ANTIBACTERIAL ACTIVITIES OF THREE MEDICINAL PLANTS ON 

ORGANISMS ASSOCIATED WITH DENTAL PLAQUE 

 

 

 

 

CLEMENT ELESESHIE NYADROH 

                                                            (10396410) 

 

 

 

 

 

 

 

THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON 

IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD 

OF MASTER OF PHILOSOPHY (MPHIL) DEGREE IN MICROBIOLOGY 

 

 

 

JULY 2014 

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DECLARATION 

It is hereby declared that the work in this thesis is original and was carried out by the 

student and supervised by the supervisors below. Work from other authors where cited 

have been duly acknowledged. This work has not been concurrently submitted in 

candidature for any degree. 

 

Student:     

 

Signature: _____________________Date:_____________________ 

Clement Eleseshie Nyadroh 

 

Principal Supervisor:  

Signature: _____________________Date:_______________________ 

Prof. Mercy J. Newman 

Department of Microbiology, University of Ghana Medical School, College of Health 

Sciences, University of Ghana.  

 

 

Co-Supervisor: 

 

Signature: ______________________Date: ________________________ 

Dr. Elizabeth S. Bannermann 

Department of Microbiology, University of Ghana Medical School, College of Health 

Sciences, University of Ghana. 

 

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ABSTRACT 

The antimicrobial properties of plants have shown promise for development of new 

drugs which might aid to overcome the increasing challenge of resistance and also the 

toxicity of the currently available antibiotics.  Hence, the aim of the study was to 

investigate the antibacterial activities of Sida acuta, Anthocleista nobilis, and 

Zanthozylum leprieurii on clinical isolates of Streptococcus mutans and Streptococcus 

sanguis and two reference strains: Streptococcus mutans (ATCC 700610) and 

Streptococcus sanguis (ATCC 10556) which are known to cause dental caries and 

periodontal diseases. 

The plants were selected based on existing traditional medicine knowledge, usage and 

interaction with herbal healers. The antibacterial activity of aqueous and ethanol extract 

was determined by agar-well diffusion method. Subsequently, Minimum Inhibitory 

Concentration (MIC) was determined by using macro broth dilution method at a 

concentration ranging between 200mg/ml and 3.125 mg/ml. Minimum Bactericidal 

Concentration (MBC) was obtained by sub-culturing the test dilution which showed no 

visible turbidity. Standard antibiotic penicillin (1.5i.u) and gentamicin (10µg) were 

used for comparison. Both the aqueous and ethanolic extract of the selected medicinal 

plants showed inhibition zones which did not differ significantly (P>0.05) against each 

tested bacteria. Among the plant parts tried on the microorganisms, the leaves of Sida 

acuta proved to be more potent than the stem bark of Anthocleista nobilis, followed by 

the stem bark of Zanthozylum leprieurii. The ethanolic extract of Anthocleista nobilis 

was more efficient in its antibacterial activity as compared to its aqueous extract. The 

inhibitory activity of Anthocleista nobilis stem bark against Streptococcus mutans was 

comparatively more than that of gentamicin (10µg) but less than penicillin (1.5i.u). The 

aqueous and ethanolic extract of Zanthozylum leprieurii did not have inhibitory activity 

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on Streptococcus sanguis strains. The ethanolic extract of Zanthozylum leprieurii 

showed better results as compared to the aqueous ones. The antibacterial activity of all 

the plant extracts were concentration dependent, increasing with increasing 

concentration (P<0.05). The results from the study support the ethnomedicinal use of 

the plants and suggest that the plants extracts have compounds with antibacterial 

properties that can be used, as phytotherapeutic agents in the developments of new 

drugs. Sida acuta and Anthocleista nobilis showed promising antibacterial activities and 

thus can be employed as an effective anti-plaque agent and can be used in the 

prevention of dental caries. 

  

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DEDICATION 

This work is dedicated to God without whom I am nothing, and also to my parents, Mr. 

Helmut K. Nyadroh and Mrs. Lucy Nyadroh, my supervisors, Prof. Mercy J. Newman 

and Dr. Elizabeth S. Bannermann for their encouragement, invaluable support, prayer, 

and love. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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ACKNOWLEDGEMENT 

I am grateful to the Most High God for his abundant Grace and Mercy that has 

sustained me, making it possible for me to complete this journey successfully. Had it 

not been Him I never would have been where I am today. To Him I give all the glory. 

My deepest appreciation goes to my supervisors, Prof. Mercy J. Newman, Dr. Elizabeth 

S. Bannerman and Dr. Daniel Tormeti for their supervision, insight, guidance and 

invaluable contribution throughout the research study. They proved indefatigable 

working around the clock to make my research the best. God richly bless you. My 

sincere appreciation goes to Dr. Mary Adamafio and Dr. Emma Vasco of the University 

of Ghana Dental School (UGDS) for their assistance rendered during the sample 

collection. May God shower you with his abundant blessings. I am much indebted to 

the staff of the Microbiology Department, Centre for Plant Medicine Research (CPMR) 

especially Dr. Daniel Boamah, Sylvester Kaminta, Salomey Acheampong, Ekow, and 

Doreen, for the assistance given me during the practical work in the laboratory. 

I am also thankful to Mr. Frederick Ayertey of the Pharmacology laboratory, Centre for 

Plant Medicine Research (CPMR) and Mr. Shadrack A. Coffie, Laboratory Manager, 

Biotechnology center, University of Ghana for their heartwarming and tireless 

assistance which made the whole thesis a wonderful piece. I am very grateful to Dr. 

Theophilus Adiku, Head of Microbiology Department, University of Ghana Medical 

School, Prof. Kingsley Twum-Danso, Dr. Eric S. Donkor and Dr. Simon K. Attah for 

their encouragement during the research, May God grant you all your heart desires. I 

am also very grateful to the Director, Staff and entire management of Centre for Plant 

Medicine Research (CPMR) for concerns and guidance that have helped me throughout 

this work. 

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Finally, I express my heartfelt appreciation to my father; Mr. Helmut K. Nyadroh and 

mother Mrs. Lucy Nyadroh for their love and support, there are no words to express 

how your words in season changed my life, kept me in check and helped me through 

this journey. May the Almighty God never turn His face away from you and may you 

increase in knowledge of him. 

 

 

 

 

 

 

 

  

 

 

 

 

 

 

 

 

 

 

 

 

  

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TABLE OF CONTENTS 

Content                      Page 

DECLARATION ............................................................................................................... i 

ABSTRACT ..................................................................................................................... ii 

DEDICATION ................................................................................................................ iv 

ACKNOWLEDGEMENT ................................................................................................ v 

TABLE OF CONTENTS ............................................................................................... vii 

LIST OF TABLES .......................................................................................................... ix 

LIST OF FIGURES .......................................................................................................... x 

LIST OF ABBREVIATIONS ......................................................................................... xi 

CHAPTER ONE ............................................................................................................... 1 

1.0 INTRODUCTION ...................................................................................................... 1 

1.1 BACKGROUND ........................................................................................................ 1 

1.2 PROBLEM STATEMENT......................................................................................... 3 

1.3 JUSTIFICATION ....................................................................................................... 4 

1.4 AIM AND OBJECTIVES .......................................................................................... 4 

1.5 SPECIFIC OBJECTIVES .......................................................................................... 4 

CHAPTER TWO .............................................................................................................. 5 

2.0 LITERATURE REVIEW ........................................................................................... 5 

2.1 Dental Plaque .............................................................................................................. 5 

2.1.1 Mechanism of Dental Plaque Formation ............................................................. 7 

2.2 Streptococcus mutans ................................................................................................. 9 

2.3 Streptococcus sanguis .............................................................................................. 10 

2.4 Herbal Medicine ....................................................................................................... 11 

CHAPTER THREE ........................................................................................................ 17 

3.0 MATERIALS AND METHODS ............................................................................. 17 

3.1 MATERIALS ........................................................................................................... 17 

3.1.1 STUDY SITE AND POPULATION ................................................................. 17 

3.1.2 STUDY DESIGN .............................................................................................. 17 

3.1.3 INCLUSION CRITERIA .................................................................................. 18 

3.1.4 EXCLUSION CRITERIA ................................................................................. 18 

3.1.5 INFORMED CONSENT ................................................................................... 18 

3.1.5 SAMPLE COLLECTION, TRANSPORT, AND STORAGE .......................... 18 

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3.1.6 TEST MICROORGANISMS AND CONTROL ANTIBIOTICS ..................... 19 

3.2 METHODS ............................................................................................................... 19 

3.2.1 Extract Preparation ............................................................................................ 19 

3.2.2 Isolation of bacteria from clinical specimen ...................................................... 20 

3.2.3 Culture and Biochemical Identification of the Microorganisms ....................... 20 

3.2.3 ANTIMICROBIAL ASSAY ............................................................................. 21 

3.2.3.1 Agar-well diffusion method ........................................................................ 21 

3.2.3.2 Determination of Minimum Inhibitory Concentration (MIC) and Minimum 

Bactericidal Concentration (MBC) of the extracts .................................................. 22 

3.2.3.3 Statistical Analysis .......................................................................................... 22 

CHAPTER FOUR .......................................................................................................... 24 

4.0 RESULTS ................................................................................................................. 24 

CHAPTER FIVE ............................................................................................................ 31 

5.0 DISCUSSION ........................................................................................................... 31 

CHAPTER SIX .............................................................................................................. 35 

6.0 CONCLUSIONS AND RECOMMENDATIONS ................................................... 35 

6.1 CONCLUSIONS ...................................................................................................... 35 

6.2 RECOMMENDATIONS ......................................................................................... 36 

REFERENCES ............................................................................................................... 37 

APPENDICES ................................................................................................................ 57 

Appendix 1: Independent sample test comparing the zone of inhibition between 

aqueous and ethanolic extract of Sida acuta .................................................................. 57 

Post- Hoc ........................................................................................................................ 58 

Appendix 2: Independent sample test comparing the zone of inhibition between 

aqueous and ethanolic extract of Anthocleista nobilis ................................................ 60 

Post- Hoc ........................................................................................................................ 61 

Appendix 3: Independent sample test comparing the zone of inhibition between 

aqueous and ethanolic extract of Zanthozylum leprieurii ........................................... 63 

Post- Hoc ........................................................................................................................ 64 

Appendix 4: Consent Form ......................................................................................... 66 

Appendix 5: The Pictures of Selected Medicinal Plants ............................................. 69 

Appendix 6: Ethical Clearance ................................................................................... 70 

 

 

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LIST OF TABLES 

Table 1: The Ethnomedicinal Uses of the Selected Plants ............................................................ 17 

Table 2: Antimicrobial activity of aqueous extract of Sida acuta against the test 

organisms ...................................................................................................................... 49 

Table 3: Antimicrobial activity of aqueous extract of Anthocleista nobilis against the 

test organisms ................................................................................................................ 50 

Table 4: Antimicrobial activity of aqueous extract of Zanthozylum leprieurii against the 

test organisms ................................................................................................................ 51 

Table 5: Antimicrobial activity of ethanolic extract of Sida acuta against the test 

organisms ...................................................................................................................... 52 

Table 6: Antimicrobial activity of ethanolic extract of Anthocleista nobilis against the 

test organisms ................................................................................................................ 53 

Table 7: Antimicrobial activity of ethanolic extract of Zanthozylum leprieurii against 

the test organisms .......................................................................................................... 54 

Table 8: The MIC values in mg/ml of the plant extracts in Mueller-Hinton Broth over a 

24 hour period ............................................................................................................... 55 

Table 9: The MBC values in mg/ml of the plant extracts on Mueller-Hinton Agar ..................... 56 

 

 

 

 

 

 

 

 

 

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LIST OF FIGURES 

Figure 1:  Antimicrobial Activity of Sida acuta Leaf Extracts on the Test Organisms ..... 26 

Figure 2: Antimicrobial Activity Anthocleista nobilis Stem bark Extract on the Test 

Organisms .......................................................................................................... 27 

Figure 3: Antimicrobial Activity of Zanthozylum leprieurii Stem bark Extract on the 

Test Organisms .................................................................................................. 28 

Figure 4: Minimum Inhibitory Concentration (MIC) of the Plant Extracts on Test 

Organisms .......................................................................................................... 29 

Figure 5: Minimum Bactericidal Concentration (MBC) Values of Respective MIC 

Concentrations of the Plants extracts on Test Organisms .................................. 30 

Figure 6: A Mean Plot of Zone of Inhibition against the Concentration of Sida acuta ..... 59 

Figure 7: A Men Plot of Zone of Inhibition against the Concentration of Anthocleista 

nobilis ................................................................................................................. 62 

Figure 8: A Mean Plot of Zone of Inhibition against the Concentration of Zanthozylum 

leprieurii............................................................................................................. 65 

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LIST OF ABBREVIATIONS 

ANOVA              Analysis Of Variance 

ATCC                  American Type Culture Collection 

API                     Analytical Profile Index 

CLSI                   Clinical and Laboratory Standards Institute 

CPMR                 Centre for Plant Medicine Research 

DMSO                 Dimethyl Sulphoxide 

LSD                     Least Significant Difference 

MBC                    Minimum Bactericidal Concentration 

MIC                     Minimum Inhibitory Concentration 

SPSS                   Statistical Package for Social Sciences 

UGDS                 University of Ghana Dental School 

WHO                  World Health Organization 

  

 

 

  

 

  

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CHAPTER ONE 

1.0 INTRODUCTION 

1.1 BACKGROUND 

Traditional medicine may be defined as ―the medicine that refers to health practices, 

approaches, knowledge and beliefs incorporating plant, and animal and mineral based 

medicines, spiritual therapies, manual techniques and exercises, applied singularly or in 

combination to treat, diagnose and prevent illnesses or maintain well-being‖ (WHO, 

2006). Traditional medicines have been described by the World Health Organization as 

one of the surest means to achieve total health care coverage of the world‘s population. 

In spite of the sidelining of traditional medicine practices in the past, current attention 

has given it a new drive into research, investments, design of programmes in this field 

in several developing countries (Bodeker, 1994). The World Health Organization 

(WHO) estimates that up to 80% of the world‘s people rely on plants for their primary 

health care (Fennell et al., 2004). Despite the exceptional progress in synthetic organic 

chemistry of the 20th century, over twenty fifth of prescribed medicines in developed 

countries are derived directly or indirectly from plants (Newman et al., 2000). Ghana 

today has dual systems of medical practice in law. These are the traditional and modern 

medical practices and are promoted to co-existence in order to reach the greatest 

number of citizens (Abbiw, 1990). Traditional medicines play a vital role where 

approximately 70% of the population uses it services (Abbiw, 1990). These services 

serve as forerunners in the primary medical care of the population due to their 

accessibility and affordability to the vast rural populace (Abbiw, 1990). Herbal 

remedies used in folk medicine provide a remarkable and still largely unexplored 

source for the development of new drugs for chemotherapy which might aid to 

overcome the increasing challenge of resistance and also the toxicity of the currently 

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available antibiotics (Saied et al., 2003). It is therefore important to carry out screening 

of these plants in order to authenticate their use in folk medicine and to reveal the active 

principles present (Saied et al., 2003). It is in this context that the leaves of Sida acuta 

and stem bark of Anthocleista nobilis and Zanthozylum leprieurii were screened for 

antibacterial activity against clinical isolates of Streptococcus mutans and 

Streptococcus sanguis, which are known to cause dental caries and periodontal diseases 

(Petersen et al., 2005). 

Sida acuta belongs to the family Malvaceae. This shrub is 30 to 100cm in height, with a 

strong taproot, stem and branches flattened at the extremities (Swarbrick, 1997). The 

leaves are slender, alternate, lancoelate, and acute. The leaves are 1.2 to 9 cm or more 

long and about 0.5 to 4 cm wide in diameter, with a pair of stipules (Swarbrick, 1997). 

Anthocleista nobilis belongs to the family Gentianaceae. It is a small to medium sized 

tree up to 18m tall; the bark is smooth, pale grey, inner bark cream yellow and 

glandular. Leaves are opposite and crowed at the end of branchelets (Olatunji, 1983). 

Zanthozylum leprieurii is a tree of about 24 metres high with large thorns. It belongs to 

the family, Rutaceae. The leaves are compound with reddish translucent glands 

scattered over the surface. The flowers are white in panicle inflorescences with lateral 

branches spike-like, and its fruits are bright orange to red with glandular pitted surface 

(Olatunji, 1983). 

 

 

 

 

 

 

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1.2 PROBLEM STATEMENT 

The increasing resistance of pathogenic bacteria to currently used chemotherapeutics 

and antibiotics, opportunistic infections in immunocompromised individuals and 

financial considerations in developing countries have necessitated the need for a global 

alternative prevention treatment options and products of oral diseases that are safe, 

effective and are affordable (Tichy and Novak, 1998). 

Despite several agents being commercially available, these chemicals can change oral 

micro-biota and have adverse side-effects such as vomiting, diarrhoea and tooth 

staining (Park, et al; 2003). For example, bacterial resistance to most (if not all) of the 

antibiotics commonly used to treat oral infections (penicillin and cephalosporin, 

erythromycin, tetracycline and derivatives and metronidazole) has been documented 

(Bidault and Grenier, 2007). In addition antibacterial agents such as cetylpyridinium 

chloride, chlorohexidine, and amine fluorides are reported to cause toxicity, teeth 

staining, and oral cancer (Knoll-Kohler and Stiebel, 2002). Hence, the search for 

alternative products continues and natural phytochemicals isolated from plants used in 

traditional medicine are considered as good alternatives to synthetic chemicals (Prabu 

et al; 2006). The three medicinal plants are selected for antimicrobial assay based on 

their ethnomedicinal and traditional use against infectious diseases, and interaction with 

herbal healers. 

 

 

 

 

 

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1.3 JUSTIFICATION  

The continual emergence of resistance to synthetic antimicrobial agents has 

necessitated the need for the search of alternative means of treating infectious diseases. 

Herbal medicine is one of the areas that have shown to be a useful alternative. The 

study evaluated the antibacterial activities of Sida acuta, Zanthozylum leprieurii, and 

Anthocleista nobilis against clinical isolates of Streptococcus mutans and Streptococcus 

sanguis which are known to cause dental plaque. These would give an insight into their 

usage pharmaceutically as phytotherapeutic agents for the prevention and treatment of 

oral diseases, which may provide alternatives to synthetic antibiotics. 

1.4 AIM AND OBJECTIVES 

To investigate the antibacterial activities of the extracts of Sida acuta, Zanthoxylum 

leprieurii and Anthocleista nobilis on Streptococcus mutans and Streptococcus sanguis 

isolated from dental plaque of patients at the University of Ghana Dental School 

(UGDS). 

1.5 SPECIFIC OBJECTIVES 

 To evaluate and compare the antibacterial activity of the aqueous and ethanolic 

extracts of Sida acuta, Zanthoxylum leprieurii and Anthocleista nobilis on 

Streptococcus mutans and Streptococcus sanguis isolated from dental plaque of patients 

at the University of Ghana Dental School. 

 To determine the Minimum Inhibitory Concentration (MIC) and Minimum 

Bactericidal Concentration (MBC) of the extracts of Sida acuta, Zanthoxylum leprieurii 

and Anthocleista nobilis against Streptococcus mutans and Streptococcus sanguis 

isolated from dental plaque of patients at the University of Ghana Dental School. 

 

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CHAPTER TWO 

2.0 LITERATURE REVIEW 

2.1 Dental Plaque 

Dental plaque found on the tooth surface generally refers to the different microbial 

community (predominantly bacteria) embedded in a matrix of polymers of bacterial and 

salivary origin (Scheie, 1994). Dental plaque develops naturally on teeth and helps in 

preventing the colonization of the enamel by exogenous organisms by forming part of 

the host defense systems (Scheie, 1994). Dental caries and periodontal diseases are 

prevalent worldwide. Dental caries affects 60-90% of school-aged children as well as 

the vast majority of adults in most industrialized countries (Petersen et al; 2005). The 

cost of treating dental caries is very expensive for government of both developing and 

developed countries. In industrialized countries the cost of treating dental caries cost 

between 5% to 10% of total health care expenditures exceeding the cost of treating 

cardiovascular diseases, cancer and osteoporosis (Sheiham, 2001). The prevalence rate 

of dental caries is high in most developing low-income countries where more than 90% 

of caries is untreated (Wang, 2002). Oral health is essential to general well-being and 

relates to the quality of life that extends beyond the craniofacial complex. There is 

significant evidence linking poor oral health to chronic conditions, systemic diseases, 

such as cardiovascular diseases, rheumatoid arthritis and osteoporosis. These have also 

been linked with poor oral health and also lead to pregnancy complications such as 

preterm low birth weight (Yeo et al; 2005). Tooth loss due to poor periodontal health 

affect about 20% of adult globally, can result in morbidity and premature death. Dental 

plaque, an example of biofilm, has a greater risk of causing diseases because it is 

usually found at protected and stagnant surfaces (Scheie, 1994). The microorganisms 

that form plaque are mainly Streptococcus mutans, Streptococcus sanguis, and 

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anaerobes (Garcia, et al; 2008).  Dental plaque is primarily composed of 

microorganisms and one gram of plaque (wet weight) contains approximately 10
11

 

bacteria (Petersen et al., 2005). More than 500 distinct species of microbial species are 

found in dental plaque (Scheie, 1994).  An individual can harbor 150 or more distinct 

species of microorganisms (Scheie, 1994). Studies estimate that about 25,000 species of 

bacteria are found in the oral cavity out of which 1,000 can exist as part of the dental 

biofilm ecosystem (Donlan and Costerton, 2002). The species abundance and 

biodiversity of microorganisms is due to the ecological factors provided by the mouth 

(Marsh et al., 2003). The main ecological factors are pH, saliva, Redox reactions and 

temperature (Marsh et al., 2011). The microorganisms prefer a pH of 7 (Neutral pH 

level). Saliva acts as a buffer, maintaining the pH at a range of 6.75 and 7.25 and also 

serves as source of nutrients for the microorganisms (Marsh et al., 2011). Gingival 

crevicular fluid also serves as nutrient source for the microorganisms (Marsh et al., 

2011). The normal temperature in the mouth is 35⁰C - 37⁰C. A significant shift in the 

dominant species in plaque occurs when there is a two degrees change in temperature 

(Marsh et al., 2011).  Aerobic bacteria undergo Redox reactions to maintain the oxygen 

level in the mouth at a semi-stable homeostatic condition (Marsh et al., 2011). Acids 

released from dental plaque as a result of the fermentation of sugars by the 

microorganisms leads to demineralization of the adjacent tooth surface and 

consequently dental caries (Scheie, 1994). Dental plaque organisms also cause irritation 

around the gums which can result in gingivitis, periodontal diseases and tooth loss 

(Scheie, 1994).  

Periodontal diseases may trigger pathways leading to cardiovascular diseases through 

direct or indirect effects of oral bacteria (Hernichel-Gorbach et al., 1996). Oral bacteria 

such as Streptococcus sanguis and Porphyromonas gingivalis induce platelet 

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aggregation, which leads to thrombus formation (Hernichel-Gorbach et al., 1996). 

These organisms have the platelet aggregation-associated protein, which is collagen-

like molecule on their surface (Herzberg et al., 1998).  There are over more than 1,000 

reported cases of endocarditis associating it onset with dental procedures or diseases 

(Drangsholt, 1998). Dental procedures such tooth extraction, endodontic treatment, root 

scaling, periodontal surgery have been reported to cause bacteremia (Drangsholt, 1998). 

Persistent dental disease is painful, and most importantly it has been linked to diabetes, 

high blood pressure, heart diseases and multiple sclerosis later in life (Taylor et al., 

2004) . Dental caries caused by dental plaque organisms also cause bad breath and foul 

taste, in some cases the infection can spread from the teeth to surrounding soft tissues 

which may leads to edentulous mouth (Baelum et al., 1997). 

 

2.1.1 Mechanism of Dental Plaque Formation  

The formation of dental plaque involves two types of bacterial adherent interactions. 

Firstly selective attachment of the bacteria to the acquired pellicle which is the inner-

layer between plaque and the substratum (such as the tooth surface) and secondly 

bacterial accumulation through specific adhesive and cohesive interactions involving 

components of the plaque matrix and direct bacterial cell contact. Organisms are 

selectively attached to the pellicle due to specific interactions involving their cell 

surface constituents and macromolecules of the salivary pellicle. Organism such as 

Streptococcus Salivarius which is prominent in the dorsum of the tongue and in saliva 

do not adsorb well to teeth. Other organisms such as Streptococcus sanguis and A. 

viscosus which are numerous in saliva adsorb easily to the pellicle and are prominent in 

developing new plaque (Scheie, 1994). 

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In the first phase of bacterial adherance, the organisms are loosely attached to the 

surface by Van deer Waal forces due to the net negative charge on both the teeth and 

bacterial surface. There is no strong attraction because of the repulsive effect of the 

negative charges. The second phase of attachment leads to a firmer bonding which 

involves the linking of the polymeric substance on the bacterium surface to the target 

surface by the formation of hydrogen, hydrophobic or ionic bonds. The adsorption of 

Streptococcus mutans involves electrostatic interactions in which the bacterial binds to 

hydroxyapatite which include calcium and phosphate groups on the mineral surface. It 

is postulated that the cell wall teichoic acid which gives the bacteria a net negative 

charge forms bridges with the calcium ions on the enamel or pellicle.  The bacteria 

attachment is via adhesins found on their surface which binds to specific receptors on 

the pellicle and other host tissues. Protein adhesins called ‗lectin‘ binds to specific 

sugars. Other adhesins which have hydrophobic moieties binds to hydrophobic 

receptors. Adhesins aids in the recognition and binding to specific receptors and 

complex macromolecules (Scheie, 1994).  

The second phase, involves bacterial accumulation. Bacterially-derived polymers and 

salivary components are both essential in this process. Streptococcus mutans 

synthesizes extracellular glucans and fructans from sucrose but not from other 

carbohydrates and this polymer synthesis helps it multiply in large masses. Recent 

studies show that certain serovars of Streptococcus mutans can form plaque in the 

absence of sucrose, however, such plaques are less tenacious to enamel than plaque 

formed in the presence of sucrose. The average range of pH in plaque is about 7.1 in 

caries free persons and about 5.5 in persons with extreme caries activities. A drop in pH 

causes the demineralization of the tooth surface. At a critical pH of 5.5 the tooth 

minerals lose calcium and phosphates into plaque. This buffering activity helps initially 

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in maintaining the pH at 5.5, a drop in the pH leads to the demineralization of the 

subsurface. A further decrease in the pH leads to demineralization of the enamel. An 

important discovery suggests that certain cariogenic and acidogenic species of 

streptococcus especially Streptococcus mutans metabolize dietary sucrose and 

synthesize glucan by using the enzyme glucosyltransferase. This enzyme is essential in 

the establishment of Streptococcus mutans in dental plaque. Certain cariogenic bacteria 

have reservoirs of carbohydrates which are obtained from intracellular polysaccharides 

which serves as a source of carbohydrate for fermentation and maintenance of acid 

production in plaque during periods when the individual‘s diet is sugar free (Scheie, 

1994). 

 

2.2 Streptococcus mutans 

Streptococcus mutans belongs to the viridans group of streptococci, and is part of the 

normal oral flora of man, and is an aetiological agent in smooth-surface dental caries 

(Hamada et al., 1980). Streptococci of the mutans group are highly acidogenic; they 

produce short-chain carboxylic acids which dissolve hard tissues such as dentine and 

enamel, and are the most cariogenic pathogens (Shaw, 1987). In addition, they produce 

insoluble extracellular polysaccharides, which improve their adherence to the tooth 

surface and encourage biofilm formation by fermenting sucrose (Shen et al., 2004). 

Biofilms can tolerate many antagonistic conditions such as variations in pH, 

antimicrobial agents, and nutrient and oxygen deprivation (Davey et al., 2000). Due to 

the considerable serological and genetic heterogeneity of human and animal 

Streptococcus mutans strains they are grouped into eight species collectively known as 

mutans streptococci with the major species in humans being  Streptococcus  mutans 

(serotype c,e,f) and Streptococcus sopranos (serotype d,g) with occasional isolation of 

Streptococcus rattus (serotype b) and Streptococcus cricetus (serotype a), 

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Streptococcus  mutans serotype c account for 70-100% of human isolates of mutans 

streptococci (Loesche, 1986). Streptococcus mutans produces bacteriocin, mutacin, 

which is active against other streptococcal species and non-streptococcal Gram-positive 

bacteria (Hamada et al., 1975). Mutacin provides an ecological advantage in the 

adverse microbial community and also facilitate the transmission of species between 

mother and child (Grönroos et al., 1998). The efficient establishment and colonization 

of Streptococcus mutans in the oral cavity is due to the production of bacteriocin 

(Roger, 1976). The production of the enzyme haemolysin which is a virulence factor is 

responsible for blood invasion and aids in supplying them with their requirement of 

iron (Kuramitisu et al., 1993). 

Virulence factors of Streptococcus mutans are mainly adhesion, acidogenicity, and acid 

tolerance. The adherence by Streptococcus mutans to teeth surfaces is by sucrose-

dependent and sucrose-independent mechanisms. The former involves extracellular 

glucosyltransferase (GTFs) for synthesis of glucans (glucose polymers) that mediate 

bacterial adhesion and contribute to biofilm formation (Van Houte, 1994). 

Streptococcus mutans has three GTFs: GTF B, GTF C, and GTF D. The former two 

synthesize primarily water-insoluble glucans, while the latter synthesizes only water-

soluble glucans; the activity of all three enzymes is required for optimal adherence of 

Streptococcus mutans (Tanzer et al., 2001). The enzymes are important virulent factors 

of Streptococcus mutans necessary for the pathogenesis of dental caries (Tanzer et al., 

2001). Streptococcus mutans is also known to cause vaginitis (Rizvi et al., 2003).  

 

2.3 Streptococcus sanguis 

Streptococcus sanguis is a member of the viridans streptococcus group. It is Gram-

positive cocci with alpha-hemolytic abilities (Zhu et al., 2001). Streptococcus sanguis 

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is an oral commensal involved in dental caries by pioneering bacteria colonization of 

the oral cavity leading to biofilm formation (Zhu et al., 2001). 

It survives by metabolizing dietary sugars into organic acids particularly, lactate. The 

accumulation of bacteria, food particles and salivary constituents around and on the 

teeth leads to the formation of ecological niche dental plaque (Nikiforuk, 1985). The 

production of acid from the fermentation of dietary sugars leads to a fall in localized pH 

levels leading to the corrosion of the enamel and beginning of dental caries (Loesche, 

1986). The frequency of Streptococcus sanguis is and its incidence is antagonistically 

related to cariogenic Streptococcus mutans by producing the enzyme sanguicin (Zhu et 

al., 2001). 

Streptococcus sanguis cause infective endocarditis and other important diseases as 

atherosclerosis in risk patients, specifically after a trauma when it enters into circulating 

blood (Okahashi et al., 2010). In animal models, the severity of infective endocarditis is 

associated with the ability of Streptococcus sanguis to adhere to and activate platelets 

(Herzberg et al., 1992).  About 60% of Streptococcus sanguis can induce human 

platelets to aggregate in vitro (Herzberg et al., 1992). 

 

2.4 Herbal Medicine 

Herbal medicines mainly used in traditional medical systems have been used in medical 

practice for thousands of years, due to their accessibility and affordability. Herbal 

medicines could therefore help to meet the health needs of majority of the populace 

who are unable to afford imported drugs or do not have access to the facilities of a 

modern hospital (krogsgaard et al., 1984). Using of plants for medicinal purposes is an 

important part of the culture and the tradition in Africa, thus 80% of the population 

depends directly on traditional medicine for their primary health care (Kirby et al., 

1996). 

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Herbal medicines are prepared from a variety of medicinal plants materials which are 

the leaves, roots, bark, and fruits and they usually contain active biological ingredients 

used for treating chronic or mild ailments or diseases (De Slmet, 1992). These 

medicinal plants can be used to manage and treat almost all known diseases of man and 

animals (Lewington, 1990). The World Health Organization (WHO) estimates that up 

to 80% of the world‘s people rely on plants for their primary health care (Fennell et al., 

2004). 

Despite the remarkable progress in synthetic organic chemistry of the twentieth century, 

over 25% of prescribed medicines in industrialized countries are derived directly or 

indirectly from plants (Newman et al., 2000).  This highlights the continuous interest in 

laboratory screening of medicinal plants, not only to determine the scientific rationale 

for their use, but also discover new active principles (Newman et al., 2000).  It has been 

estimated that between 35,000 and 70,000 different species of plants have been used by 

various people of the world with the commercial value of plant-derived drugs by 

western drugs standing at about 40 billion United State dollars every year (Lewington, 

1990).  According to WHO about 74% of the 119 plant derived pharmaceutical 

medicines are used in modern medicine in ways that correlate directly with their 

traditional uses as plant medicine in native cultures (WHO, 2003). 

The sophistication of herbal remedies varies around the world with technological 

advancement of countries that produce them and use them. These remedies usually 

ranges from teas and crude tablets used in traditional medicine to concentrated, 

standardized extracts produced in modern pharmaceutical industries under a physician‘s 

prescription and supervision (Chowa, 1996). In Europe, the herbal medicines are 

mainly grouped into three (3) categories. The first and the most rigorously controlled 

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are prescription drugs, which include injectable forms of phytomedicine and those used 

to treat life-threatening diseases. The second category is the over-the-counter (OTC) 

phytomedicines and the third category is traditional herbal remedy  products that  have 

not been clinically tested but judged safe on the basis of generations of use in the 

treatment of diseases without serious incidents (Chowa, 1996).   

A special feature of plants is their ability and capacity to produce a large number of 

organic chemicals of high structural diversity called secondary metabolites (Shah, 

2005). Screening of compounds obtained from medicinal plants for their 

pharmacological assay has indeed been the vast source of innumerable therapeutic 

agents representing molecular diversity engineered by nature (Shah, 2005).  The use of 

plant derived new compounds that are not based on existing synthetic antimicrobial 

agents is one of the ways to prevent antibiotic resistance of pathogenic species (Shah, 

2005).  Phytomedicines have shown a great promise in the treatment of infections 

including viral infections (Cowan, 1999).   

A lot more of the plants needs to be scientifically studied, even though some herbal 

medicines have withstood scientific testing. Most people patronize herbal medicines 

because they feel they are innocuous and less expensive than orthodox medicines, 

however, they can be hazardous if they are not used appropriately. For instance, herbal 

medicines have been reported to be responsible for about 35% of cases of acute renal 

failures in some African countries (Bull et al., 1989). 

Addae-Mensah (1992), has reported that even though Croton membranaceous has 

diterpenes and alkaloids which possess anticancer and anti-ulcer properties, the same 

plant has phorbol esters which are known to be co-carcinogenic and can cause cancer of 

the oesophagus. He also reports that Crotalaria, Senecio, Cynoglossum and 

Heliotropium genera contains pyrrolizidine alkaloids found to be highly hepato-toxic. 

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Scientific investigations into herbal medicines must therefore involve the therapeutic 

effects as well as the conceivable long-term toxic effects which may not manifest 

themselves until it is too late. 

The World Health Organization (WHO) since May 1978 has been making a study of 

medicinal plants and this has promoted the identification of about 20,000 species of 

medicinal plants with a more detailed investigation of a shortlist of 200 species. A 

larger number of these plants have their origins from the world‘s tropical forest and 

they are extensively used in traditional medicine, which play a major role in 

maintaining the health and welfare of both rural and city dwellers in developing 

countries (Robin, 2005). 

Medicinal Plants are vital products found in the forest areas throughout the world 

especially in South Asia, from the plains to the high Himalayas and the tropical and 

subtropical belts (Komen 1991; Robin 2005). However, tropical forests are the source 

of a large proportion of the world‘s recognized medicinal plants. India has documented 

more than 2,500 plant species as having medicinal value, Sri Lanka about 1,400 and 

Nepal around 700. In Ghana, about 2,000-2,500 medicinal plants are used as medicinal 

plants and are found mostly at high altitudes particularly in stressful environments 

where they grow very slowly and cannot live elsewhere. Others are however, more 

largely distributed and adapt easily to different ecological conditions (Abbiw, 1990). 

Medicinal plants in Africa and drugs derived from them are the back-bone of several 

food and pharmaceutical industries or treatment in primary health care and constitute a 

big fortune of great economic and strategic value (Luc van-puyvelde, 1988). Ghana has 

a particularly strong tradition in medicinal plants as they play a vital cultural and 

economic role in poverty alleviation, due to the fact that people of significant status 

both in villages and urban areas use these plants (Abbiw, 1990). Another reason for the 

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growing popularity of herbal medicine in Ghana is that many people believe they are 

safer and ―more natural‖ than pharmaceutical products (Dwuma-Badu, 1986). 

Many medicinal plants of Africa are still under investigation for their chemical 

components and bioactivity against microorganisms. Table 1, indicates the 

ethnomedicinal uses of Sida acuta, Anthocleista nobilis, and Zanthozylum leprieurii 

which holds an enormous promise for the development of antimicrobial agents.  

 

 

 

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Table 1: The Ethnomedicinal Uses of the Selected Plants 

 

 

 

Plant Name 

(Family) 

Local Name Traditional Medicinal Use References 

Sida acuta 

(Malvaeceae) 
Wirewood 

(Obraneatuto -

Akan) 

Used to treat asthma, renal 

inflammation, colds, fever, headache, 

ulcers, mouth ulcers worms 

 

Leaves used to treat diarrhoea, malaria, 

and dental diseases 

 

Haemorrhoids, fevers, impotency, 

gonorrhoea, and rheumatism.  In 

mixture  as  aphrodisiac  and  for  boils 

and eye cataracts 

 

(Coee and 

Anderson, 1996) 

 

 

Nacoulma et al., 

(1996) 

 

(Dash,  1991;  Pal  

and  Jain, 

1998) 

Anthocleista  nobilis 

(Gentianaceae) 

 

 

 

 

(Wudifo kete – 

Akan) 

Leaves used to treat abdominal pains of 

uterine origin 

 

The bark and roots are used to treat 

constipation, periodontal diseases, 

menstrual pains, fever, and stomach-

ache 

 

 

 

 

Olatunji, (1983) 

 

 

 

 

Zanthozylum leprieurii 

(Rutaceae) 

(Oyaa-Akan) The bark, leaves, and roots are used for 

a variety of treatments including 

toothache, coughs, leprous ulcerations, 

rheumatism, lumbago, stomach ache, 

urinary and venereal diseases 

 

 

Stem bark is used to treat dental 

diseases, kidney pain, arthritis, skin 

infections, dysentery, intestinal worms, 

back pain, post-partum pain syphilitic 

sores, rheumatic pains, bleeding gums, 

and leprous ulcers 

 

 

 

Olatunji, (1983) 

 

Oliver-Bever, 

(1982) 

 

 

 

 

 

 

 

Adesina, (1987) 

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CHAPTER THREE 

3.0 MATERIALS AND METHODS 

3.1 MATERIALS 

3.1.1 STUDY SITE AND POPULATION  

Clinical sample collection started from February and ended in April 2014. A total of 

twenty (20) dental plaque samples were collected from the gingival side of the canine 

and molar teeth of each consented patient using sterile scalers and curettes at the 

University of Ghana Dental School (UGDS), Korle-Bu. The isolation and identification 

of organisms from dental plaque was performed at the Department of Microbiology, 

University of Ghana Medical School (UGMS), Korle-Bu.  

The evaluation of the antimicrobial properties of the selected medicinal plants was done 

at the Microbiology Department of the Centre for Plant Medicine Research (CPMR) at 

Mampong-Akwapim. 

 

3.1.2 STUDY DESIGN  

 An experimental study was used in carrying out this research which is  aimed at 

evaluating the antimicrobial properties of leaves of Sida acuta, the bark of Anthocleista 

nobilis and Zanthoxylum leprieurii from traditional herbal healers who use these herbs 

to cure various diseases at Koforidua in the Eastern region against Streptococcus 

mutans and Streptococcus sanguis that cause dental plaque. A simple random sampling 

technique was employed in the research in which only patients with dental plaque were 

recruited. Samples from consented patients who met the inclusion criteria were taken 

for analysis. 

 

 

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3.1.3 INCLUSION CRITERIA  

 Consented patients with dental plaque 

 All plaque samples that show growth of organisms. 

 

3.1.4 EXCLUSION CRITERIA  

 Patients who did not consent to the study were excluded. 

 

3.1.5 INFORMED CONSENT  

The study was approved by the Ethical and Protocol Review Committee, University of 

Ghana Medical School (UGMS), College of Health Science, Korle-Bu. The protocol 

identification number of the ethical clearance for this study was MS-Et/M.4 – P 

3.3/2013-2014 with reference number MS-AA/C.2/Vol.18
A
. Consent was also sought 

from the Dean, University of Ghana Dental School (UGDS), Korle-Bu. 

 

3.1.5 SAMPLE COLLECTION, TRANSPORT, AND STORAGE 

The dental plaque samples were collected from the gingival side of the canine and 

molar teeth of consented patients who attended the University of Ghana Dental School 

(UGDS) Clinic, Korle-Bu using sterile scalers and curettes. The samples were placed 

into sterile tubes containing 5ml Brain Heart Infusion Broth (BHI, Difco, Detroit, USA) 

which was sealed tightly labeled and transported immediately to the laboratory and 

incubated at 37⁰C for 18hours. 

The medicinal plants included in the study, Sida acuta, Anthocleista nobilis and 

Zanthoxylum leprieurii, were collected from traditional herbal healers in Koforidua, 

Eastern region who use these plants to cure various diseases. The plants were 

authenticated by a Taxonomist from the Plant Development Department (PDD) of the 

Centre for Plant Medicine Research (CPMR) and voucher specimens (Sida acuta − 

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Voucher No: CPMR 7210, Anthocleista nobilis − Voucher No: CPMR 0610, 

Zanthozylum leprieurii − Voucher No: CPMR 50) of each plant kept at the Centre for 

Plant Medicine Research (CPMR) herbarium. 

 

3.1.6 TEST MICROORGANISMS AND CONTROL ANTIBIOTICS 

Six clinical strains were used in the study: Streptococcus mutans W7, Streptococcus 

mutans W13, Streptococcus mutans W11, Streptococcus sanguis W14, Streptococcus 

sanguis W18, and Streptococcus sanguis W20.  Another two reference strains of 

Streptococcus mutans (ATCC 700610) and Streptococcus sanguis (ATCC 10556) were 

also tested. Penicillin (1.5i.u) and Gentamicin (10µg) antibiotics (Himedia, India) were 

used as positive controls. 

 

3.2 METHODS 

3.2.1 Extract Preparation  

The Plants collected were sun dried for one week and ground in sterile mortar and 

pestle to fine powder. The powders were kept in transparent sealed plastic containers 

and stored until used. For the ethanol extraction 500g of the pulverized plant was 

macerated in 5 litres of 70% ethanol for 24 hours. The extract was then filtered using 

Whatman No. 1 filter paper and evaporated using a rotary evaporator and then 

lyophilized at the College of Agriculture, University of Ghana, Legon. 

For the aqueous extraction, 500g of the pulverized plants was dissolved in 5 litres of 

distilled water and boiled using a hot plate at high temperature at 100
o
C for 30 minutes 

and then low temperature at 60
o
C for 15 minutes for concentration. The suspension was 

then filtered using Whatman No. 1 filter paper and the filtrate was lyophilized at the 

College of Agriculture, University of Ghana, Legon. 

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3.2.2 Isolation of bacteria from clinical specimen 

The dental plaque samples were cultured in Brain Heart Infusion broth (BHI, Difco, 

Detroit, USA) and then incubated at 37ºC for 18 hours. After bacterial enrichment, the 

turbid broth media were cultured onto Blood Agar using streak plate method and 

incubated at 37ºC in 5% CO2 for 48 hours. 

 

3.2.3 Culture and Biochemical Identification of the Microorganisms 

The cultured bacteria were examined for macroscopic characteristics such as colour, 

size, morphology and haemolysis on Blood agar.  Differential media of Mitis Salivarius 

agar (MSA) aided in distinguishing viridans streptococcus from other streptococcus. 

Gram reaction showed Gram positive cocci in short or long chains. The slide catalase 

test was done using a drop of hydrogen peroxide (H2O2) on a microscope slide to 

distinguish Streptococcus species which were catalase negative from Staphylococcus 

species. Subsequently, optochin test was performed to distinguish viridans 

Streptococcus from pneumococci which are optochin (ethylhydrocupreine 

hydrochloride) sensitive. A disc (5 µg) of optochin was applied to the surface of an 

inoculated blood agar and incubated aerobically at 35-37
o
C for 18-24 hours. The 

viridans streptococci were resistant showing an inhibition zone size of less than 10 mm. 

For biotyping, Analytical Profile Index 20 Strep (bioMérieux, France), an identification 

system for Streptococcaceae was used. The API 20 Strep strip contains 20 microtubes 

containing dehydrated substrates for the demonstration of enzymatic activity or the 

fermentation of sugars. The enzymatic test was inoculated with a dense suspension of 

the organism (turbidity greater than 4 McFarland) from a pure culture. During 

incubation, metabolism produces colour changes that were either spontaneous or 

revealed by the addition of reagents. The fermentation tests were inoculated with an 

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enriched medium which rehydrates the sugar substrates. Fermentation of carbohydrates 

is detected by a shift in the pH indicator. The reactions were read according to the 

reading table and the identification was obtained by referring to the Analytic Profile 

Index. 

 

3.2.3 ANTIMICROBIAL ASSAY 

3.2.3.1 Agar-well diffusion method 

The spreading method of Cruickshank et al., (1980) and agar - well diffusion method 

was used. Each control strain and clinically isolated organism of Streptococcus mutans 

and Streptococcus sanguis was inoculated individually into 6ml of peptone broth 

(Sigma, P0556, Sigma-Aldrich Inc., USA) and the density was adjusted to 0.5 

McFarland turbidity standards resulting in a suspension of 1.5×10
8
 colony forming 

units. 

Mueller Hinton  agar with 5% sheep blood  (Oxoid, CM0337, Oxoid Ltd, United 

Kingdom)  was seeded with the test organisms and the plate made to dry for some few 

minutes. After, which wells were made in the agar using sterile cork borer measuring 

6mm in diameter. About 100µl of the prepared 200mg/ml, 150mg/ml, 100mg/ml, 

50mg/ml, 25mg/ml, 12.5mg/ml, 6.25mg/m, 3.125mg/ml of aqueous and ethanolic 

extracts of Sida acuta, Zanthoxylum leprieurii and Anthocleista nobilis were dispensed 

into the labeled wells. The plates were then kept in the refrigerator for one hour for the 

extract to diffuse into the medium. The aqueous extract was dissolved in 10mls sterile 

distilled water and the ethanolic extract was reconstituted in 10 ml of 20% Dimethyl 

Sulfoxide (DMSO).  Penicillin (1.5i.u) and gentamicin (10µg) antibiotics (Himedia, 

India) were used as positive controls to compare the zones of inhibition with that of the 

extracts. Sterile distilled water and 5% DMSO was used as negative control.  The plates 

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were incubated at 37ºC in 5% CO2 for 24−48 hours. Analysis was done in duplicate.  

Diameters of the zones of inhibition in the duplicate plates were measured by 

calculating the difference between cork borer (6mm) and the zone of inhibition (Singh 

et al., 2002). 

 

3.2.3.2 Determination of Minimum Inhibitory Concentration (MIC) and Minimum 

Bactericidal Concentration (MBC) of the extracts 

The Minimum Inhibitory Concentration (MIC) was determined using the macro broth 

dilution method according to methods describe by CLSI, (2006). The plant extracts was 

diluted from concentrations ranging from 200 to 3.125 mg/ml in Mueller Hinton broth 

(Liofilchem, Italy). To each dilution tube 0.1 ml of bacterial inoculum was seeded. 

Control tubes with no bacterial inoculation were simultaneously maintained. Tubes 

were incubated at 35−37°C for 24 hours. The lowest concentration of the extract that 

produced no visible bacterial growth (turbidity) was recorded as the Minimum 

Inhibitory Concentration (CLSI, 2006). The Minimum Bactericidal Concentration was 

determined by sub-culturing the test dilution (which showed no visible turbidity) on to 

freshly prepared blood agar media. The plates were incubated for 18-42 h at 37 °C. The 

highest dilution that yielded no single bacterial colony on the Mueller agar plates was 

taken as the MBC.  

 

 

 

 

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3.2.3.3 Statistical Analysis 

 tudent T-test was used to compare the significant difference between aqueous and 

ethanolic extracts (Appendix 9   11). One-way ANOVA was used for the comparison 

of the means followed by post hoc L D.  P≤0.05 was considered statistically 

significant. Results were expressed as mean ± SD (Standard Deviation) data, using 

Statistical Package for Social Scientist (SPSS) version 20.0 statistical analysis software. 

Tables and graphical displays were also used to illustrate data. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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CHAPTER FOUR 

4.0 RESULTS 

The susceptibility of the test organisms to the aqueous and ethanolic extracts of the 

individual medicinal plants namely, Sida acuta, Anthocleista nobilis and Zanthozylum 

leprieurii was studied. The isolates that showed zones of inhibition equal to or more 

than 7mm were considered susceptible. Figures 1–3 below summarizes the antibacterial 

activity of the aqueous and ethanolic extracts of the individual medicinal plants on the 

test organisms. 

The aqueous extract of Sida acuta showed significant zones of inhibition against the 

test organisms. The maximum zone of inhibition was produced against the standard 

strain S. sanguis (ATCC 10556) as shown in Figure 1 with a mean inhibition zone of 

21.5mm which was greater than the positive controls; Penicillin and Gentamicin which 

produced inhibition zones of 20mm and 12mm respectively. The aqueous extract of 

Anthocleista nobilis exhibited diverse antimicrobial activity against all the test 

organisms. The maximum zone of inhibition was produced against clinical strains of S. 

mutans and S. sanguis (ATCC 10556) with zones of inhibition 15.7mm and 15mm 

respectively which were higher than Gentamicin (positive control) which had mean 

inhibition zone of 12mm. However the zone of inhibition was lower than that of 

Penicillin (positive control) with a mean inhibition zone of 20mm as shown in Figure 2 

and Table 3. The aqueous extract of Zanthozylum leprieurii did not show any inhibitory 

activity on S. sanguis strains (Table 4) although, there was a small zone of inhibition 

against S. mutans strains.  The ethanolic extracts of the various plants demonstrated the 

highest inhibitory effects on most of the organisms. It was observed that the inhibitory 

activities of the plant extracts increased with increasing concentration. The ethanolic 

extract of Sida acuta leaves inhibited the growth of the clinical strains of S. mutans at 

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inhibition zone size of 22.7mm. This zone size was greater than penicillin and 

gentamicin (positive control) with inhibition zone sizes of 20mm and 12mm 

respectively.  The ethanolic extract of Anthocleista nobilis exhibited inhibitory activity 

against the test organisms. The highest inhibitory zone of this extract was observed at 

200mg/ml with the range of inhibition zone size of 17‒ 13.3mm as shown in Table 3 

and Figure 2. The ethanolic extract Zanthozylum leprieurii did not show any inhibitory 

activity on S. sanguis strains (Table 7), however, there were smaller zones of inhibition 

recorded against S. mutans strains. Minimum Inhibitory Concentration and Minimum 

Bactericidal Concentration of the extract against the microorganisms are shown in 

Figures 4 and 5. For instance, the MIC and MBC value of Sida acuta leaves for S. 

mutans clinical strains were both 3.125mg/ml. Anthocleista  nobilis, Zanthozylum 

leprieurii, penicillin and gentamicin had MIC values of 50mg/ml, 100mg/ml, 

6.25mg/ml, and 50mg/ml respectively, while their corresponding MBC values were 

50mg/ml, 100mg/ml, 6.25mg/ml, and 50mg/ml. The MIC values of 100mg/ml, 

100mg/ml, 3.125mg/ml, and 50mg/ml were obtained for Sida acuta, Anthocleista 

nobilis, Penicillin, and Gentamicin respectively against S. sanguis clinical strains, while 

their corresponding MBC values were 100mg/ml, 100mg/ml, 6.25mg/ml, and 50mg/ml 

(Figure 4 and 5).   

The MIC values of Anthocleista nobilis against the test organisms were found to be 

greater than 3.125mg/ml. From the experiment it was shown that the Sida acuta leaves 

performed best followed by Anthocleista nobilis stem bark and Zanthozylum leprieurii 

stem bark. 

 

 

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Figure 1:  Antimicrobial Activity of Sida acuta Leaf Extracts on the Test 

Organisms 

  

 

 

 

 

0

5

10

15

20

25

30

S
. 

m
u

ta
n
s

S
. 

sa
n

g
u
is

S
. 

m
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s 

(A
T

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C

 7
0

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6

1
0
)

S
. 

sa
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is

 (
A

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 1

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5
6

)

S
. 

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S
. 

sa
n

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is

S
. 

m
u

ta
n
s 

(A
T

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 7
0

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6

1
0
)

S
. 

sa
n

g
u
is

 (
A

T
C

C
 1

0
5

5
6

)

AQUEOUS ETHANOL

Zo
n

e
 o

f 
In

h
ib

it
io

n
 

(m
m

) 

Test Organisms 

200mg/ml

150mg/ml

100mg/ml

50mg/ml

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       Figure 2: Antimicrobial Activity Anthocleista nobilis Stem bark Extract on the 

Test Organisms 

 

 

 

 

 

 

0

5

10

15

20

25

S
. 

m
u

ta
n
s

S
. 

sa
n

g
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is

S
. 

m
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(A
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 7
0

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6

1
0
)

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. 

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 (
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 1

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. 

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. 

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. 

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(A
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)

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. 

sa
n

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is

 (
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 1

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6

)

AQUEOUS ETHANOL

Zo
n

e
 o

f 
In

h
ib

it
io

n
 

(m
m

) 

Test Organisms 

200mg/ml

150mg/ml

100mg/ml

50mg/ml

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Figure 3: Antimicrobial Activity of Zanthozylum leprieurii Stem bark Extract on 

the Test Organisms 

 

 

 

 

 

 

 

0

2

4

6

8

10

12

14

S
. 

m
u

ta
n
s

S
. 

sa
n

g
u
is

S
. 

m
u

ta
n
s 

(A
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 7
0

0
6

1
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)

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. 

sa
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 (
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. 

m
u

ta
n
s

S
. 

sa
n

g
u
is

S
. 

m
u

ta
n
s 

(A
T

C
C

 7
0

0
6

1
0
)

S
. 

sa
n

g
u
is

 (
A

T
C

C
 1

0
5

5
6

)

AQUEOUS ETHANOL

Zo
n

e
 o

f 
in

h
ib

it
io

n
 

(m
m

) 

Test Organisms 

200mg/ml

150mg/ml

100mg/ml

50mg/ml

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Figure 4: Minimum Inhibitory Concentration (MIC) of the Plant Extracts on Test 

Organisms 

 

 

 

 

0

20

40

60

80

100

120

S
. 
m

u
ta

n
s

S
. 
sa

n
g

u
is

S
. 
m

u
ta

n
s 

(A
T

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 7
0
0

6
1

0
)

S
. 
sa

n
g

u
is

 (
A

T
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 1

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5
5

6
)

Clinical strains Control strains

M
in

im
u

m
 I

n
h

ib
it

o
r
y

 C
o

n
c
e
n

tr
a

ti
o

n
 

(m
g

m
l)

 

Test Organisms 

Sida acuta

Anthocleista nobilis

Zanthozylum leprieurii

Penicillin

Gentamicin

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Figure 5: Minimum Bactericidal Concentration (MBC) Values of Respective MIC 

Concentrations of the Plants extracts on Test Organisms 

 

 

 

0

20

40

60

80

100

120

S
. 
m

u
ta

n
s

S
. 
sa

n
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u
is

S
. 
m

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(A
T

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C

7
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6

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0

)

S
. 
sa

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is

 (
A

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 1

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5
5

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)

Clinical strains Control strains

M
in

im
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m
 B

ac
te

ri
ci

d
al

 C
o

n
ce

n
tr

at
io

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(m
g/

m
l)

 

Test Organisms 

Sida acuta

Anthocleista
nobilis
Zanthozylum
leprieurii
Penicillin

Gentamicin

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CHAPTER FIVE 

5.0 DISCUSSION 

The increasing threat and spread of antibiotic resistance by a wide range of common 

pathogens has led to increased investigations into traditional medicinal plants as 

alternatives (Nostro et al., 2000). Many published works also show the efficacy of 

traditional medicinal plants against microorganisms (Evans et al., 2002). According to 

Tshibangu et al. (2002), plants have given western pharmacopoeia about 7000 different 

pharmaceutically essential compounds and a number of top-selling medicines of 

modern time, examples of which are artemisinin, taxol, camptothecin, and quinine.  

In the present study, antibacterial activity of aqueous and ethanolic extracts of Sida 

acuta, Anthocleista nobilis and Zanthozylum leprieurii was effective against clinically 

isolated cariogenic bacteria. Among the three plants, the leaves of Sida acuta were 

found to be more active than the stem bark of Anthocleista nobilis and Zanthozylum 

leprieurii. This is probably why substantial research has been done on the leaves of 

Sida acuta as compare to the other plants. 

The leaves of Sida acuta displayed good antibacterial activity against Streptococcus 

mutans and Streptococcus sanguis which are both Gram positive microorganisms. 

These findings were similar to studies done by Oboh et al. (2007) in which the leaves 

of Sida acuta was found to be effective against three Gram positive bacteria; 

Streptococcus faecalis, Staphylococcus aureus, and Bacillus subtilis (MIC values 

between 5-10 mg/ml). Anani et al. (2000) studied the antimicrobial activity of the 

aqueous and ethanolic extracts of the leaves of Sida acuta. They also used the Macro 

broth dilution method to determine the MIC values and obtained similar MIC (3.125 

mg/ml) values against Staphylococcus aureus and Bacillus subtilis as was obtained in 

this research. Similar work done by Coee and Anderson (1996), Ekpo and Etim (2009), 

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and Oboh et al. (2007) showed that the extract of Sida acuta had preferential and 

specific activity against Gram positive bacteria such as Staphylococcus aureus, 

Streptococcus faecalis, and Bacillus subtilis. They also recorded MIC values lower than 

50mg/ml. This confirms the present studies in which leaves of Sida acuta have 

significant antibacterial activities against Streptococcus mutans and Streptococcus 

sanguis. The high sensitivity of Gram positive bacteria could be attributed to the cell 

wall structure which has an outer peptidoglycan layer which is not an effective barrier 

as compared to Gram negative bacteria. The slightly higher potency of the ethanolic 

extract over the aqueous extract in the agar diffusion assay agrees with the reports of 

Essien et al. (2009). Limited studies have been done on the antimicrobial effects of the 

plant towards cariogenic bacteria. Studies done by Essien et al. (2009) suggest that the 

antimicrobial property of Sida acuta is attributed to the presence of alkaloids and 

flavonoids. The mechanism of action of the alkaloids is attributed to their ability to 

exert cytotoxic action via the inhibition of DNA synthesis and stabilization of 

topoisomerase II DNA covalent complexes (Rabe and Vastoden, 2000). Flavonoids 

obtained from Sida acuta inhibited in vitro Streptococcus mutans (Batsista et al., 1994). 

This report provides new evidence for efficacy against Streptococcus mutans and 

Streptococcus sanguis. 

The stem bark of Anthocleista nobilis showed antibacterial activity against 

Streptococcus mutans and Streptococcus sanguis with MIC values between 50 – 100 

mg/ml. These findings were similar to studies done by (Annan and Dickson, 2008) in 

which the aqueous and ethanolic stem bark of Anthocliesta nobilis was effective against 

two Gram positive bacteria (Staphylococcus aureus and Bacillus subtilis). They 

obtained MIC values less than 500 mg/ml.  

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(Akinyemi and Ogundare, 2012) also detected that another species Anthocleista 

djalonensis, was effective against Staphylococcus aureus, Salmonella typhi, and 

Escherichia coli. Therefore the antibacterial activity of Anthocleista nobilis obtained in 

this research agrees with the antimicrobial activity displayed by other Anthocleista 

species. Studies done by Kraus (1995) suggest that the antimicrobial property of 

Anthocleista nobilis is attributed to the presence polyphenols. Polyphenol is capable of 

carrying mass destruction to the cell membrane of Gram positive bacteria (Herrera-

Arellano et al., 2004). 

Zanthozylum leprieurii stem bark was the least effective against the test organisms. The 

aqueous and ethanolic extract had significant activity against Streptococcus mutans but 

did not have any inhibitory activity on Streptococcus sanguis. This could be due to the 

inherent resistant factor of the isolate and the likely previous exposure of the organism 

to other antimicrobial drugs or agents as a result of drug abuse in the population. The 

findings were similar with studies done by (Oshomo and Idu, 2012) in which the 

aqueous and ethanolic extract of Zanthozylum zanthoxylloides, another species inhibited 

the growth of Streptococcus mutans, Bacillus subtilis, and Staphylococcus aureus. 

Similar studies done by (Agyare et al. 2014) have showed that the plant extract is 

effective against Staphylococcus aureus, Streptococcus pyogenes and Bacillus subtilis 

which are both Gram positive organisms. Yao et al. (2005) reported antibacterial 

efficacy of ethanol extract from stem bark of Zanthozylum nitidum growing in China 

against three oral pathogenic bacteria (Streptococcus mutans, Actinomyces naeslundii, 

and Actinobacillus actinomycetes micomtans). Wan et al. (2005) also observed that 

toothpaste containing Zanthozylum nitidum decreased the incidence of dental plaque 

and gingivitis. The efficacy of Zanthozylum armatum as used by the local inhabitants in 

Iran for the management of dental caries was confirmed by Negi and coworkers (Negi 

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et al. 2012) Therefore, the antibacterial activity displayed by Zanthozylum leprieurii 

obtained in this research falls in line with the antibacterial activity displayed by other 

Zanthozylum species. This infers that Zanthozylum leprieurii possesses antibacterial 

properties. However, none of the previous studies have addressed the antimicrobial 

effects on Zanthozylum leprieurii against Streptococcus mutans and Streptococcus 

sanguis. Previous studies attribute the antimicrobial activity of Zanthozylum leprieurii 

to the presence of alkaloids, flavonoids, terpenoids, and saponins (Sodipo et al., 1991; 

Stephen et al., 2009).  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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CHAPTER SIX 

6.0 CONCLUSIONS AND RECOMMENDATIONS 

6.1 CONCLUSIONS 

The study was done to investigate the antibacterial activities of the extracts of Sida 

acuta, Zanthozylum leprieurii and Anthocleista nobilis on Streptococcus mutans and 

Streptococcus sanguis. The result from the study shows that all the medicinal plants 

possessed antibacterial activities against the test microorganisms. Sida acuta and 

Anthocleista nobilis showed promising antibacterial activities and thus can be employed 

as an effective anti-plaque agent and can be used in the prevention of dental caries.  

From this study, it was observed that the ethanolic extracts exhibited high inhibitory 

activities on the test organisms than aqueous extracts. This could be due to the ability of 

ethanol to extract more of the essential oils and secondary plant metabolites which are 

believed to exert antibacterial activities on the test microorganisms.  The results from 

the study support the ethnomedicinal use of the plants and suggest that the plant 

extracts have compounds with antibacterial properties that can be used as antimicrobial 

agents in the developments of new drugs.  

 

 

 

 

 

 

 

 

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6.2 RECOMMENDATIONS  

Based on the findings from this study, it is recommended that: 

1. Evaluation of the phytochemical properties and pharmacological studies of the 

medicinal plants should be conducted to determine their relative safety as a possible 

antimicrobial agent. 

2. Toxicity studies should be carried out on the medicinal plants to determine their 

safety indices. 

3. Clinical trials of the medicinal plants should be explored to determine the potential of 

these medicinal plants in the treatment of serious human and animal infectious diseases. 

 

 

 

 

 

 

 

 

 

 

 

 

 

  

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Table 2: Antimicrobial activity of aqueous extract of Sida acuta against the test 

organisms 

 

Concentration 

     (mg/ml) 

Mean Zone of Inhibition (mm) ± SD 

S. mutans S. sanguis S. mutans 

(ATCC 

700610) 

S. sanguis 

(ATCC 10556) 

200 19.2±0.2 18.7±0.0 14.5±0.3 21.5±0.7 

150 15.5±0.1 14.0±0.0 10.0±0.0 14.5±0.7 

100 11.2±0.1 7.7±0.0 7.5±0.3 7.5±0.7 

50 9.5±0.2 0±0.0 0±0.0 0±0.0 

25 7.5±0.2 0±0.0 0±0.0 0±0.0 

12.5 7.3±0.0 0±0.0 0±0.0 0±0.0 

6.25 0±0.0 0±0.0 0±0.0 0±0.0 

3.125 0±0.0 0±0.0 0±0.0 0±0.0 

Controls 

Penicillin(1.5i.u) 20.3±0.0 20.0±0.0 20.0±0.0 21±0.0 

Gentamicin 

(10µg) 

12.3±0.0 17.0±0.0 17.0±0.0 16±0.0 

Distilled water 0±0.0 0±0.0 0±0.0 0±0.0 

 

 

 

 

 

 

 

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50 

 

 

Table 3: Antimicrobial activity of aqueous extract of Anthocleista nobilis against 

the test organisms 

 

 Mean Zone of Inhibition (mm) ± SD 

Concentration 

     (mg/ml) 

S. mutans S. sanguis S. mutans 

(ATCC 

700610) 

S. sanguis 

(ATCC 10556) 

200 15.7±0.0 12±0.0 14±0.0 15±0.0 

150 11.2±0.3 9.7±0.0 12.5±0.7 12±0.0 

100 8.3±0.0 8.0±0.0 13±0.0 10±0.0 

50 7.5±0.2 0±0.0 7.5±0.7 0±0.0 

25 0±0.0 0±0.0 0±0.0 0±0.0 

12.5 0±0.0 0±0.0 0±0.0 0±0.0 

6.25 0±0.0 0±0.0 0±0.0 0±0.0 

3.125 0±0.0 0±0.0 0±0.0 0±0.0 

Controls 

Penicillin(1.5i.u) 20.3±0.0 20.0±0.0 20.0±0.0 21±0.0 

Gentamicin 

(10µg) 

12.3±0.0 17.0±0.0 17.0±0.0 16±0.0 

Distilled water 0±0.0 0±0.0 0±0.0 0±0.0 

 

 

 

 

 

 

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51 

 

Table 4: Antimicrobial activity of aqueous extract of Zanthozylum leprieurii 

against the test organisms 

 

 Mean Zone of Inhibition (mm) ± SD 

Concentration 

     (mg/ml) 

S. mutans S. sanguis S. mutans 

(ATCC 

700610) 

S. sanguis 

(ATCC 10556) 

200 8.2±0.1 0±0.0 8.5±0.7 0±0.0 

150 7.0±0.0 0±0.0 7±0.0 0±0.0 

100 0±0.0 0±0.0 0±0.0 0±0.0 

50 0±0.0 0±0.0 0±0.0 0±0.0 

25 0±0.0 0±0.0 0±0.0 0±0.0 

12.5 0±0.0 0±0.0 0±0.0 0±0.0 

6.25 0±0.0 0±0.0 0±0.0 0±0.0 

3.125 0±0.0 0±0.0 0±0.0 0±0.0 

Controls 

Penicillin(1.5i.u) 20.3±0.0 20.0±0.0 20.0±0.0 21±0.0 

Gentamicin 

(10µg) 

12.3±0.0 17.0±0.0 17.0±0.0 16±0.0 

Distilled water 0±0.0 0±0.0 0±0.0 0±0.0 

 

 

 

 

 

   

  

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52 

 

Table 5: Antimicrobial activity of ethanolic extract of Sida acuta against the test 

organisms 

 

Concentration 

     (mg/ml) 

Mean Zone of Inhibition (mm) ± SD 

S. mutans S. sanguis S. mutans 

(ATCC 

700610) 

S. sanguis 

(ATCC 10556) 

200 22.7±0.1 18.8±0.2 22.5±0.7 15.5±0.3 

150 18.2±0.2 13±0.0 18.5±0.7 10±0.0 

100 16.2±0.2 8.7±0.0 12±0.0 7.5±0.3 

50 14.3±0.0 7.7±0.0 9.5±0.7 0±0.0 

25 12.7±0.2 0±0.0 0±0.0 0±0.0 

12.5 10.8±0.0 0±0.0 0±0.0 0±0.0 

6.25 9.5±0.0 0±0.0 0±0.0 0±0.0 

3.125 7.8±0.1 0±0.0 0±0.0 0±0.0 

Controls 

Penicillin(1.5i.u) 20.3±0.0 20.0±0.0 20.0±0.0 21±0.0 

Gentamicin 

(10µg) 

12.3±0.0 17.0±0.0 17.0±0.0 16±0.0 

Distilled water 0±0.0 0±0.0 0±0.0 0±0.0 

 

 

 

 

 

 

 

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53 

 

Table 6: Antimicrobial activity of ethanolic extract of Anthocleista nobilis against 

the test organisms 

 

Concentration 

     (mg/ml) 

Mean Zone of Inhibition (mm) ± SD 

S. mutans S. sanguis S. mutans 

(ATCC 

700610) 

S. sanguis 

(ATCC 10556) 

200 17.3±0.0 13.3±0.0 21.5±0.7 14±0.0 

150 11±0.0 10±0.0 18±0.0 12.5±0.3 

100 8.7±0.0 7.7±0.0 10±0.0 11±0.0 

50 7.0±0.0 0±0.0 8±0.0 7±0.0 

25 0±0.0 0±0.0 0±0.0 0±0.0