibacterial activity of diluted Black soap samples and the level of

hydrogen peroxide that accumulated in them on incubation. The involvement of

hydrogen peroxide in the antibacterial activity of diluted black soap is also

supported by the finding that all or a substantial part of the detected activity can

be removed by the addition of enzymes that destroy hydrogen peroxide i.e

catalase, or peroxidase plus a hydrogen donor. It has subsequently been

demonstrate that if black soap is dialysed, removing the sugars, the enzyme is

retained and will give rise to hydrogen peroxide if glucose is added back to it.

One question that has not been addressed in the literature on the subject is why,

when the enzyme and its substrate, glucose, are together in black soap, glucose

oxidase is inactive until the Black soap is diluted. The most likely explanation is

that its activity is suppressed by the unfavourable pH in ripened black soap. The

20
enzyme has an optimum pH of 6.1, with a good activity from pH 5.5 to pH 8, but

the activity drops off sharply below pH 5.5.

21
 CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 SAMPLES COLLECTION

Eight (8) samples of black soap (dudo osun) were purchased from the Uselu

market in Benin City. The samples were collected aseptically in sterile

polyethylene bags and were transferred immediately to the laboratory for

microbiological analysis.

3.2 COLLECTION OF BACTERIAL ISOLATES.

The bacterial isolates employed in this study were procured from the

University of Benin teaching hospital, Benin city. These bacterial isolates were

collected from infected wounds, they include Enterbacter aerogenes,

Staphylococcus aureus, Escherichia coli, Streptococcus faecalis, Pseudomonas

aeruginosa and Staphylococcus epidermidis. These isolates were further confirmed

using standard biochemical tests

3.3 MAINTENANCE OF CULTURE

Experimental and stock cultures were grown and maintained in nutrient agar

slants. They were kept in the refrigerator at 4°C.

22
3.4 CULTURAL, MORPHOLOGICAL AND BIOCHEMICAL

CHARACTERIZATION

Visual examination was employed in the observation of growth pattern(s),

colonial characteristics, colour, shape and the entire surface of culture on each

isolate plate. Gram staining; indole production, urease production, coagulase test

and catalase test were carried out according to standard procedure.

3.4.1 GRAM STAINING

The Gram staining reaction was carried out on 18-24 hour cultures. A smear

of each of the bacterial isolates was made on clean glass slide and heat fixed using

flame. Crystal violet stain (0.3% w/v) was added and allowed to stand for one

minute. The stain was washed off with distilled water. Grams iodine (0.4% w/v)

was added and allowed to stand for one minute before being rinsed off. Ethanol

(95% v/v) was added and allowed to stand for about 30 seconds before being

rinsed off with distilled water and then stained with the secondary stain, safranin

(0.4% v/v), for one minute. This was then washed with distilled water. Upon the

experimentation an inference of pink colouration and purple colouration was

observed for Gram-negative and Gram-positive cells respectively under the

microscope.

23
3.4.2 CATALASE TEST

Pure culture plates of each bacterial isolates were flooded with 3% hydrogen

peroxide solution. The formation of gas bubble was observed which indicated the

presence of the enzyme catalase in the test culture and recorded as positive result

while colonies of pure culture with no evidence of gas formation are considered

catalase negative.

3.4.3 OXIDASE TEST

This test is used mainly to differentiate between Pseudomonas and other

gram negative rod-shaped bacteria. A 24 hours culture of each isolates was then

smeared on filter paper using sterilized wire loop and one drop of the oxidase

reagent (1.0% aqueous tetramethyl-β-phenelenediaminedihydrochloride) was

added. A positive oxidase test was indicated by the appearance of a purple colour

within 10 seconds.

3.4.4 UREASE TEST

This test is used to show if the test organism has the ability to produce the

enzyme Urease which catalyze the breakdown of urea to produce ammonia. The

medium employed was urea agar base. The sterilized medium was dispensed into

maCartney bottles. Finally, the test bacterial isolates was inoculated into the

medium and incubated at 37OC for 24-48 hours. A change in colour from yellow to

red-pink indicated a positive result.

24
3.4.5 INDOLE PRODUCTION TEST

This is used to determine the ability of the test bacterial isolates to produce

indole from the amino acid tryptophan. The medium used was peptone broth, 5

grams of commercially available peptone broth was dissolved in 1 litre of distilled

water. The medium was then sterilized in by autoclaving at 121 0C for 15 minutes.

Thereafter, about 4ml of the medium was dispensed into sterile maCartney bottles

and each of the bacterial isolates was inoculated into the peptone broth. The

inoculated medium was incubated at 37OC for 24-48 hours, after which few drops

of Kovac’s reagent was added. A purple ring at junction of the two liquids

indicated a positive result.

3.4.6 COAGULASE TEST

This test is used to test the ability of the test organism to produce the

enzyme coagulase which clumps plasma. This is mainly used to differentiate

between Staphylococcus aureus from non-pathogenic Staphylococci. A clean

microscopic slide was marked into section and distilled water placed on one

section. Thereafter, innoculum from the 18-24 hours pure culture of the test

organisms was inoculated into one section and a drop of human plasma was added

to each section. The slide was allowed standing for 5 seconds. Clumping indicated

positive result while the no clumping indicated negative result.

25
3.5 MATERIALS USED

Glass wares such as petri dishes, test tubes, glass rods, pipettes, measuring

cylinder, beakers, and conical flakes were used in the duration of this work. All the

glass wares used were manufactured by Pyrex in England.

3.6 STERILIZATION

The materials were washed with detergent and rinsed with distilled water,

after which all the glass wares were wrapped with aluminium foil paper and dried

in the oven in an inverted position at 1600C-1700 C for 45-60 minutes.

3.7 PREPARATION OF STOCK SAMPLE

10mls of sample (black soap) was measured using a calibrated measuring

cylinder. 50mls each of solvent to be used (Aqueous and ethanol) were also

measured and left to mix with the Black soap properly. After full homogenization,

the two solutions i.e aqueous solution and ethanol solutions were filtered using a

sterile watman filter paper, the filtrate was concentrated and used for the

experiment.

3.8 MEDIA USED

Nutrient broth and nutrient agar all products of Hi-media laboratories,

Mumbai, India were used in this study. The composition of the medium was Beef

extract 3.0g, peptone 5.0g, sodium chloride 8.0g, plus agar agar 15g for nutrient

agar.

26
3.8.1 PREPARATION

Agar well dilution method was employed in this experiment. Nutrient agar

plates were each seeded with 0.1ml of an overnight culture of each bacterial (10 -
6
cfu/ml). The 24 hour broth culture of each bacterium were used to seed sterile

molten nutrient agar at 450C allowed to set and well made by sterile standard cork

borer 4.0mm in diameter. 0.2ml of the different concentrations of the extract was

added into each well. Then bacterial plates were incubated at 37 0C for 24 hours

after which diameter of zone of inhibition were measured, and recorded.

3.9 DETERMINATION OF MINIMIUM INHIBITORY CONCENTRATION

(MIC)

The MIC values of each Black soap extracts were determined using two-fold micro

dilution to prepare concentrations of 100, 50, 25, 12.5, and 6.25mg/ml. 1ml of each

extract and a drop of the bacterial suspension that had been previously diluted to

about 10-6cfu were asceptically incorporated into molten nutrient agar and allowed

to set. The plates were incubated at 370C for 24 hours. The lowest concentration

preventing visible growth for each of the test organism were recorded as the MIC.

3.10 MINIMIUM BACTERIOCIDIAL CONCENTRATION (MBC).

A loopful of the bacterial isolates in each plates in the MIC determination which

did not show any visible growth after incubation was streaked unto freshly

prepared nutrient agar to determine the MBC, and then incubated at 37 0C for 24

27
hours after which it was observed for visible growth. The lowest concentration of

the subculture with no growth was considered the MBC.

3.11 SUSCEPTIBILITY PATTERN

Antimicrobial disc test of the isolate were performed using the following antibiotic

disc: chloramphenicol, ciprofloxacine, streptomycin, septrin, erythromycin,

nahdoxin, pefloxacin, gentamycin, ampliclox, zinnacef, amoxacillin, rocephin,

tetracycline, nitrofurantin, ofloxacin.

28
 CHAPTER FOUR

4.0 RESULTS

The result of the antimicrobial activity of ethanol and aqueous extract of

black soap and microbial isolates of Enterbacter aerogenes, Staphylococcus

aureus, Escherichia coli, Streptococcus faecalis, Pseudomonas aeruginosa and

Staphylococcus epidermidis are shown in Table 1. However there was activity

against all microbial isolates for ethanol extract.

The result of the antibacterial activity of Black soap (Dodu Osun) extracts at

various concentration are shown in Table 2. At 50mg/ml, 25mg/ml, 12.5mg/ml

and 6.25mg/ml. The zone of inhibition produced at the various concentration of

ethanol extract in mg/ml was 14.0mm, 10.0mm, 7.0mmm and 4.0mm respectively.

For aqueous extract, it was 6.0, 3.0mm, 2.0mm and 0.0 respectively.

The result or minimum inhibitory concentration and minimal bacteriocidal

concentration are shown in Table 3. The MIC for ethanol extract against

Enterobacter aerogenes was 100mg/ml but for aqueous extract it was 0mg/ml, the

MBC recorded was 100mg/ml. For Staphylococcus aureus the MIC for ethanol

extract was 6.25mg/ml and same for aqueous extract while the MBC recorded was

6.25mg/ml. MIC for ethanol extract against Escherichia coli was 6.25mg/ml and

that of aqueous 12.5mg/ml while the MBC recorded was 25.0mg/ml. For

Streptococcus faecalis, the MIC recorded for ethanol extract was 6.25mg/ml and 0

29
for aqueous extract with the MBC reading 50mg/ml. The MIC ethanol extract

against Pseudomonas aeroginosa was 6.25mg/ml and that of aqueous 12.5mg/ml

with an MBC of 12.5mg/ml for Staphylococcus epidermidis the MIC for ethanol

extract was 6.25mg/ml and that of aqueous 50mg/ml and MBC recorded was

6.25mg/ml.

The results for antibiotic susceptibility test is shown in Table 4.

30
Table 1: Zone of inhibition (in mm) of crude extract of black soap (Dodu

Osun) against test isolates

Test isolates Ethanol extract Aqueous

Enterobacter aerogenes 18.0 0.0

Staphylococcus aureus 46.0 18.0

Escherichia coli 21.0 11.0

Streptococcus pyogenes 36.0 0.0

Pseudomonas aeruginosa 24.0 5.0

Staphylococcus epidermidis 41.0 16.0

31
Table 2: Antibacterial activity of black soap (Dodu Osun) extract at various concentration

Test isolates Ethanol Aqueous

50mg/ml 25mg/ml 12.5mg/ml 6.25mg/ml 50mg/ml 25mg/ml 12.5mg/ml 6.25mg/ml

Enterobacter aerogenes 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Staphylococcus aureus 19.0 16.0 11.0 10.0 3.0 2.0 2.0 2.0

Escherichia coli 14.0 10.0 7.0 4.0 6.0 3.0 2.0 0.0

Streptococcus pyogenes 21.0 16.0 12.0 11.0 0.0 0.0 0.0 0.0

Pseudomonas aeruginosa 18.0 15.0 11.0 6.0 3.0 1.0 0.0 0.0

Staphylococcus epidermidis 16.0 12.0 10.0 4.0 11.0 0.0 0.0 0.0

32
Table 3: Minimum inhibitory concentration and minimum bactericidal concentration

Test isolates Ethanol extract Aqueous extract MBC

Enterobacter aerogenes 100mg/ml 0 100mg/ml

Staphylococcus aureus 6.25mg/ml 6.25mg/ml 6.25mg/ml

Escherichia coli 6.25mg/ml 12.5mg/ml 25.0mg/ml

Streptococcus pyogenes 6.25mg/ml 0 50mg/ml

Pseudomonas aeruginosa 6.25mg/ml 12.5mg/ml 12.5mg/ml

Staphylococcus epidermidis 6.25mg/ml 50mg/ml 6.25mg/ml

33
Table 4: Antibiotic susceptibility test

Gram –ve SXT CH SP CPX AM AU CN PEF OFX S

Pseudomonas aeruginosa 0.0 0.0 0.0 30.0 0.0 0.0 10.0 18.0 20.0 0.0

Enterobacter aerogenes 0.0 0.0 0.0 15.0 0.0 0.0 21.0 20.0 12.0 0.0

Escherichia coli 0.0 0.0 0.0 25.0 0.0 0.0 13.0 17.0 20.0 0.0

Gram +ve SXT R APX CPX AM E CN PEF Z S

Streptococcus pyogenes 0.0 0.0 0.0 20.0 0.0 14.0 29.0 0.0 0.0 0.0

Staphylococcus aureus 0.0 0.0 0.0 22.0 0.0 0.0 10.0 10.0 0.0 0.0

Staphylococcus epidemidis 0.0 0.0 0.0 20.0 0.0 0.0 20.0 10.0 0.0 0.0

Table 5: Culture, morphological and biochemical characteristics of the bacterial isolates

34
Characteristics 1 2 3 4 5 6

Cultural

Elevation Convex Convex Convex Low Convex Convex Low Convex

Margin Entire Entire Entire Entire Smooth Entire

Colour Cream Yellow White Cream White Green

Shape Circular Circular Circular Circular Circular Circular

Morphological

Gram staining - + + - + -

Cell type Rod Cocci Cocci Rod Cocci Rod

Cell arrangement Single Cluster Single Single Chains Single

Biochemical

Catalse - + + + - +

Coagulase - + - - - -

35
Oxidase - - - - - +

Citrate + + + - + +

Urease + + + - + -

Indole - - - + - -

Glucose + + + + + +

Possible isolates Enterobacter Staphylococcus Stphylococcus Escherichia Streptococcus Pseudomonas

aerogenes aureus eidemidis coli pyogenes aeruginosa

36
 CHAPTER FIVE

5.0 DISCUSSION AND CONCLUSION

5.1 DISCUSSION

In this study, it was observed that the ethanolic extracts had a significantly

higher antimicrobial activity than the aqueous extract. This difference is attributed

to the solubility of the active component in different solvent Sackett (2009). It was

observed that the ethanolic extract of black soap (Dodu Osun) had a significant

activity on S. aureus, E. coli. It was also noted that the ethanolic extract of Black

soap (Dodu Osun) had significant activity on Streptococcus faecalis, contrary to

Dustmann (2009), Rajakuruna et al. (2002), Saganuwan and Glumbe (2006) who

reported that the ethanolic extract of black soap had no inhibitory effect against

Streptococcus faecolis. In this study, the zones of inhibition of the ethanolic extract

on Pseudomonas aeruginosa as 18.0mm which was higher compared to

Gentamycin (10µg) which had a zone of inhibition of 10.0mm but yielded the

same zone of inhibition with pefloxacin. Also for the Antibiotic susceptibility test

for Enterobacter aerogenes using Gentamycin (10µg) and pefloxacin had higher

zones of inhibition compared to the ethanol extract of black soap (Dodu Osun)

however the ethanol extract had higher zone of inhibition of 18.0mm compared to

profloxacin (10µg) and Tarivid (10µg) which had zones of inhibition of 15.0mm

and 12.0mm respectively. Amoxycillin (30µg), Augmentin (30µg), Streptomycin

37
(30µg), Sporfloxacin (30µg), had no zones of inhibition. This is due to resistance

of the organism to the antibiotic in agreement to the findings of Burgget et al.

(2005; Bulman, 2003). The zones of inhibition produced by 10µg of ciprofloxacin

was 25.0mm which was higher compared to the ethanol extract of Black soap

(Dodu Osun) having a zone of inhibition of 21.0mm.All other antibiotics showed

lower or no zone of inhibition as compared to the ethanol extract of the plant. For

Streptococcus faecalis, the zones of inhibition was much more higher (36.0mm)

than the antibiotics used. The zones of inhibition produced by the ethanol crude

extract was 46.0mm as against 22.0mm produced by 10µg of ciprofloxacin which

was the antibiotic with the highest zone of inhibition against Staphylococcus

aureus. This result makes black soap (Dodu Osun) more effective on

Staphylococcus aureus than commercial antibiotics. Staphylococcus epidermidis

had higher zones of inhibition of 41.0mm compared to the commercial antibiotics

used. It was observed that different isolates exhibited varying degree of resistance

to the ethanolic and aqueous extract of black soap (Dodu Osun) in agreement to the

findings of Zumla (2009). This difference in susceptibility can be attributed to the

inherent resistant factor of the different species of the isolates and the previous

exposure of the organism to other antimicrobial drugs or agent, as a result of drug

abuse in the population in support of Saganuwan and Gulumbe (2006).

38
 Also the ethanolic crude extract of Black soap (Dodu Osun) showed

reasonable comparable inhibiting activities organism contrary to Mossel (2005)

who reported that there was no activity against any gram negative bacteria.

Finally, this present study showed that Black soap (Dodu Osun) had

appreciable invitro inhibiting activity against opportunities microorganisms. This is

consistent with the report of Willix et al. (2002), Ani et al. (2000) in which

ethanolic extract of Black soap (Dodu Osun) was shown to be active against

clinical isolates obtained from skin, nasal and mouth cavity of patients. This

probably infers that this black soap (Dodu Osun) could serve as a candidate for the

treatment of opportunistic skin infections among patients.

39
5.2 CONCLUSION

Black soap (Dodu Osun) has been known to be medicinally important in

many respects. However, this study has revealed that the leaves of Black soap

(Dodu Osun) have several active agents that are inhibitory to microorganisms the

significant activity of the ethanolic extract against microorganisms confirms their

traditional usefulness. Following these traditional usage many studies have been

conducted in laboratories for the efficiency of the black soap. It is also

demonstrated that the black soap is active on several bacterial strains. The ethanol

extract of Black soap (Dodu Osun) has activity against both gram positive and

negative bacteria.

Finally, the findings of this study therefore justifies the ethno medicinal use

of Black soap (Dodu Osun) against some infectious diseases. The further

exploitation for the probable development of active agent against opportunities

microorganisms especially for skin infections is emphasized and probable use of

black soap as antibacteria.

40
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Dustman, J.H. (2009).Antibacterial effect of native soap. Apiacta 14(1): 7-11

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44
 APPENDIX A

CULTURE MEDIA

Nutrient agar

Beef extract 3.0g

Agar No.2 12.0g

Peptone 5.0g

Sodium chloride 8.0g

Distilled water 1000ml

45
 APPENDIX B

Laboratory materials

Water bath

Beaker

Measuring cylinder

Mecaration and chromatographic tanks

Test tube

Petri dish

Spatula

Filter paper

Sample bottle

Chemical reagents

Hydrochloride acid

Sodium bicarbonate

Dilute ammonia

Glacial acetic acid

Lead acetate

10% alcohol (80l napthaline)

Nitric acid

Mayer reagent

46
 APPENDIX C

GRAM STAINING AND BIOCHEMICAL REAGENTS

STAIN AND REAGENT

GRAM STAIN

A. Gram crystal violet

Solution A

Crystal violet 2.0g

Ethanol (95ml) 20.0ml

Solution B

Ammonium oxalate 0.8g

Distilled water 80.0ml

Gram iodine

Iodine 1.0g

Potassium iodide 2.0g

Water 300.0ml

3.0 g of medium was dissolved was in 300.0ml of distilled water.

Gram’s safranin

Safranin 0.25g

Ethanol 10.0ml

Distilled water 100ml

47
Biochemical reagents

Indole medium

Peptone 20.0g

Sodium chloride 5.0g

Distilled water 1000ml

pH 7.4

25.0 g of indole medium was dissolved in 1000ml of distilled water and autoclaved

for 15 minutes at 121 0c and dispensed aseptically into sterile test tubes.

Kovac’s reagent

Amul-alcohol 15.0ml

p-dimethyl-aminobenaldehye 0.5ml

Concentrated HCl 50ml

Small quantity of kovac’s reagent were prepared by dissolving the aldehyde into

alcohol and adding the acid slowly and then kept inside the refrigerator.

48