ANTIMICROBIAL ACTIVITY OF HIBISCUS SABDARIFFA AGAINST CLINICAL ISOLATES OF BACTERIA PRISCILLA ADELAIDE NAA AGOWA LOVELL-ANTIAYE (10397194) Department of Microbiology, University of Ghana Medical School College of Health Sciences, Korle- Bu Accra- Ghana THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL MICROBIOLOGY DEGREE JULY 2014 University of Ghana http://ugspace.ug.edu.gh i 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:_________________ Priscilla Adelaide Naa Agowa Lovell-Antiaye Principal Supervisor: Signature: ____________________________________Date:_________________ Prof. Mercy J. Newman Clinical Microbiology Department, School of Biomedical and Allied Health Sciences, University of Ghana Co-Supervisor: Signature: ____________________________________Date:_________________ Dr. Elizabeth S. Bannerman Clinical Microbiology Department, School of Biomedical and Allied Health Sciences, University of Ghana University of Ghana http://ugspace.ug.edu.gh ii ABSTRACT There have been several reports on the antimicrobial activity of different herbal extracts. Medicinal plants are believed to be the best source for obtaining a variety of drugs. Hibiscus sabdariffa L. is a medicinal plant known for its nutritional delicacy and medicinal properties such as the treatment of diseases like hypertension, coughs, biliousness, boils and wounds. Currently, the main therapy for bacterial infections is the use of synthetic antibiotics. However, the misuse and overuse of antibiotics has become the key factor for the emergence of drug resistant strains of several groups of microorganisms. Hence, the need to investigate the antimicrobial properties of medicinal plants. The objectives of the study were to investigate the in vitro inhibitory activity of Hibiscus sabdariffa calyces, leaves and roots preparation against some clinical isolates of bacteria. Secondly, to assess the antimicrobial property of Hibiscus sabdariffa against the clinical resistant strains of bacteria. Lastly, to compare the Hibiscus sabdariffa aqueous and ethanolic extract effects on clinical isolates of bacteria. The antibacterial activities of H. sabdariffa calyces, leaves and roots extracts (12.5, 25, 50, 100, 200mg/ml) were tested against clinical isolates of bacteria (Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella sp., Salmonella typhi, Staphylococcus aureus); with reference strains of Escherichia coli ATCC 25922, Proteus mirabilis ATCC 49565, Salmonella typhi ATCC 19430, Klebsiella pneumoniae ATCC 33495, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 25923; and two each Extended Spectrum Beta-Lactamases and Methicillin-Resistant University of Ghana http://ugspace.ug.edu.gh iii Staphylococcus aureus strains using Agar Diffusion and Minimum Inhibitory Concentration Methods. The calyces of the plant gave the highest inhibitory effect (P <0.05) followed by the leaves. The roots did not exhibit antibacterial activity against the test bacteria (P>0.05). The calyces extract was potent against all the test organisms except Proteus mirabilis which was resistant to all the plant extracts. Staphylococcus aureus showed the highest susceptibility to the calyces extract. The aqueous leaves extracts were active against Pseudomonas aeruginosa and Escherichia coli. The resistant strains especially MRSA were highly susceptible to the calyces extract with Minimum Inhibitory Concentration Methods values of 6.25mg/ml. Ethanolic extract of the calyces had a slightly higher antibacterial activity while the leaves were effective when extracted by water. Therefore, the plant’s calyces and leaves can be a source for the production of antibiotics which could significantly inhibit the growth of bacteria. It is recommended that more studies should be done using different solvents for extracting only the important compounds from the H. sabdariffa plant. Purification measures would also be necessary. Additionally, there is a need to investigate the phytochemical compounds from the H. sabdariffa plant part extracts. University of Ghana http://ugspace.ug.edu.gh iv DEDICATION This work is dedicated to God and my inspiring family especially the loving memory of my father, Mr. Robinson Lovell- Antiaye, who zealously believed in the pursuit of higher education and longed for this time in my life. University of Ghana http://ugspace.ug.edu.gh v ACKNOWLEDGEMENT I thank the Almighty God who strengthened me and endowed me with wisdom throughout my course of study. He has made this dream a reality. All glory is given to Him. I would like to express my sincere gratitude to my supervisors Professor Mercy J. Newman and Dr. Elizabeth S. Bannerman for their valuable advice, inspiration and enthusiasm towards my research and thesis writing. I am grateful to the staff of Biotechnology Centre, School of Agriculture, University of Ghana, Legon for their contributions towards the research work. I also appreciate the diverse ways the staff of Centre for Plant Medicine Research (CPMR), Mampong Akwapem contributed to the completion of my research work. I am thankful to the staff of the Microbiology Unit, Central Laboratory, Korle-Bu Teaching Hospital for their technical support and encouragement during this study. Special thanks go to the staff of the Department of Microbiology, University of Ghana Medical School for their contributions towards the research work. I would like to show appreciation to my husband Flt Lt Kwaku Gyamfi Bediako- Dinkunim, my mother, Mrs. Hannah Lovell- Antiaye, and sister, Mrs. Agnes Mahama, for their support and prayers. I also acknowledge the financial and emotional supports of my uncle, Mr. Matthew Antiaye and my entire family. Finally, I am indebted to my friends Mary, Noah and Shadrach for their encouragements towards my masters’ programme. University of Ghana http://ugspace.ug.edu.gh vi TABLE OF CONTENTS DECLARATION………………………………………………………………………..…..i ABSTRACT .......................................................................................................................... ii DEDICATION ..................................................................................................................... iv ACKNOWLEDGEMENT ..................................................................................................... v TABLE OF CONTENTS ..................................................................................................... vi LIST OF FIGURES ............................................................................................................ viii LIST OF PLATES ................................................................................................................ ix LIST OF APPENDICES ....................................................................................................... x LIST OF ABBREVIATIONS ............................................................................................. xii CHAPTER ONE .................................................................................................................... 1 1.0 INTRODUCTION ................................................................................................... 1 1.1 Background.................................................................................................................. 1 1.2 Problem Statement....................................................................................................... 2 1.3 Significance of Study .................................................................................................. 2 1.4 Hypothesis ................................................................................................................... 3 1.5 Aim .............................................................................................................................. 3 1.6 Specific Objectives ...................................................................................................... 3 CHAPTER TWO ................................................................................................................... 5 2.0 LITERATURE REVIEW ............................................................................................ 5 2.1 Medicinal Plants .......................................................................................................... 5 2.1.1 Uses of Medicinal plants ...................................................................................... 5 2.1.2 Plants as antimicrobials ........................................................................................ 6 2.1.2.1 Brief history of plants as antimicrobials ........................................................ 7 2.1.2.2 Present use of plants as antimicrobials .......................................................... 8 2.2 Plants with Promising Antimicrobial Activity ............................................................ 9 2.3 Hibiscus sabdariffa Plant .......................................................................................... 10 2.3.1 Local and Foreign Names ................................................................................... 11 2.3.2 Uses .................................................................................................................... 12 2.3.2.1 Nutritional use ............................................................................................. 12 University of Ghana http://ugspace.ug.edu.gh vii 2.3.2.2 Medicinal use............................................................................................... 13 CHAPTER THREE ............................................................................................................. 17 3.0 METHODOLOGY ........................................................................................................ 17 3.1 Study Sites ................................................................................................................. 17 3.2 Study design .............................................................................................................. 17 3.3 Plant and Specimen collection and storage ............................................................... 17 3.4 Laboratory Analysis .............................................................................................. 19 3.4.1 Culture interpretation and Biochemical Identification Test ............................... 19 3.4.2 Preparation of extracts ........................................................................................ 20 3.4.3 Ethanolic Extraction ........................................................................................... 21 3.4.4 Aqueous Extraction ............................................................................................ 21 3.4.5 Evaluation of the antimicrobial activity of Hibiscus sabdariffa ........................ 22 3.4.5.1 Preparation of extract stock solution ........................................................... 22 3.4.5.2 Agar diffusion method ................................................................................. 22 3.4.5.3 Micro-dilution method................................................................................. 23 3.5 Ethics ......................................................................................................................... 25 3.6 Statistical Analysis of Data ....................................................................................... 25 CHAPTER FOUR ............................................................................................................... 26 4.0 RESULTS ...................................................................................................................... 26 4.1 Antimicrobial activity of the extracts (Agar diffusion method) ................................ 26 4.2 MIC of the extracts .................................................................................................... 31 CHAPTER FIVE ................................................................................................................. 34 5.0 DISCUSSION............................................................................................................ 34 5.1 LIMITATIONS ......................................................................................................... 39 CHAPTER SIX ................................................................................................................... 40 CONCLUSION AND RECOMMENDATIONS ................................................................ 40 6.0 CONCLUSION ......................................................................................................... 40 6.1 RECOMMENDATIONS .......................................................................................... 40 REFERENCES .................................................................................................................... 42 APPENDICES ..................................................................................................................... 49 University of Ghana http://ugspace.ug.edu.gh viii LIST OF FIGURES Figure 4.1: Antimicrobial activity of different concentrations of aqueous and ethanol calyces extract of H.sabdariffa against the clinical isolates of bacteria .............................. 27 Figure 4.2: Antimicrobial activity of different concentrations of aqueous and ethanol calyces extract of H.sabdariffa against the control ATCC strains of bacteria. ................... 28 Figure 4.3: Antimicrobial activity of H. sabdariffa calyces against clinical resistant strains of bacteria ............................................................................................................................ 29 Figure 4.4: Antimicrobial activity of H.sabdariffa leaves against clinical isolates of bacteria ................................................................................................................................ 29 Figure 4.5: Minimum Inhibitory Concentration of aqueous and ethanol calyces extract of H. sabdariffa on the test bacteria. ........................................................................................ 33 University of Ghana http://ugspace.ug.edu.gh ix LIST OF PLATES Plate 1:A, B, C showing the antimicrobial susceptibility test of H. sabdariffa leaves, calyces and roots against some test bacteria ........................................................................ 31 Plate 2:Intact calyces of the Hibiscus sabdariffa plant ....................................................... 53 Plate 3:Roots of Hibiscus sabdariffa ................................................................................... 53 Plate 4:Leaves of Hibiscus sabdariffa ................................................................................. 54 University of Ghana http://ugspace.ug.edu.gh x LIST OF APPENDICES Appendix 1: Preparation of reagents and chemicals ........................................................... 49 Appendix 2:Preparation of culture media ............................................................................ 49 Appendix 3: Pictures of H. sabdariffa plant parts…………………………………………53 Appendix 4: Mean zone of inhibition of Hibiscus sabdariffa calyces extract concentration effect on the clinical isolates of bacteria ............................................................................. 55 Appendix 5:Mean zone of inhibition of Hibiscus sabdariffa calyces extract concentration effect on the control ATCC strains of bacteria .................................................................... 56 Appendix 6: Mean zone of inhibition of Hibiscus sabdariffa calyces extract concentration effect on four resistant strains of bacteria. ........................................................................... 57 Appendix 7:Mean zone of inhibition of Hibiscus sabdariffa leaves extract concentration effect on the clinical isolates of bacteria. ............................................................................ 58 Appendix 8:Mean zone of inhibition of Hibiscus sabdariffa leaves extract concentration effect on the control ATCC strains of bacteria .................................................................... 59 Appendix 9:Mean zone of inhibition of Hibiscus sabdariffa leaves extract concentration effect on four resistant strains of bacteria. ........................................................................... 60 Appendix 10:Mean zone of inhibition of Hibiscus sabdariffa roots extract concentration effect on the clinical isolates of bacteria ............................................................................. 61 Appendix 11:Mean zone of inhibition of Hibiscus sabdariffa roots extract concentration effect on the control ATCC strains of bacteria .................................................................... 62 Appendix 12:Mean zone of inhibition of Hibiscus sabdariffa roots extract concentration effect on four resistant strains of bacteria. ........................................................................... 63 University of Ghana http://ugspace.ug.edu.gh xi Appendix 13:Clinical Test Microorganisms To Reference Antibiotics (Agar Diffusion Test) ..................................................................................................................................... 64 Appendix 14:Control ATCC strains to reference antibiotics (Agar Diffusion Test) .......... 65 Appendix 15:Minimum Inhibitory Concentration of some selected test bacteria .............. 65 Appendix 16-19: All statistical tables ................................................................................. 67 Appendix 20: Ethical Clearance..........................................................................................75 University of Ghana http://ugspace.ug.edu.gh xii LIST OF ABBREVIATIONS ATCC American Type Culture Collection BP British Pharmacopoeia CLED Cystine Lactose Electrolyte Deficient CLSI Clinical and Laboratory Standards Institute CPMR Centre for Plant Medicine Research DCA Deoxycholate Citrate Agar DMSO Dimethyl sulfoxide ESBL Extended Spectrum Beta-Lactamases GHS Ghana Health Service INT Iodonitrotetrazolium Chloride KBTH Korle- Bu Teaching Hospital LF Lactose Fermenters MDR Multidrug Resistance MHB Mueller-Hinton Broth MIC Minimum Inhibitory Concentration MRSA Methicillin-Resistant Staphylococcus aureus University of Ghana http://ugspace.ug.edu.gh xiii NLF Non-Lactose Fermenters UNESCO United Nations Educational, Scientific and Cultural Organization WHO World Health Organization University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE 1.0 INTRODUCTION 1.1 Background According to WHO 2008, “Traditional medicine is the sum total of knowledge, skills and practices based on the theories, beliefs and experiences, indigenous to different cultures that are used to maintain health, as well as to prevent, diagnose, improve or treat physical and mental illnesses”. Herbal medicine is the most common form of traditional medicine that uses plants or other plant materials as active ingredients (WHO, 2008). WHO report 2008 estimates that about 80% of the population in Asia and Africa depend on traditional medicine for primary health care. In Ghana, the doctor to population ratio in 2009 was 1:11,929. Greater Accra region had the best doctor to population ratio of 1:5,103 with the Northern region having the worst of 1:50,751 (GHS, 2010). This suggests that some people especially those in areas with low doctor to population ratio mostly rely on medicinal plants which are more accessible and less costly to them. Hibiscus sabdariffa is a medicinal plant that belongs to the Malvaceae family, commonly found in the tropics and subtropics. It is known as roselle, sorrel, karkade and Florida cranberry (Morton, 1987). Locally, the plant is called Rarna, Sule (Hausa), Sakpa (Ga) Eυema (Ewe) (Dokosi, 1998). The H. sabdariffa plant is consumed as food and also useful in herbal medicine (Cisse et al., 2009). The young shoots and leaves are used as vegetables and potherbs. The calyces used to make beverages and jams. The calyces are also used as colouring matter (Dokosi, 1998). Hibiscus sabdariffa is medicinally used as a University of Ghana http://ugspace.ug.edu.gh 2 laxative, an anticarcinogenic, an antihypertensive, and a cholesterol lowering medicine. It exhibits great antioxidant activity, lowers hepatoxicity and reduces fever. In some parts of Africa it is used as a remedy for abscesses, bilious conditions, cough, sores, wounds, dysuria, and scurvy (Morton, 1987). According to Fullerton et al. (2011), Hibiscus sabdariffa also holds a great promise as an antimicrobial agent. There is therefore the need for more research to be done to assess the effectiveness of this medicinal plant. 1.2 Problem Statement Currently, the main therapy for bacterial infections is the use of synthetic antibiotics. However, the misuse and overuse of antibiotics has become the key factor for the emergence of drug resistant strains of several groups of microorganisms. Drug resistance is now a global public health threat that involves all major microbial pathogens and antimicrobial drugs (Stuart and Bonnie, 2004). Therefore, researchers are now turning their attention to herbal products, investigating for new hints to develop better drugs against resistant strains (Braga et al., 2005). Plant-based therapeutics are known to be easily biodegradable, having no or minimal adverse side effects, and being easily accessible at low prices as such there is a high demand for it in both developing and developed countries (Fullerton et al., 2011). There is therefore the need to find new herbal antimicrobial agents in this era of rapid global spread of resistant isolates to commonly used antibiotics. 1.3 Significance of Study The emergence of resistant strains of microbes to commonly used synthetic drugs has become a global concern. Some reports have indicated that herbal products may help University of Ghana http://ugspace.ug.edu.gh 3 alleviate this problem. In Africa, most people use herbal products to cure ailments. However, there are few published reports of effectiveness of these products. It is certain that some of these herbal products will provide the solution to drug resistance. There is therefore the need to investigate the antimicrobial properties of medicinal plants. In view of the above stated reasons, the study was intended to investigate the in vitro inhibitory activity of Hibiscus sabdariffa preparation against some clinical isolates of bacteria. Secondly, it assessed the Hibiscus sabdariffa aqueous and ethanolic extract and its effects on some clinical isolates of bacteria. Lastly, the study may provide useful information on the antimicrobial property of Hibiscus sabdariffa against the resistant strains of clinically isolated bacteria. 1.4 Hypothesis The study hypothesizes that Hibiscus sabdariffa plant has antimicrobial properties that are effective against some clinical isolates of bacteria. 1.5 Aim This research work aims to determine the antimicrobial activity of Hibiscus sabdariffa leaves, calyces and roots against clinical isolates of selected bacteria. 1.6 Specific Objectives The specific objectives of this work are: 1. To evaluate the antimicrobial activity of Hibiscus sabdariffa leaves, calyces and roots extracts against some clinical isolates of bacteria from KBTH. 2. To assess antimicrobial effect of the extracts of Hibiscus sabdariffa leaves, calyces and roots against typed resistant clinical isolates. University of Ghana http://ugspace.ug.edu.gh 4 3. To compare the antimicrobial activity of aqueous and ethanolic extracts of Hibiscus sabdariffa leaves, calyces and roots against clinical isolates of bacteria from KBTH. University of Ghana http://ugspace.ug.edu.gh 5 CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 Medicinal Plants “A medicinal plant is any plant in which one or more of its organ, contains a substance that can be used for therapeutic purpose or which is a precursor for synthesis of useful drugs” (Sofowora, 1982). Plants are acknowledged to be the richest bio-resource of drugs for traditional and modern medicines, as well as pharmaceutical precursors and chemical entities for synthetic drugs (Sukhdev et al., 2008). 2.1.1 Uses of Medicinal plants According to the World Health Organization (WHO) almost 80% of the human population depends on medicinal plants. This indicates the importance of plants for therapeutic purposes. Medicinal plants, since ancient times as described in Vedas and Bible texts, have been used by different cultures as a source of medicine. UNESCO (1996) observed that in most developing countries the use of traditional medicine and medicinal plants is a basis for health maintenance. In the industrialized world, the use of medicinal plants has been traced to the extraction and development of several drugs and chemotherapeutics from these plants (UNESCO, 1998). Traditional information on medicinal plants has provided many important drugs such as the antimalarial drugs Quinine and Artemisinin derived from Cinchona sp. and Artemesia annua plants respectively (Joy et al., 2001). University of Ghana http://ugspace.ug.edu.gh 6 Traditional medicine can historically be traced to every region. The practice of traditional medicine is deeply entrenched in a specific social-cultural context, which varies between communities (Rukangira, 2001). In Ghana, both rural and urban regions consider medicinal plants as important. Studies by Irvine (1961) and Abbiw (1990) have emphasized the importance of medicinal plants in Ghana. The use of herbal drugs is generally associated with quality and availability of health care resources. In rural Ghana, the low doctor to population ratio compels inhabitants to rely mostly on medicinal plants which are more accessible and less costly to them (Abbiw, 1996). Therefore, herbal medicines are generally the first line of treatment in rural areas, where as in the urban areas, herbal medicines are used for persistent problems for which patients cannot find a cure from modern medicine. 2.1.2 Plants as antimicrobials Over the last ten years, lots of researches have been aimed at the discovery of antimicrobial agents from plants. One of the key reasons for these studies is the emergence of multidrug resistance (MDR) bacteria to drugs which are currently available, which can be life threatening. Another reason is the discovery that, besides plant extracts possessing antimicrobial agents against a wide range of microorganisms; they also have other medicinal benefits (González- Lamothe et al., 2009). Plants naturally produce a substance known as phytoanticipins which is constantly being synthesized by plants that act as chemical barriers to microorganisms. Phytoalexin is also a plant material which is produced in response to many stimuli, and can impede microbial attack (VanEtten et al., 1994). Studies are underway to discover if these substances could contribute to the production of new antimicrobials (González- Lamothe et al., 2009). University of Ghana http://ugspace.ug.edu.gh 7 Additionally, antibacterial plant products such as phenolic acids, polyphenols, phenanthrenes, flavonoids, and terpenoids have been studied by a number of researchers but currently, no product has been accepted for systemic use against bacterial infections, partly due to the non-specific mode of action of these purified compounds. Also, the presence of a mixture of different compounds with variable degrees of bioactivity and cytotoxicity has made it challenging to isolate specific active components from plant extracts (Rios and Recio, 2005). Gram negative bacteria are the most common pathogens of plants. However, purified plant products show low activity against such organisms. Gram positive bacteria, on the other hand, are often sensitive to these biologically active plant products and this suggests that the susceptibility of Gram negatives and Gram positives organisms to purified plant products is due to their cell wall and membrane arrangement. This also suggests that antibacterial activity against Gram negative bacteria can be executed by the use of a mixture of plant natural compounds. Advances in the understanding plant products and their mode of action by experimental methods will provide evidence of the bioactivities of plant extracts that could enhance the probability of some therapeutic applications (González- Lamothe et al., 2009). 2.1.2.1 Brief history of plants as antimicrobials The concept that plants contain therapeutic agents is prehistoric. Worldwide, humans have used infusions of indigenous plants for different purposes (Cowan, 1999). Since the creation of the world, plants have been used as food by human beings and some animals. It is assessed that there are 250,000 to 500,000 species of plants (Borris, 1996) of which only about 1 to 10% is used as food, and more than 80,000 are medicinal (Joy et al., 2001). In University of Ghana http://ugspace.ug.edu.gh 8 the late fifth century B.C, Hippocrates stated 300 to 400 medicinal plants (Moerman, 1996). The Bible also mentions about 30 medicinal plants such as frankincense and myrrh. The fall of ancient civilizations led to the loss of the pharmaceutical plant documents which averted the improvements of medicinal plants (Stockwell, 1988). However, the Arabs and Asians are discovering their antiquated works on medicinal plants to build upon them. In North America, the use of medicinal plants can be traced to the Native Americans who used plants extensively for medicinal purposes (Weiner, 1980). The answer to the harmful medical practices, in the 19th century, such as the use of mercury baths to treat syphilis and hallucinogens as tuberculosis “cure” as observed by Oliver Wendell Holmes, led to the consideration of plants for therapeutic purposes (Holmes, 1861). The usage of plants in medicine is gaining popularity. For example, in cancer treatment, plants with antileukaemic alkaloids, vinblatine and vincristine such as Catharanthus roseus syn. Vinca roseus are used (Nelson, 1982). As an antimicrobial agent, plants are being targeted for the production of novel drug compounds for the treatment of infectious diseases. Some examples of drugs of plant origin known for their antimicrobial properties are isoquinoline alkaloid emetine, an amoebicidal drug, obtained from the underground part of Cephaelis ipecacuanha. Quinine from the bark of Cinchona tree is used as an antimalarial drug (Iwu et al., 1999). 2.1.2.2 Present use of plants as antimicrobials Today, crude forms of plant materials used in traditional healing practices are being modified and used as commercially available drugs. It is estimated that 50% Western drugs are derived from plant materials (Robbers, 1996). The advantages of using plant derived University of Ghana http://ugspace.ug.edu.gh 9 medicines are that they are safer than synthetic drugs, affordable and available, and they offer great therapeutic value. Therapeutic Benefit The use of natural products as antimicrobials arises from microbial sources. Penicillin discovery led to later discoveries of streptomycin and aureomycin. Most of the clinical antibiotics are produced by soil microorganisms or fungi. Plants such as Lichens have bacteriostatic and antifugicidal properties and garlic (Allium sativum) produces allinine, which is an antibacterial agent (Trease and Evans, 1972). Economic Benefit Over the last decade, there has been a surge of interest in natural products globally. This is due to the belief that natural products are effective than conventional medicines. Herbal products worldwide have increased jobs employment and revenue to countries. In the United States, botanical products have reached $3.1 billion of the $10.4 billion dollar dietary supplement industry (NBJ, 1998). International trade of Hibiscus sabdariffa calyces (roselle) has also increased over the last ten years, with 15,000 tonnes per year entering the world market. In 1998, United States and Germany, the large importers, paid 1200–1700 US$ per tonne for Egyptian and Sudanese roselle. This further goes to highlight the economic benefit of plants to countries that cultivate them (McClintock and El Tahir, 2004). 2.2 Plants with Promising Antimicrobial Activity A number of plants are believed to possess biological activity against a wide variety of microorganisms. Many of these plants are believed to be found in Africa such as Garcinia kola (Guttiferae), Acanthospermum hispidum, Nauticlea latifolia, Bridelia atroviridis and University of Ghana http://ugspace.ug.edu.gh 10 Zanthoxylum gilletis, Phyllanthus niruri, Cassia alata, Ageratum conyzoides, and Sida acuta. These medicinal plants have been studied and are known to have anti-infective activity against Gram positive and Gram negative organisms (Hoffman et al., 2004; Agyare et al., 2006). Many medicinal plants of Africa are still under investigation for their chemical component and bioactivity against microorganisms. One plant that holds an enormous promise of an antimicrobial agent is the Hibiscus sabdariffa plant (Fullerton et al., 2011). 2.3 Hibiscus sabdariffa Plant The Hibiscus sabdariffa plant belongs to the Malvaceae family. There are two main types of the plant: H. sabdariffa var. sabdariffa and H. sabdariffa var. altissima. The latter is a tall unbranched plant about 300-480cm high grown for fibre. Hibiscus sabdariffa var. sabdariffa is a bushy shrub with red or pale green stems and red or pale yellow inflated edible calyces (Dokosi, 1998). Hibiscus sabdariffa plant is believed to have originated from Africa specifically Sudan. It was then introduced to India and America in the 17th century, and Sri Lanka, Thailand, Malaysia and Java at the start of the 20th century. The plant is distributed throughout the tropics and subtropics especially in tropical Africa, Asia and some parts of India. In tropical Africa, it is especially common in the West and Central Africa. However, truly wild plants of Hibiscus sabdariffa have been collected in Ghana, Niger, Nigeria and Angola (McClintock and El Tahir, 2004). It is an annual plant usually grown mainly in April or in late August and requires about four months to mature. The plant is cultivated from the seed or some cut plant parts (Stephens, 2012). University of Ghana http://ugspace.ug.edu.gh 11 Hibiscus sabdariffa is an erect, slightly branched herb. The plant may have a smooth or hispid stem with glabrous, long-stalked, palmate leaves. The palmately lobed leaves are divided into 3-5 lobes with serrate margins. The pedunculate flowers are large and coloured red to yellow. The ½ to 1½ inches in diameter red calyces are fleshy when matured. The root is a deep penetrating tap root (Dokosi, 1998). Figure 2.3. Hibiscus sabdariffa plant (Retrieved, from http://toptropicals.com/cgi- bin/garden_catalog/cat.cgi?uid=Hibiscus_sabdariffa, November 19, 2013) 2.3.1 Local and Foreign Names The local names of Hibiscus sabdariffa plant indicate that the plant is very popular among the people of Northern, Western and certain parts of the southern Ghana. The plant is called Digbemre or Injamgbam (Dagbani); Rarna or Sule (Hausa); Bito (Moshi); Riaripari University of Ghana http://ugspace.ug.edu.gh 12 (Guan -Krachi); Nangana (Frafra); Tingyanbam (Konkomba); Sakpa (Ga); Eυema (Ewe) (Dokosi, 1998). Hibiscus sabdariffa has diverse foreign names which suggest the presence of the plant in several parts of the world. In most French-speaking West African countries, Hibiscus sabdariffa is commonly known as Bissap. In Nigeria: Zobo; Sierra Leone: Sour – sour; Sudan: Furundu, Sudan Tea; Japan, Malaysia, West Indies the plant is known as Roselle; Arabia, Egypt, Switzerland: Karkarde; Guinea, Jamaica: Red Sorrel; India: Mesta; Iran: Sour Tea; Thailand: Thai Red; Florida: Florida cranberry (Morton, 1987). 2.3.2 Uses 2.3.2.1 Nutritional use The plant H. sabdariffa var. sabdariffa is mainly used as a vegetable and for preparation of beverage. The calyces of Hibiscus sabdariffa are mainly used for the preparation of beverages, especially as tea and coffee substitute for people who are sensitive to stimulants (Irvine, 1961). It is also used in preserves, jelly, juice, or as a sauce (Stephens, 2012) and also in making roselle wine, gelatin, pudding and cakes. The calyces can be used for colouring food and drinks (Duke and Atchley, 1984). In Ghana, a drink known as Sooboro (Hausa) is made by boiling the calyces in water, sweetened with sugar and may flavored with ginger and other local spices. The leaves and calyces are also used as a cooked vegetable or used to make a soup. In Senegal, sauces made with the leaves and calyces, and eaten with tuber and cereals. Additionally, in the United States of America, the leaves are used for salads, and the red fleshy calyx used in fruit salads. Also, during the dry season, University of Ghana http://ugspace.ug.edu.gh 13 especially in Côte d’Ivoire, the dried calyces are ground to powder and used in sauces. The plant is used as feed for livestock after the harvest of calyces. The leaves of Hibiscus sabdariffa are also used in soups in Northern Ghana (Dokosi, 1998). In many West African countries, the green leaves of the plant are eaten as a potherb. The H. sabdariffa leaves are added to a soup prepared with groundnut and “dawadawa” (ground, fermented seeds of Parkia clappertoma). The dried leaves are kept in pots, and used in soup preparation during the dry season. When cooking, the water is changed many times to reduce the sliminess of the potherb. This water may be used in preparing millet or guinea-corn porridge which keeps a few more days in good condition than that prepared with ordinary water. This proposes preservation properties of the leaves. The Hibiscus sabdariffa seeds are eaten roasted as a snack or ground into meal (McClintock and El Tahir, 2004). The seeds are used as groundnut substitute in times of scarcity. The fermented seeds are used as a substitute for “dawadawa” (Dokosi, 1998). The Nigerians ferment roselle seeds to make ‘sorrel meat’ cake. The oil of roselle seed is extracted and used for cooking. However, the seed oil is claimed to contain some toxic substances and may be better used in the soap and cosmetics industries. In Chad, Tanzania and China, the oil is used for soap and cosmetics. This oil is also used as a substitute for castor oil. The residue, after removal of the oil, is used as food, either in soup or mixed with beans and other ingredients into cakes (McClintock and El Tahir, 2004). 2.3.2.2 Medicinal use Hibiscus sabdariffa has been found to possess several health benefits. H. sabdariffa is medically used as an antiseptic, antibilious, antiscorbutic, aphrodisiac, astringent, diuretic, University of Ghana http://ugspace.ug.edu.gh 14 emollient, purgative, sedative and tonic. It is also a folk remedy for abscesses, cough, and dysuria (Perry, 1980). The calyces of plant are used to lower high blood pressure. The calyces of Hibiscus sabdariffa have been found to contain gossypetin, anthocyanin, glycoside, hibiscin and citric acid, which confer the diuretic, choleretic and antihypertensive properties of the plant (McKay et al., 2010). In Nigeria, the red calyces are soaked in water and the liquid, drunk as a tonic. The leaves and seeds are used in the treatment of scurvy, which suggests the presence of vitamin C. It may also have antioxidant properties (Duke & Atchley, 1984). The mucilaginous leaves are used in pharmaceutical and cosmetics industries. Angolans used it as emollient and a soothing cough remedy. In Senegal, the leaves are used in the treatment of conjunctivitis. Leaves are applied as a poultice to heal sores and ulcers especially in Central Africa (McClintock and El Tahir, 2004). The seeds of Hibiscus sabdariffa also act as an aphrodisiac (Irvine, 1961). After oil extraction of Hibiscus sabdariffa seeds, the residue can be moistened and applied to wounds. Taiwanese regard the seed as diuretic, laxative and tonic. In India, a decoction of the seeds was given to relieve dysuria and many cases of dyspepsia and debility. The roots of Hibiscus sabdariffa are also used in treating syphilitic sores or chancre. The roots also act as aperients and tonic (Mehta, 1994). The constituents of the plant include citric, tartaric acids, hibiscin, and malic acids, delphinidin, anthocyanin hibiscitrin, gossypitrin, sabdaritrin and cyanidin. The antimicrobial activity of the plant can be linked to the flavonoids (Badreldin et al., 2005). Flavonoids are polyphenolic compounds which have been known to possess antimicrobial University of Ghana http://ugspace.ug.edu.gh 15 activity (Cowan, 1999). The antimicrobial mechanisms of flavonoids can be classified as the inhibition of nucleic acid synthesis, cytoplasmic membrane function, and energy metabolism (Cushnie and Lamb, 2005). The H. sabdariffa plant has also exhibited antifungal, antiparasitic and antibacterial actions. Studies indicate that an ethanol extract of the dried leaves of the plant has been shown to have an in vitro inhibitory effect against some fungi that include Aspergillus fumigatus, Rhizopus nigricans and Trichophyton (Guerin and Reveillere, 1984). An aqueous extract of dried sepals of H. sabdariffa was active against Schistosoma mansoni (Elsheikh et al., 1990). In Ghana, little work has been done on its medicinal use. Frimpong (2008) studied the suitability of Hibiscus sabdariffa calyx extract as colouring agent for paediatric syrups. She found out that the colour conformed to the BP standard. The study also revealed that the plant has antimicrobial properties with methanolic extracts showing a better antimicrobial activity than the aqueous extract. According to Sharaf et al., 1966, the colouring matter of the calyces is said to be lethal to Mycobacterium tuberculosis. Other works done by Sharaf et al., 1966, similarly showed that the aqueous extracts of H. sabdariffa prevented the growth of Pasteurella, Pseudomonas, Proteus and Streptococcus bacteria. Oboh and Elusiyan (2004) also studied the nutrient composition and antimicrobial activity of sorrel drinks against P. aeruginosa, Lactobacillus sp., Bacillus sp., and Corynebacterium and found out that the aqueous extracts of H. sabdariffa inhibited the growth of P. aeruginosa, Lactobacillus sp., Bacillus sp., and Corynebacterium sp. University of Ghana http://ugspace.ug.edu.gh 16 Fullerton et al. (2011) determined the antimicrobial activity of sorrel (Hibiscus sabdariffa) on Escherichia coliO157:H7 isolated from food, veterinary, and clinical samples. Ethanolic extract of Hibiscus sabdariffa calyces was studied and showed to have antimicrobial agents effective in inhibiting E. coli O157:H7. The Hibiscus sabdariffa plant therefore holds a great promise as an antimicrobial agent. University of Ghana http://ugspace.ug.edu.gh 17 CHAPTER THREE 3.0 METHODOLOGY 3.1 Study Sites The study was carried out at the Centre for Plant Medicine Research (CPMR), Mampong- Akuapem, Eastern Region; and the Microbiology Department, Central Laboratory, Korle- Bu Teaching Hospital (KBTH), Greater Accra Region. The CPMR focuses on research into herbal medicines. The centre has an extraction laboratory as well as a microbiology laboratory in which the preparation of the plant extract and the microbial analysis of these extracts were done. The Korle-Bu Teaching Hospital, the third largest hospital in Africa and the leading national referral centre in Ghana, aided in the clinical isolation of the selected bacteria in their well-equipped bacteriology laboratory (Retrieved, from http://kbth.gov.gh/index.php?id=126,March 28, 2014). 3.2 Study design This study was a quantitative experimental research. 3.3 Plant and Specimen collection and storage The fresh Hibiscus sabdariffa leaves and roots were acquired from a farm in Kpotame, Sogakope in the Volta Region of Ghana while the Hibiscus sabdariffa calyces were obtained from the Madina local market in the Greater Accra Region. All the plant parts were authenticated by CPMR botanist. Samples of the leaves, calyces and roots of the plant (Voucher number: CPMR 0214) are being kept in the CPMR herbarium. University of Ghana http://ugspace.ug.edu.gh 18 Of the twenty eight different types of bacteria used for this study, six are American Type Culture Collection (ATCC) namely: Escherichia coli ATCC 25922, Proteus mirabilis ATCC 49565, Salmonella typhi ATCC 19430, Klebsiella pneumoniae ATCC 33495, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 25923; eighteen are clinical isolates, three each of Escherichia coli, Proteus mirabilis, Salmonella typhi, Klebsiella sp., Pseudomonas aeruginosa and Staphylococcus aureus, which were obtained from the Microbiology Department, Central Laboratory, Korle-Bu Teaching Hospital (KBTH) from December 2013 to February 2014. Lastly, two strains each of Methicillin-Resistant Staphylococcus aureus (MRSA) and ESBL (Extended Spectrum Beta-Lactamases) isolates were received from Beverly Egyir who worked on ‘Antimicrobial Resistance, Epidemiology and Population Structure of Staphylococcus aureus in Ghana’ and Noah Obeng-Nkrumah, a PhD student, who is working on the ‘Molecular Epidemiology of Beta-Lactamse producing E.coli and Klebsiella pneumoniae’; both with Clinical Microbiology Department, School of Biomedical and Allied Health Sciences, University of Ghana. Gram staining and biochemical tests of indole, urease, citrate, oxidase, and Triple Sugar Iron were performed on samples to confirm the identification of Escherichia coli, Proteus mirabilis, Salmonella typhi, Klebsiella sp. and Pseudomonas aeruginosa. For the confirmation of Staphylococcus aureus; Gram staining, catalase and coagulase tests were done. Confirmed isolates of Escherichia coli, Klebsiella sp. and Staphylococcus aureus were purified on Cystine Lactose Electrolyte Deficient (CLED) agar; and that of Salmonella typhi, Proteus mirabilis, and Pseudomonas aeruginosa on MacConkey agar. The pure cultures were then stocked by inoculating in Eppendorf tubes containing 2mls of University of Ghana http://ugspace.ug.edu.gh 19 Mueller Hinton agar (Oxoid, United Kingdom) slants. The inoculated Eppendorf tubes were incubated at 35-37°C for 18-24 hours, and then kept frozen at a temperature of -20oC. 3.4 Laboratory Analysis 3.4.1 Culture interpretation and Biochemical Identification Test The collected isolates were thawed, and cultured on the agar plates. Differential media of CLED, DCA, and MacConkey agars aided in distinguishing Gram negative bacteria that are lactose fermenters (LF) and non-lactose fermenters (NLF). Escherichia coli, Proteus mirabilis, Salmonella typhi, Klebsiella sp., with the exception of Pseudomonas aeruginosa, were identified using biochemical and microbiological procedures, as defined by Cowan and Steel (1993), as Enterobacteriaceae. Pseudomonas aeruginosa was identified by oxidase test. The bacteria oxidize the reagent, tetramethyl-p-phenylenediamine dihydrochloride, to a deep purple colour within 10 seconds. The test was performed by soaking a piece of filter paper with a few drops of oxidase reagent, a colony of the bacteria was picked with a wooden loop and smeared on the filter paper; the inoculated area of paper was observed for colour change to deep purple colour (Gordon and McLeod, 1928). Isolated colonies from pure GNR bacteria were picked using sterile bacteriological straight wire and inoculated into tubes of peptone water (Liofilchem, Roseto Degli Abruzzi, Italy), urea agar slants (Liofilchem, Roseto Degli Abruzzi, Italy) citrate slants (Liofilchem, Roseto Degli Abruzzi, Italy), Triple Sugar Iron agar slants (Liofilchem, Roseto Degli Abruzzi, Italy) and motility test media. Inoculated biochemical media were incubated aerobically at 35-37oC for 18 to 24 hours. Biochemical reactions were examined after University of Ghana http://ugspace.ug.edu.gh 20 incubation. Drops of Kovac’s reagent (Oxoid, Hampshire, UK) were added to peptone water cultures for reactions suggestive of indole production. For each isolate, reactions of biochemical tests were compared to that of reference strains for species identification. For Staphylococcus aureus, Gram staining showed Gram positive cocci in clusters. The slide catalase test was done using a drop of hydrogen peroxide (H2O2) on a microscope slide. Staphylococcus produces the enzyme catalase, which breaks down the H2O2 to oxygen and water; as such bubbles of oxygen were released. The tube coagulase test was performed, with negative and positive controls, to help identify Staphylococcus aureus which produces the enzyme coagulase. One millilitre of diluted plasma (1 part of plasma to 9 parts of peptone water) was pipetted in three test tubes labelled positive control, negative control and test. The positive tube was inoculated with standard Staphylococcus aureus ATCC 25923 strain. The negative test tube was not inoculated, and the last test tube with the test organism under investigation. The tubes were incubated aerobically at 35- 37oC for 18-24 hours. The test and positive control showed clotting of tube contents or fibrin clot which confirms Staphylococcus aureus. 3.4.2 Preparation of extracts Extraction Techniques used were Maceration and Decoction. Macroscopic evaluation in Hibiscus sabdariffa One kilogram each of Hibiscus sabdariffa calyces, leaves and roots were examined macroscopically for any foreign matter such as moulds, insects and undesirable plant parts. University of Ghana http://ugspace.ug.edu.gh 21 Preliminary Preparation One kilogram each of Hibiscus sabdariffa calyces, leaves and roots were washed and dried in a shed at the CPMR, Mampong- Akuapem for about two weeks. The completely dried plant parts were then pulverized. 3.4.3 Ethanolic Extraction Sukhdev (2008) method for the extraction of plant compound was followed. One hundred and fifty grams of dried Hibiscus sabdariffa calyces, leaves and roots were weighed out using an analytical weighing scale. One and half litres of 70% ethanol was added to it and left to macerate for 5 days at room temperature, with occasional stirring. After 5 days, the 70% ethanol was decanted and filtered through a Whatman No. 1 filter paper. The marc was pressed, filtered and added to the first filtrate. The extracts were then concentrated under reduced pressure using the Eyela rotary evaporator N-1200 (Tokyo Rikakikai Company Limited, Japan); after which they were freeze-dried and altered into powdered form at the College of Agriculture, University of Ghana, and Legon. 3.4.4 Aqueous Extraction Two hundred and fifty grams each of the dried Hibiscus sabdariffa leaves, roots and calyces were brought to boil in 2.5 litres of distilled water on a hot plate for about thirty minutes and then strained. The aqueous extracts were filtered using Whatman No. 1 filter paper and subsequently cooled for 45 minutes. After cooling, the extracts was freeze-dried and re-constituted into powder at the College of Agriculture, University of Ghana, Legon. University of Ghana http://ugspace.ug.edu.gh 22 3.4.5 Evaluation of the antimicrobial activity of Hibiscus sabdariffa 3.4.5.1 Preparation of extract stock solution A concentration of 200mg/ml aqueous extracts of the Hibiscus sabdariffa leaves, roots and calyces was prepared by dissolving 2g of the powdered extracts in 10ml of sterile distilled water. Similarly, 2g of ethanolic extract was dissolved in 10ml of 5% Dimethyl sulfoxide (DMSO). This concentration was chosen based on preliminary susceptibility test on these bacteria using these extracts. Alternatively, other concentrations of the extract were made by use of the following formula: C1/ C2= V1/V2, Where V1 = volume of stock reagent C1 = concentration of the stock reagent V2 = final volume needed C2= final concentration needed (Efiok, 1993). Sterilization of the prepared extract solutions was done by using 0.22μm membrane filtration (Millex GP filter unit, Millipore, Ireland) and stored in aliquots at 2-8oC. 3.4.5.2 Agar diffusion method The disc diffusion method (Clinical and Laboratory Standards Institute, CLSI) was used to screen for antibacterial activity of Hibiscus sabdariffa calyces, leaves and roots. About University of Ghana http://ugspace.ug.edu.gh 23 three to four isolated colonies of similar morphology were picked from 18-24 hour agar plate of pure cultures of Escherichia coli, Proteus mirabilis, Salmonella typhi, Klebsiella sp., Pseudomonas aeruginosa and Staphylococcus aureus using a sterile loop; and then inoculated individually into 4mls peptone broth (Sigma, P0556, Sigma-Aldrich Inc., USA). The density of the inoculum was adjusted to 0.5 McFarland turbidity standard, resulting in a suspension of 1×107 colony forming units. Mueller Hinton agar plates (Oxoid, CM0337, Oxoid Ltd, United Kingdom) were seeded with the test organisms and the plates left to dry for some few minutes. After drying, wells were made in the agar using sterile cork borer measuring 5 mm in diameter. Eighty millilitres of the prepared serial concentrations of 12.5, 25, 50, 100 and 200mg/ml of aqueous and ethanolic extract of leaves, roots and calyces were dispensed into the labelled wells. The plates were then kept in the refrigerator for one hour for the extract to diffuse into the medium. Ciprofloxacin (5μg) and amikacin (30μg) 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 were used as negative controls. The plates were incubated aerobically overnight at 37C. The following day, the zones of inhibition were measured in millimetres. Analysis was done in duplicate. 3.4.5.3 Micro-dilution method The Minimum Inhibitory Concentration was determined for active plant extracts against some selected susceptibility bacteria. Ciprofloxacin and amikacin (MAST Diagnostics, Bootle ,United Kingdom) were used to perform the micro-dilution test. Twenty five microlitres stock solution of 800μg/ml ciprofloxacin and 3.2mg/ml of amikacin were prepared by following manufacturer’s instruction. The stock solutions were stored at 2-8oC University of Ghana http://ugspace.ug.edu.gh 24 for a maximum of 7 days. Mueller Hinton broth (Liofilchem, Roseto Degli Abruzzi Italy) was used as the diluent for the Minimum Inhibitory Concentration (MIC) test because it acted as a medium for the growth of the bacteria. Each test isolate was emulsified in the Mueller-Hinton broth (MHB) and incubated aerobically for 8-12 hours at 35-37oC. The inoculum was then adjusted to 0.5 McFarland standard. The suspension was further diluted to provide an inoculum of 1×105cfu/ml. The MIC dilution method as described by Eloff (1998) was followed. For each active plant extract, 96-well sterile microtitre plates (Sumilon Multi Well Plate, MS-30964, S.B. Medical. Co. Ltd, Japan) were labelled with appropriate plant extract dilutions in rows. A two fold serial dilution of the extract with an initial concentration of 100mg/ml was used. One hundred microlitres of the diluted extract was dispensed into the wells in rows. One hundred microlitres of the inocula were then added to each of the dilution to make a final volume of 200µl in each well. Un-inoculated wells of extract and broth were used to determine sterility. Reference antibiotic dilutions of initial concentration of 20μg/ml ciprofloxacin (MAST Diagnostics, Bootle, United Kingdom) and 80μg/ml amikacin (Mast Diagnostics, Bootle, United Kingdom) were tested against test bacteria in each row. Preparations were covered and incubated aerobically at 35-37oC for 18 to 24 hours. After incubation, 40 µl of 0.2 mg/ml INT (Iodonitrotetrazolium Chloride, Fluka, Austria) was added to each of the wells after incubation. Microtitre plates were examined after 30 to 120 minutes incubation. Bacterial growth was indicated by a red colour of INT reduced to the formazan. The lowest concentration at which a decrease in the red colour was apparent compared to the next dilution was taken as the MIC value. MICs were recorded as the least dilution which inhibited visible growth of the bacteria. University of Ghana http://ugspace.ug.edu.gh 25 3.5 Ethics Clinical isolates were assigned arbitrary numbers. This study received ethical clearance from the Ethical and Protocol Review Committee of the University of Ghana Medical School (UGMS). 3.6 Statistical Analysis of Data Results were analyzed with Genstat Ninth Edition Statistical Software and SPSS version 16.0 to address the objectives of the study. The mean zones of inhibition for each isolate were analyzed and the mean difference between the extract types was compared using Tukey’s Studentized range test and ANOVA at a significance level of P < 0.05. University of Ghana http://ugspace.ug.edu.gh 26 CHAPTER FOUR 4.0 RESULTS 4.1 Antimicrobial activity of the extracts (Agar diffusion method) The outcome of this study conducted to evaluate the antimicrobial activity of Hibiscus sabdariffa (leaves, calyces and roots) extracts using six clinical and control ATCC strains of bacteria (Escherichia coli, Salmonella typhi, Klebsiella sp., Proteus mirabilis, Pseudomonas aeruginosa and Staphylococcus aureus); and also four resistant strains (Two ESBL and two MRSA organisms) are shown in Figure 4.1 to 4..4 and Appendix 4 to Appendix 12. Of six concentrations (0, 12.5, 25, 50, 100 and 200mg/ml), extracts of 50,100 and 200mg/ml had significant inhibitory results in each of the plant part’s extract. The differences among growth inhibition at low concentrations (0, 12.5 and 25mg/ml) extracts against all the test microorganisms were not significant because the plant extracts at that concentration had no microbial inhibitory effect. This investigation also revealed that antibacterial activity increased significantly (P<0.05) with increase in extract concentration (Figure 4.1 to 4.3 and Appendix 4 to Appendix 6). The antimicrobial activity of the calyces of Hibiscus sabdariffa showed higher growth inhibition than the leaves and roots. The roots of the plant tested against all the test bacteria at various concentrations had no microbial growth inhibition. However, only aqueous extract of the leaves was more potent in terms of growth inhibition against Pseudomonas aeruginosa and E. coli (Figure 4.4 and Appendix 7, Appendix 8). Additionally, this research revealed that only Proteus mirabilis was resistant to all the H. University of Ghana http://ugspace.ug.edu.gh 27 sabdariffa plant extracts at all the test concentrations as compared to the other test bacteria. At 200mg/ml ethanol extract of calyx, S. aureus recorded the highest mean zone inhibition of clinical isolates (18.33±0.3), as well as that for resistant strain MRSA 5038 (24±0.3) and ATCC strain (23.00±0.6) (Figure 4.1 to 4.3 andAppendix 4 to 6). Among the Gram negative bacteria, the aqueous calyx showed peak growth inhibition (16.67±0.3) against clinical isolate Pseudomonas aeruginosa (Fig 4.1, Appendix 4); the ethanol calyx extract showed high growth inhibition against clinical isolate E. coli (16.67±0.3) (Fig 4.1, Appendix 4). Figure 4.1: Antimicrobial activity of different concentrations of aqueous and ethanol calyces extract of H.sabdariffa against the clinical isolates of bacteria 0 5 10 15 20 25 E . co li K le b . sp . P s. a er u g in o sa S . ty p h i S . a u re u s P .m ir a b il is E . co li K le b . sp . P s. a er u g in o sa S . ty p h i S . a u re u s P .m ir a b il is AQUEOUS ETHANOL Z o n e o f in h ib it io n ( m m ) 200mg/ml 100mg/ml 50mg/ml University of Ghana http://ugspace.ug.edu.gh 28 Figure 4.2: Antimicrobial activity of different concentrations of aqueous and ethanol calyces extract of H.sabdariffa against the control ATCC strains of bacteria 0 5 10 15 20 25 30 E . co li A T C C 2 5 9 2 2 K . p n eu m o n ia e A T C C 3 3 4 9 5 P s. a er u g in o sa A T C C 2 7 8 5 3 S . ty p h i A T C C 1 9 4 3 0 S . a u re u s A T C C 2 5 9 2 3 P . m ir a b il is A T C C 4 9 5 6 5 P . m ir a b il is A T C C 4 9 5 6 5 E . co li A T C C 2 5 9 2 2 K . p n eu m o n ia e A T C C 3 3 4 9 5 P s. a er u g in o sa A T C C 2 7 8 5 3 S . ty p h i A T C C 1 9 4 3 0 S . a u re u s A T C C 2 5 9 2 3 Aqueous Ethanol Z o n e o f In h ib it io n ( m m ) 200mg/ml 100mg/ml 50mg/ml University of Ghana http://ugspace.ug.edu.gh 29 Figure 4.3: Antimicrobial activity of H.sabdariffa calyces against clinical resistant strains of bacteria Figure 4.4: Antimicrobial activity of H.sabdariffa leaves against clinical isolates of bacteria 0 5 10 15 20 25 30 ESBL C13EU ESBL C16E5 MRSA 744 MRSA 5038 ESBL C13EU ESBL C16E5 MRSA 744 MRSA 5038 Aqueous Ethanol Z o n e o f in h ib it io n ( m m ) 200mg/ml 100mg/ml 50mg/ml 0 2 4 6 8 10 12 14 E . co li P s. a er u g in o sa K le b . sp . S . ty p h i S . a u re u s P . m ir a b il is P . m ir a b il is E . co li K le b . S p P s. a er u g in o sa S . ty p h i S . a u re u s Aqueous Ethanol Z o n e o f in h ib it io n (m m ) 200mg/ml 100mg/ml 50mg/ml University of Ghana http://ugspace.ug.edu.gh 30 In Figure 4.3, the water extract of calyces against ESBL C13EU recorded the lowest mean zone of inhibition at all the concentrations. However, plant extracts documented high inhibitory effect against MRSA 5038. In comparing the antimicrobial activity of aqueous and ethanol extracts of Hibiscus sabdariffa calyces, the results of the Agar Diffusion Assay against five control ATCC strains and clinical isolates (Escherichia coli, Klebsiella pneumoniae, Salmonella typhi,, Pseudomonas aeruginosa and Staphylococcus aureus); as well as four resistant strains (ESBL C13EU, ESBL CI6E5, MRSA 744, MRSA 5038) are seen in Figure 4.1 to 4.3 and Appendix 4 to 6. In regards to the extraction type of calyx against all the microorganisms at various concentrations, there was no significant (P >0.05) difference between the aqueous and ethanol extract. However, interaction between Staphylococcus aureus and ethanol extraction had higher growth inhibition than the other bacteria (Figure 4.1 to 4.3). Generally, the inhibitory effect of the antimicrobial activity of the aqueous and ethanol extracts of the Hibiscus sabdariffa seems to have an effect on both Gram positive and Gram negative bacteria. This suggests the broad spectrum nature of the plant against bacteria. Antimicrobial activity of different concentrations of aqueous and ethanol leaf extract of H.sabdariffa against the clinical isolates of bacteria showed that the aqueous extract produced the highest zone of inhibition of 12mm against Ps. Aeruginosa, and E.coli 9mm at 200mg/ml (Figure 4.4). There was a difference between the aqueous and ethanol extract University of Ghana http://ugspace.ug.edu.gh 31 type. The inhibitory activity of the leaves at 200mg/ml against test organisms was less than the reference antibiotics; Ciprofloxacin and Amikacin (Appendix13 and 14). Five percent DMSO and sterile distilled used as control showed no activity against the test organisms. A. H. sabdariffa leaves showing inhibition against Pseudomonas aeruginosa ATCC 27853 at 200, 100, 50, 25 and 12.5 mg/ml with negative and positive control. B. H. sabdariffa calyces showing inhibitory effects against clinical isolate E.coli at 200, 100, 50, 25 and 12.5 mg/ml with negative and positive control. C. H. sabdariffa roots showing inhibitory effects against Proteus mirabilis ATCC49565 at 200, 100, 50, 25 and 12.5 mg/ml with negative and positive control. Plate 1: A, B, C showing the antimicrobial susceptibility test of H. sabdariffa leaves, calyces and roots against some test bacteria University of Ghana http://ugspace.ug.edu.gh 32 4.2 MIC of the extracts The results of the MIC values of the Hibiscus sabdariffa calyces extract determined against selected susceptible bacteria are shown in Fig 4.5. Generally, the MIC of the ethanol and aqueous extract for the various organisms ranged between 6.25 and 50mg/ml; higher concentrations of 50mg/ml Hibiscus sabdariffa calyces extracts were required to inhibit Pseudomonas aeruginosa, Staphylococcus aureus, ESBL C16ES, ESBL CI3EU in comparison to the other isolates. The extraction type had no influence on the MIC test against the selected bacteria. In Fig 4.5, the MIC values of the H. sabdariffa calyces aqueous and ethanol extract against some selected test bacteria are shown. The MIC of the ethanol extract for the different control ATCC organisms was 25mg/ml and that for the aqueous extract ranged between 25 and 50 mg/ml. Additionally, the MIC values of the H. sabdariffa calyces both aqueous and ethanol extract against clinical isolates of bacteria ranged between 25 and 50 mg/ml. Unpredictably, the MRSA 744 and MRSA 5038 showed a more robust activity with MIC values of 6.25 and 12.5mg/ml respectively. University of Ghana http://ugspace.ug.edu.gh 33 Figure 4.5: Minimum Inhibitory Concentration of aqueous and ethanol calyces extract of H. sabdariffa on the test bacteria. 0 10 20 30 40 50 60 E . co li P s a er u g in o sa S a lm o n el la t yp h i S . a u re u s K le b si el la s p . E . co li P s a er u g in o sa S a lm o n el la t yp h i S . a u re u s K le b si el la s p . E S B L C 1 3 E U E S B L C 1 6 E 5 M R S A 7 4 4 M R S A 5 0 3 8 CLINICAL CONTROL RESISTANT M IC ( m g /m l) AQUEOUS ETHANOL University of Ghana http://ugspace.ug.edu.gh 34 CHAPTER FIVE 5.0 DISCUSSION The therapeutic use of plants especially as antimicrobials has been reported by many scientists (Cowan, 1999; Sharaf et al., 1966, González-Lamothe et al., 2009). Reports of antimicrobial activity of H. sabdariffa show various levels of microbial growth inhibition against Gram positive and Gram negative bacteria. This indicates the broad spectrum nature of the plant extract. Studies show that the plant is effective against E.coli 0157:H7 (Fullerton et al., 2011) as well as pathogenic bacteria of Pseudomonas aeruginosa and Escherichia coli (Khalaphallah and Wagdi, 2014), and Klebsiella pneumoniae (Alshami and Alharbi, 2014). In the present study, antimicrobial activity of Hibiscus sabdariffa calyces and leaves extracted by water and ethanol extracts was effective against some clinical isolates of bacteria. Among the three plant parts extract of H. sabdariffa, calyces extract were found to be more active than the leaves and roots extracts. This is probably why much work has been done on the calyces as compared to the other parts. The calyces extract were notably effective against Escherichia coli, Salmonella sp., Klebsiella sp., Pseudomonas aeruginosa and Staphylococcus aureus.These findings were similar to studies done by Sharaf et al. (1966), Oboh and Elusiyan (2004) who found H. sabdariffa plant to be effective against Pseudomonas aeruginosa; Frimpong (2008) also detected the plant extract to be effective against S. aureus, E. coli and Pseudomonas aeruginosa. Recently, Khalaphallah and Wagdi (2014) have showed the extract of the plant is effective against Pseudomonas aeruginosa, E. coli and Bacillus subtilis. University of Ghana http://ugspace.ug.edu.gh 35 On the contrary, all the H. sabdariffa extracts were not effective against Proteus mirabilis. Sharaf et al. (1966), however, discovered that the H. sabdariffa plant prevented the growth of Proteus. In this study, the P. mirabilis resistance to the plant extract could be due to the strains of Proteus included in these researches, the concentrations of the extracts and methodology used at that time which was different to the current CLSI method used. The swarming phenomenon of Proteus mirabilis also is presumed to be a factor of its supposed resistance since CLSI (2012) stated that “Strains of Proteus spp. may swarm into areas of inhibited growth around certain antimicrobial agents. With Proteus spp., ignore the thin veil of swarming growth in an otherwise obvious zone growth inhibition”. Yet in this case the swarming was not a thin envelope; and probably the presence of certain constituents in the plant extract could have encouraged it to swarm the more. The variation in the degrees of antimicrobial activities of the extracts on the isolates is presumed to be due to differences in responses by the isolates to different active compounds present in the plant. There is little literature about the antimicrobial property H. sabdariffa plant though some scientists have attributed it to the presence of the secondary metabolites (alkaloids, flavonoids, phenolics, and biterpenoids) in the extract (VanEtten et al., 1994; Badreldin et al., 2005; Rios and Recio, 2005; Olaleye, 2007). Flavonoids are phenolic compounds; C6– C3 unit linked to an aromatic ring which is known to be synthesized by plants in response to microbial infection, and so it is not astonishing that they are antimicrobial substances against a wide array of microorganisms. Cowan (1999), reported that the number of hydroxyl groups on the phenol group are thought to be related to their relative toxicity to microorganisms, with evidence that increased hydroxylation results in increased toxicity. University of Ghana http://ugspace.ug.edu.gh 36 The mechanism of action of the extract may be by inhibition of electron transport, protein translocation, phosphorylation steps, and other enzyme-dependent reactions, followed by an increase in plasma membrane permeability and finally ion leakage from the bacterial cells (Walsh et al., 2003) and may be related to the permeability of the bacteria cell surface to the extracts (Cowan,1999). H. sabdariffa calyces also contain proanthocyanidins. Proanthocyanidins is recognized to either combine to bacterial cells P-fimbriae or transform the structural entity of P- fimbriae and inhibit formation of the adhesion of P-fimbriated E. coli to the uroepithelium, also inhibiting the formation of biofilm in vitro (Gupta et al., 2012). The fimbriae of Klebsiella sp., Salmonella sp., Pseudomonas aeruginosa also adhere to the epithelial cells and bind to specific protein receptors to initiate infection; by inference, proanthocyanidins in H. sabdariffa may also prevent the biofilm formation by these bacteria. The same effect is seen in H. sabdariffa calyces through a recent study done by Alshami and Alharbi (2014) who discovered that H. sabdariffa calyces extract may inhibit biofilm formation capacity against uropathogenic isolates. Biofilm forming bacteria also are known to be associated with long term persistence and resistance to antibiotics. The effectiveness of H. sabdariffa against these bacteria gives a basis for the potency against resistant strains such as ESBL in this present study. The high potency of H. sabdariffa against these bacteria gives scientific basis for it uses in folk medicine in the treatment of abscesses, bilious conditions, cough, dysuria and scurvy (Morton, 1987; Perry, 1980 Ross, 2003). University of Ghana http://ugspace.ug.edu.gh 37 Both the agar diffusion test and MIC using the micro-dilution method were used in the study to generate quantitative results that provided a clearer interpretation. The study revealed that MIC gave the minimum concentration of the extract that can inhibit microbial growth. The susceptibility of the microorganisms against the extract was better determined by MIC, with values ranging from 6.25mg/ml to 50mg/ml. According to Allen et al. (1991), low levels of antimicrobial activity of plant extracts are not detectable in agar disc diffusion method. MIC is a reliable and appropriate method that has been identified as more accurate than agar disc diffusion (Eloff, 1998). This could explain the variation in the MIC and agar diffusion test in which low concentration of the extracts (25mg/ml) gave an inhibitory effect in Salmonella sp. and Staphylococcus aureus as compared to the Agar diffusion test which gave an inhibitory effect at 100mg/ml. The slightly higher potency of the ethanolic extract over the aqueous extract in the Agar Diffusion assay agrees with the reports of Frimpong (2008) and Khalaphallah and Wagdi (2014) where ethanol extracts also showed a higher growth inhibition as compared with water extract. This may be attributed to the fact that ethanol was able to extract constituents from Hibiscus sabdariffa calyces with more potent anti-microbial activity than water. This detected difference may be due to insolubility of the active compounds in water. The MIC values for both the ethanol and water extract was generally the same suggesting that both extracts were generally the same with few non- polar compounds from the ethanol one. According to Sharaf et al. (1966) the coloring matter of the calyces is said to be lethal and this colouring matter may have been fully extracted in both water and ethanol. University of Ghana http://ugspace.ug.edu.gh 38 The study also shows that the S. aureus even MRSA is very susceptible to the calyces. Most plant products are more active against Gram positive bacteria than Gram negative bacteria (González-Lamothe et al., 2009). Gram negative bacteria are the most common pathogens of plants. However, Gram positive bacteria, on the other hand, are often sensitive to biologically active plant products and this proposes that the susceptibility of Gram negatives and Gram positives organisms to plant products is due to their cell wall and membrane arrangement (Rocío et al., 2009) and their permeability to the extracts. Nair and Chanda (2006), also found similar effects, and reported that ATCC strains of Gram positive bacteria were more sensitive than Gram-negative ones toward the plant extracts. The aqueous leaves extract was active against only Pseudomonas aeruginosa and Escherichia coli. The results show the possible phytochemical compounds present in the dark green to red leaves. The little colouring matter could also be a factor. In folk medicine, the leaves of Hibiscus sabdariffa are also used in soups in Northern Ghana. The water extract of the leaves used in soups keeps a few more days in good condition than that prepared with ordinary water. This proposes antimicrobial/ preservative properties of the leaves (Dokosi, 1998). Studies indicate that leaves of the plant have an in vitro inhibitory effect against some microorganisms (Guerin and Reveillere, 1984). In the present study, aqueous leaves extract activity against only Pseudomonas aeruginosa and Escherichia coli might be that these bacteria strains were susceptible to the leaves constituents. Ewansiha (2014) confirms the antimicrobial property of the aqueous extract of H. sabdariffa leaves against Klebsiella pneumoniae, Salmonella typhi and Shigella dysenteriae but resistant to the hexane extract. University of Ghana http://ugspace.ug.edu.gh 39 The Hibiscus sabdariffa roots extract, in this study, did not prevent the growth of all the test microorganisms. This is similar to investigations that show that Petroleum ether extracts of leaves, stem and root of the plant that did not show any activity at all in E. coli, Pseudomonas sp., S. aureus, P. vulgaris, Salmonella spp. (Arvind and Alka, 2011). These disparities are perhaps due to phytochemical constituents which are extracted differently based on the polarity of solvent used (Ewansiha, 2014). Additionally, Alo et al. (2012) has also reported that the inactivity of plant extracts may be due to age of plant, extracting solvent, method of extraction and time of harvesting of plant materials. The results of the present study support the use of H. sabdariffa and the importance of the use of plant extracts worldwide for therapeutic purposes since the prolonged use of antibiotics has led to drug resistant clinical isolates. Additionally, very few antimicrobials are produced yearly. The discovery of antimicrobial plant extracts would be an effective means of controlling clinical infections. 5.1 LIMITATIONS The calyces used in the study were not from the same plant as the roots and leaves since the ones harvested at the time of research was not matured and so the calyces were bought from another source. Despite the above reason, the results of this study offer basic information needed to exploit the possibility of using H. sabdariffa as an antimicrobial agent against clinical isolates. University of Ghana http://ugspace.ug.edu.gh 40 CHAPTER SIX CONCLUSION AND RECOMMENDATIONS 6.0 CONCLUSION Microbiological tests revealed that Hibiscus sabdariffa plant extract has antibacterial properties thus verifying folklore medicine in the treatment of abscesses, bilious conditions, cough, dysuria and scurvy (Morton, 1987; Perry, 1980 Ross, 2003). The extraction type of aqueous and ethanol generally had the same potency with ethanol extracts having a slightly better antimicrobial activity than aqueous extract in the agar diffusion test. It can be concluded from this research that, of all the plant parts tested, H. sabdariffa calyces showed the best antimicrobial activity against all of the test organisms except Proteus mirabilis. The leaf extracts followed with activity seen in E. coli and Pseudomonas aeruginosa only. The roots, however, showed no antimicrobial activity against all the test bacteria. Therefore, the plant’s calyces and leaves may be used for the production of antimicrobial agents which will significantly inhibit the growth of bacteria. 6.1 RECOMMENDATIONS Further studies using solvents other than water and ethanol for extracting the important compounds from the H. sabdariffa plant as well as purification measures would be necessary. Also, investigations of the plant extracts against more pathogenic bacteria as well as fungi. University of Ghana http://ugspace.ug.edu.gh 41 Investigations done on the active constituents of the plant and side effects in addition to toxicology have to be studied. Additionally, there is a need to study the phytochemical compounds from various part extracts of the plant. The stem and seeds of the Hibiscus sabdariffa plant has to be experimented on also. Finally, studies should be done in vivo to assess the clinical efficacy of the extract. University of Ghana http://ugspace.ug.edu.gh 42 REFERENCES 1. Abbiw, D.K. (1990). Useful plants of Ghana: West African uses of wild and cultivated plants. Intermediate Technology Publications, London and Royal Botanic Gardens, Kew, London. Pp 98-212. 2. Abbiw, D.K. (1996). Misuses and abuses in self-medication with medicinal plants: the case of Erythrophleum in Ghana. The Biodiversity of African plants. Springer Publications, Netherlands. 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Nair, R., & Chanda, S. (2006). Activity of some medicinal plants against certain pathogenic bacterial strains. Indian J Pharmacol. 38:142–144. 44. Nelson, R. (1982). The comparative clinical pharmacology and pharmacokinetics of vindesine, vincristine and vinblastine in human patients with cancer. Med. Pediatr. Oncol. 10:115–127. 45. Nutrition Business Journal (NBJ). (1998). Industry overview. Sept. 1998. 46. Oboh, G., & Elusiyan, C.A. (2004). Nutrient composition and antimicrobial activity of sorrel drinks (soborodo). J Med Food 7(3):340-342. 47. Olaleye, M.T. (2007). Cytotoxicity and antibacterial activity of Methanolic extract of Hibiscus sabdariffa. Journal of Medicinal Plants Research:1(1): 009- 013. University of Ghana http://ugspace.ug.edu.gh 47 48. Perry, L. M. (1980). Medicinal Plants of East and South East Asia. M.T Press, Cambridge. Pp. 100-104. 49. Rios, J.L., & Recio, M.C. (2005). Medicinal plants and antimicrobial activity. J. Ethnopharm 100: 80-84. 50. Robbers, J., Speedie M., & Tyler, V. (1996). Pharmacognosy and Pharmacobiotechnology. Williams and Wilkins, Baltimore. Pp. 1–14. 51. Ross, I.A. (2003). Hibiscus sabdariffa. In: Medicinal plants of the world. New Jersey: Humana Press; 267-275. 52. Rukangira, E. (2001). Medicinal plants and traditional medicine in Africa: Constraints and Challenges. Sustainable Development International; 4:179–184. 53. Sharaf, A., Geneidi, A., & Negm, S. (1966). Further study on the antibacterial effect of H. sabdariffa. Path Microbiol 29(1):120-125. 54. Sofowora, A. (1982). Medicinal plants and traditional medicine in Africa, John Wiley and Sons Ltd. New York. Pp 256-257. 55. Stephens, J.M. (2012). Horticultural Sciences Department, Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida, Gainesville FL 32611. http://edis.ifas.ufl.edu 56. Stockwell, C. (1988). Nature’s pharmacy. Century Hutchinson Ltd., London, United Kingdom. 57. Stuart, B.L., & Bonnie, M. (2004). Antibacterial resistance worldwide: causes, challenges and responses. Nature Medicine 10, S122 - S129. 58. Sukhdev, S.H., Suman, P.S.K., Gennaro, L., & Dev, D.R. (2008). Extraction Technologies for Medicinal and Aromatic Plants. United Nations Industrial University of Ghana http://ugspace.ug.edu.gh 48 Development Organization and the International Science and High Technology, Trieste, Italy. P 7. 59. Trease, G., & Evans, W. (1972). Pharmacognosy, Univ. Press, Aberdeen, Great Britain. P 161-163. 60. UNESCO (1996). Culture and Health, Orientation Texts – World Decade for Cultural Development 1988 – 1997, Document CLT/DEC/PRO – 1996, Paris, France. P 129. 61. UNESCO (1998). FIT/504-RAF-48 Terminal Report: Promotion of Ethnobotany and the Sustainable Use of Plant Resources in Africa, Paris, France. P 60. 62. VanEtten, H.D., Mansfield, J.W., Bailey, J.A., & Farmer, E.E. (1994). Two classes of plant antibiotics: Phytoalexins versus phytoanticipins. Plant Cell 6: 1191-1192. 63. Walsh, S.E., Maillard, J.Y., Russel, A.D., Catrenich, C.E., Charbonneau, A.L., & Bartolo, R.G. (2003). Activity and mechanism of action of selected biocidal agents on Gram -positive and -negative bacteria. J Appl Microbiol 94: 240–247. 64. Weiner, M. A. (1980). Earth medicine-earth food: plant remedies, drugs and natural foods of the North American Indians. Macmillan, New York, N.Y. 65. World Health Organization. (2008). Media Centre, Fact Sheet No. 134. University of Ghana http://ugspace.ug.edu.gh 49 APPENDICES APPENDIX 1 Preparation of reagents and chemicals A: Preparation of 5% DMSO Stock solution A 5ml of DMSO (Sigma, D5879, Germany) was measured into a 100ml volumetric flask and diluted with sterile distilled water up to the 100ml mark. The solution was stored at 2- 8˚C. B: Preparation of INT stock solution INT (Iodonitrotetrazolium chloride, Fluka Biocheika, 58030, Sigma- Aldrich, Austria) stock was prepared by dissolving 2mg of INT crystals in 10ml of sterile distilled water. The stock solution was kept in a refrigerator (2-8˚C) in a brown sterile bottle. APPENDIX 2 Preparation of culture media A: Bacteriological Peptone (Sigma- Aldrich, P0556, Germany) i. Formula (g/litre) Peptone 10.0g Sodium chloride 5.0 pH 7.2+ 0.2 ii. Preparation This medium was prepared according to manufacturer’s standard. The medium was heated until completely dissolved and then distributed into capped test tubes before autoclaving at 121 ºC for 15 minutes. University of Ghana http://ugspace.ug.edu.gh 50 B: Mueller- Hinton Agar (Oxoid, CM0337, Oxoid Ltd, England) i. Composition (g/litre) Beef, dehydrated infusion 300 Casein hydrolysate 17.5 Starch 1.5 Agar 17.0 pH 7.4+0.2 ii. Preparation This medium was prepared by the producer’s instruction. It was boiled to dissolve completely after which it was sterilized by autoclaving at 121ºC for 15 minutes. After sterilization, the medium was allowed to cool to 50ºC and poured aseptically. Approximately 25ml volumes were dispensed into an 90mm sterile Petri Dishes and allowed to set. The agar plates were stored at 2-8ºC. Sterility check was done on each batch of plates by incubating a plate at 37 ºC aerobically for 18-24 hours. The media quality was checked by inoculating randomly selected media with control bacteria. C: Mueller Hinton broth i. Composition(g/litre) Beef infusion solids 2.0 Acid hydrolyzed casein 17.5 Starch 1.5 Calcium ions 0.05 Magnesium ions 0.02 pH 7.3 ± 0.1 University of Ghana http://ugspace.ug.edu.gh 51 ii. Preparation Broths were prepared according to the manufacturer’s instruction (Liofilchem, Italy). The mixture was warmed until completely dissolve and then dispensed into appropriate bottles before autoclaving at 121ºC for 15 minutes. D: MacConkey Agar i. Composition (g/ litre) Peptone 20.0 Lactose 10.0 Bile salts 5.0 Neutral red 0.075 Agar 12.0 pH 7.4± 0.2 ii. Preparation Media was prepared according to the manufacturer’s instruction (Liofilchem, Italy). After sterilization, the media was cooled to about 55˚C, and then approximately 25ml volumes were dispensed into 90mm sterile Petri dishes and allowed to set. E: Cystine Lactose Electrolyte Deficient (CLED) Agar i. Composition (g/litre) Peptone 4.0 Lab- Lemco 3.0 Tryptone 4.0 Lactose 10.0 University of Ghana http://ugspace.ug.edu.gh 52 L-cystine 0.128 Bromothymol blue 0.02 Agar 15.0 pH 7.3±0.2 ii. Preparation Manufacturer’s standard (Liofilchem, Italy) was followed. The medium was boiled to dissolve completely. Sterilization was done at 121˚C for 15 minutes. After the medium had cooled, it was mixed and dispensed aseptically into sterile petri dishes and allowed to set. Sterility and media quality checks were done on them. F: Blood and Chocolate Agar i. Composition (g/litre) `Lab-Lemco' powder 10.0 Peptone Neutralised 10.0 Sodium chloride 5.0 Agar 15.0 pH 7.3 + 0.2 ii. Preparation Blood agar (Liofilchem, Italy) was prepared according to manufacturer’s instruction. The dehydrated blood agar base was heated to dissolve entirely. After sterilization at 121˚C for 15minutes, the medium was cooled to 50˚C and 7% sterile sheep blood was added to the agar based, mixed well and poured aseptically into Petri dishes. University of Ghana http://ugspace.ug.edu.gh 53 Chocolate agar: A double strength blood agar base was prepared. Sterile 2% w/v haemaglobin powder added to the double strength agar base, mixed and poured aseptically into 90mm Petri dishes. APPENDIX 3: Pictures of H. sabdariffa plant parts Plate 2: Intact calyces of the Hibiscus sabdariffa plant Plate 3: Roots of Hibiscus sabdariffa University of Ghana http://ugspace.ug.edu.gh 54 Plate 4: Leaves of Hibiscus sabdariffa University of Ghana http://ugspace.ug.edu.gh 55 APPENDIX 4: Mean zone of inhibition of Hibiscus sabdariffa calyces extract concentration effect on the clinical isolates of bacteria Clinical Isolates Extraction type Zone of inhibition (mm) 200mg/ml 100mg/ml 50mg/ml 25mg/ml 12.5mg/ml 0mg/ml E. coli Aqueous `13.33±0.3a 10.33±0.3a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a Kleb.sp. Aqueous 12.00±0.0b 5.00±0.0b 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a Ps. aeruginos Aqueous 16.67±0.3c 14.00±0.0c 12.00±0.6b 5.00±0.0a 5.00±0.0a 5.00±0.0a S. typhi Aqueous 14.67±0.3d 5.00±0.0 b 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a S. aureus Aqueous 17.00±0.0e 14.00±0.6c 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a P. mirabilis Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a P. mirabilis Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a E. coli Ethanol 16.67±0.3c 12.33±0.3d 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a Kleb.sp. Ethanol 14.33±0.6d 11.00±0.6e 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a Ps. aeruginosa Ethanol 16.33±0.3c 13.33±0.6d 9.33±0.3c 5.00±0.0a 5.00±0.0a 5.00±0.0a S. typhi Ethanol 15.67±0.3c 13.33±0.3d 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a S. aureus Ethanol 18.33±0.3f 14.33±0.3c 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a (Data are mean ± SEM values) abcdef Different letters denote differences in zones of inhibition for 200mg/ml, 100mg/ml and 50mg/ml of calyces extract University of Ghana http://ugspace.ug.edu.gh 56 APPENDIX 5: Mean zone of inhibition of Hibiscus sabdariffa calyces extract concentration effect on the control ATCC strains of bacteria Control Strains Extraction type Zone of inhibition (mm) 200mg/ml 100mg/ml 50mg/ml 25mg/ml 12.5mg/ml 0mg/ml E. coli ATCC 25922 Aqueous 14.00±0.6a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a K. pneumoniaeATCC 33495 Aqueous 11.33±0.3b 7.66±0.3 b 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a Ps. aeruginosaATCC 27853 Aqueous 15.33±0.3c 12.33±0.3c 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a S. typhiATCC 19430 Aqueous 16.33±0.3c 10.00±0.0d 5.0±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a S. aureusATCC 25923 Aqueous 18.00±0.6d 14.00±0.6e 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a P. mirabilisATCC 49565 Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a P. mirabilisATCC 49565 Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a E. coliATCC 25922 Ethanol 18.00±0.6d 13.33±0.6c 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a K. pneumoniaeATCC 33495 Ethanol 15.00±0.6c 11.00±0.0d 5.0±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a Ps. aeruginosaATCC 27853 Ethanol 16.67±0.3c 12.33±0.3c 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a S. typhi ATCC 19430 Ethanol 18.67±0.3d 14.33±0.3e 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a S. aureus ATCC 25923 Ethanol 23.00±0.6e 15.33±0.3e 12.67±0.3b 5.00±0.0a 5.00±0.0a 5.00±0.0a (Data are mean ± SEM values) abcde Different letters denote differences in zones of inhibition for 200mg/ml, 100mg/ml and 50mg/ml of calyces extract. University of Ghana http://ugspace.ug.edu.gh 57 APPENDIX 6: Mean zone of inhibition of Hibiscus sabdariffa calyces extract concentration effect on four resistant strains of bacteria. Resistant Strains Extraction type Zone of inhibition (mm) 200mg/ml 100mg/ml 50mg/ml 25mg/ml 12.5mg/ml 0mg/ml ESBL C13EU Aqueous 14.00±0.6a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a ESBL C16E5 Aqueous 17.00±0.6b 11.00±0.6b 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a MRSA 744 Aqueous 19.33±0.3c 12.33±0.3c 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a MRSA 5038 Aqueous 18.00±0.6b 14.67±0.3d 10.00±0.0b 5.00±0.0a 5.00±0.0a 5.00±0.0a ESBL C13EU Ethanol 16.33±0.3e 11.33±0.3b 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a ESBL C16E5 Ethanol 21.33±0.3f 16.00±0.0e 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a MRSA 744 Ethanol 16.33±0.3e 10.33±0.3b 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a MRSA 5038 Ethanol 24.00±0.3g 20.33±0.3f 15.67±0.3c 5.00±0.0a 5.00±0.0a 5.00±0.0a (Data are mean ± SEM values) abcdefg Different letters denote differences in zones of inhibition for 200mg/ml, 100mg/ml and 50mg/ml of leaves extract. University of Ghana http://ugspace.ug.edu.gh 58 APPENDIX 7: Mean zone of inhibition of Hibiscus sabdariffa leaves extract concentration effect on the clinical isolates of bacteria. Clinical Isolates Extraction type Zone of inhibition (mm) 200mg/ml 100mg/ml 50mg/ml 25mg/ml 12.5mg/ml 0mg/ml E. coli Aqueous 9.00±0.6b 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a Ps. aeruginosa Aqueous 12.33±0.9c 9.33±0.3b 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a Kleb.sp. Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a S. typhi Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a S. aureus Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a P. mirabilis Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a P. mirabilis Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a E. coli Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a Kleb.sp. Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a Ps. aeruginosa Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a S. typhi Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a S. aureus Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a (Data are mean ± SEM values) ‘abc’ Different letters denote differences in zones of inhibition for 200mg/ml, 100mg/ml and 50mg/ml of leaves extract University of Ghana http://ugspace.ug.edu.gh 59 APPENDIX 8: Mean zone of inhibition of Hibiscus sabdariffa leaves extract concentration effect on the control ATCC strains of bacteria Control strains Extraction type Zone of inhibition (mm) 200mg/ml 100mg/ml 50mg/ml 25mg/ml 12.5mg/ml 0mg/ml E. coli Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a Ps. aeruginosa Aqueous 10.67±0.3b 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a K.pneumomiae Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a S. typhi Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a S. aureus Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a P. mirabilis Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a P. mirabilis Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a E. coli Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a K.pneumoniae Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a Ps. aeruginosa Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a S. typhi Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a S. aureus Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a (Data are mean ± SEM values) ‘ab’ Different letters denote differences in zones of inhibition for 200mg/ml, 100mg/ml and 50mg/ml of leaves extract University of Ghana http://ugspace.ug.edu.gh 60 APPENDIX 9: Mean zone of inhibition of Hibiscus sabdariffa leaves extract concentration effect on four resistant strains of bacteria. Resistant Strains Extraction type Zone of inhibition (mm) 200mg/ml 100mg/ml 50mg/ml 25mg/ml 12.5mg/ml 0mg/ml ESBL C13EU Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a ESBL C16E5 Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a MRSA 744 Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a MRSA 5038 Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a ESBL C13EU Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a ESBL C16E5 Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a MRSA 744 Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a MRSA 5038 Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a (Data are mean ± SEM values) ‘a’ Different letters denote there is no differences in zones of inhibition for 200mg/ml, 100mg/ml and 50mg/ml of roots extract University of Ghana http://ugspace.ug.edu.gh 61 APPENDIX 10: Mean zone of inhibition of Hibiscus sabdariffa roots extract concentration effect on the clinical isolates of bacteria Clinical Isolates Extraction type Zone of inhibition (mm) 200mg/ml 100mg/ml 50mg/ml 25mg/ml 12.5mg/ml 0mg/ml E. coli Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a Ps. aeruginosa Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a Kleb.sp. Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a S. typhi Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a S. aureus Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a P. mirabilis Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a P. mirabilis Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a E. coli Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a Kleb.sp. Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a Ps. aeruginosa Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a S. typhi Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a S. aureus Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a (Data are mean ± SEM values) ‘a’ Different letters denote there is no differences in zones of inhibition for 200mg/ml, 100mg/ml and 50mg/ml of roots extract University of Ghana http://ugspace.ug.edu.gh 62 APPENDIX 11: Mean zone of inhibition of Hibiscus sabdariffa roots extract concentration effect on the control ATCC strains of bacteria Control strains Extraction type Zone of inhibition (mm) 200mg/ml 100mg/ml 50mg/ml 25mg/ml 12.5mg/ml 0mg/ml E. coli Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a Ps. aeruginosa Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a K.pneumomiae Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a S. typhi Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a S. aureus Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a P. mirabilis Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a P. mirabilis Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a E. coli Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a K.pneumoniae Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a Ps. aeruginosa Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a S. typhi Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a S. aureus Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a (Data are mean ± SEM values) ‘a’ Different letters denote there is no differences in zones of inhibition for 200mg/ml, 100mg/ml and 50mg/ml of roots extract University of Ghana http://ugspace.ug.edu.gh 63 APPENDIX 12: Mean zone of inhibition of Hibiscus sabdariffa roots extract concentration effect on four resistant strains of bacteria. Resistant Strains Extraction type Zone of inhibition (mm) 200mg/ml 100mg/ml 50mg/ml 25mg/ml 12.5mg/ml 0mg/ml ESBL C13EU Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a ESBL C16E5 Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a MRSA 744 Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a MRSA 5038 Aqueous 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a ESBL C13EU Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a ESBL C16E5 Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a MRSA 744 Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a MRSA 5038 Ethanol 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a 5.00±0.0a (Data are mean ± SEM values) ‘a’ letters denote differences in zones of inhibition for 200mg/ml, 100mg/ml and 50mg/ml of roots extract. University of Ghana http://ugspace.ug.edu.gh 64 APPENDIX 13: Clinical Test Microorganisms To Reference Antibiotics (Agar Diffusion Test) Mean Zone of Inhibition (mm) Test organisms Standard Antibiotics Ciprofloxacin(5µg) Amikacin (30µg) Escherichia coli 9 19 Pseudomonas aeruginosa 25 25 Proteus mirabilis 31 21 Klebsiella sp. 9 22 Salmonella typhi 32 29 Staphylococcus aureus 30 25 Bacteria tested in MHA medium University of Ghana http://ugspace.ug.edu.gh 65 APPENDIX 14:Control ATCC strains to reference antibiotics (Agar Diffusion Test) Zone of Inhibition (mm) Test organisms Standard Antibiotics Ciprofloxacin(5µg) Amikacin (30µg) Escherichia coli ATCC 25922 28 25 Proteus mirabilis ATCC 49565 33 22 Salmonella typhi ATCC 19430 28 22 Klebsiella pneumoniae ATCC 33495 25 22 Pseudomonas aeruginosa ATCC 27853 28 26 Staphylococcus aureus ATCC 25923 28 24 Bacteria tested in MHA medium University of Ghana http://ugspace.ug.edu.gh 66 APPENDIX 15:Minimum Inhibitory Concentration of some selected test bacteria Test organisms Ethanol Aqueous Ciprofloxacin Amikacin (mg/ml) (μg/ml) Escherichia coli 25 25 >40 >80 Escherichia coli (ATCC 25922) 25 - 20 >80 Salmonella typhi 25 25 >40 >80 Salmonella typhi (ATCC 19430) 25 - >40 >80 Klebsiella sp. 25 - >40 >80 Klebsiella pneumoniae (ATCC 33495) 25 25 40 80 Pseudomonas aeruginosa 50 50 40 >80 Pseudomonas aeruginosa (ATCC 27853) 25 50 40 >80 Staphylococcus aureus (ATCC 25923) 25 50 >40 80 Staphylococcus aureus 50 25 >40 80 ESBL C13EU 50 - 40 >80 ESBL CI6E5 50 50 >40 >80 MRSA 744 6.25 6.25 >40 40 MRSA 5038 12.5 12.5 >40 40 Ciprofloxacin- Initial concentration of 20μg/ml; Amikacin - Initial concentration 80μg/ml University of Ghana http://ugspace.ug.edu.gh 67 University of Ghana http://ugspace.ug.edu.gh 67 APPENDIX 16-19: All statistical tables APPENDIX 16 Independent sample test comparing the zone of inhibition between aqueous and ethanolic extract of Hibiscus sabdariffa calyces against clinical isolates of bacteria ANOVA comparing the zone of inhibition of the concentration of Hibiscus sabdariffa calyces against clinical isolates of bacteria Mean zone of inhibition (mm) Sum of Squares df Mean Square F Sig. Between Groups 366.704 2 183.352 6.909 .003 Within Groups 875.712 33 26.537 Total 1242.416 35 t-test for Equality of Means t df Sig. (2- tailed) Mean Difference 95% Confidence Interval of the Difference Lower Upper Mean zone of inhibition (mm) Equal variances assumed -.581 34 .565 -1.16556 -5.24031 2.90920 University of Ghana http://ugspace.ug.edu.gh 68 Post-Hoc Multiple comparisons Mean zone of inhibition (mm) LSD (I) Concentratio n (mg/ml) (J) Concentration (mg/ml) Mean Difference (I- J) Std. Error Sig. 95% Confidence Interval Lower Bound Upper Bound 200 mg/ml 100 mg/ml 3.52917 2.10304 .103 -.7495 7.8078 50 mg/ml 7.80583* 2.10304 .001 3.5272 12.0845 100 mg/ml 200 mg/ml -3.52917 2.10304 .103 -7.8078 .7495 50 mg/ml 4.27667 2.10304 .050 -.0020 8.5553 50 mg/ml 200 mg/ml -7.80583* 2.10304 .001 -12.0845 -3.5272 100 mg/ml -4.27667 2.10304 .050 -8.5553 .0020 *. The mean difference is significant at the 0.05 level. University of Ghana http://ugspace.ug.edu.gh 69 APPENDIX 17 Independent sample test comparing the zone of inhibition between aqueous and ethanolic extract of Hibiscus sabdariffa calyces against resistant isolates of bacteria ANOVA comparing the zone of inhibition of the concentration of Hibiscus sabdariffa calyces against resistant isolates of bacteria Mean zone of inhibition (mm) Sum of Squares df Mean Square F Sig. Between Groups 511.891 2 255.945 16.688 .000 Within Groups 322.082 21 15.337 Total 833.973 23 t-test for Equality of Means t df Sig. (2-tailed) Mean Difference 95% Confidence Interval of the Difference Lower Upper Mean zone of inhibitio n (mm) Equal variances assumed -1.025 22 .317 -2.51667 -7.60932 2.57599 University of Ghana http://ugspace.ug.edu.gh 70 Post-Hoc Multiple comparisons Mean zone of inhibition (mm) LSD (I) Concentrat ion (mg/ml) (J) Concentration (mg/ml) Mean Difference (I- J) Std. Error Sig. 95% Confidence Interval Lower Bound Upper Bound 50 100 -5.65000* 1.95814 .009 -9.7222 -1.5778 200 -11.31250* 1.95814 .000 -15.3847 -7.2403 100 50 5.65000* 1.95814 .009 1.5778 9.7222 200 -5.66250* 1.95814 .009 -9.7347 -1.5903 200 50 11.31250* 1.95814 .000 7.2403 15.3847 100 5.66250* 1.95814 .009 1.5903 9.7347 *. The mean difference is significant at the 0.05 level. University of Ghana http://ugspace.ug.edu.gh 71 APPENDIX 18 Independent sample test comparing the zone of inhibition between aqueous and ethanolic extract of Hibiscus sabdariffa calyces against control ATCC strains of bacteria ANOVA comparing the zone of inhibition of the concentration of Hibiscus sabdariffa calyces against control ATCC strains of bacteria Mean zone of inhibition (mm) Sum of Squares df Mean Square F Sig. Between Groups 763.852 5 152.770 7.053 .000 Within Groups 649.844 30 21.661 Total 1413.695 35 t-test for Equality of Means t df Sig. (2-tailed) Mean Difference 95% Confidence Interval of the Difference Lower Upper Mean zone of inhibition (mm) Equal variances assumed -.891 34 .379 -1.89278 -6.21078 2.42523 University of Ghana http://ugspace.ug.edu.gh 72 Post-Hoc Multiple comparisons Mean zone of inhibition (mm) LSD (I) Concentratio n (mg/ml) (J) Concentratio n (mg/ml) Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval Lower Bound Upper Bound 200 mg/ml 100 mg/ml 4.11917 2.24744 .076 -.4533 8.6916 50 mg/ml 8.30250* 2.24744 .001 3.7300 12.8750 100 mg/ml 200 mg/ml -4.11917 2.24744 .076 -8.6916 .4533 50 mg/ml 4.18333 2.24744 .072 -.3891 8.7558 50 mg/ml 200 mg/ml -8.30250* 2.24744 .001 -12.8750 -3.7300 100 mg/ml -4.18333 2.24744 .072 -8.7558 .3891 *. The mean difference is significant at the 0.05 level. University of Ghana http://ugspace.ug.edu.gh 73 APPENDIX 19 Independent sample test comparing the zone of inhibition of aqueous extract of Hibiscus sabdariffa leaves against test bacteria ANOVA comparing the zone of inhibition of the concentration of Hibiscus sabdariffa leaves against two test bacteria. t-test for Equality of Means t df Sig. (2-tailed) Mean Difference 95% Confidence Interval of the Difference Lower Upper Mean zone of inhibition (mm) Equal variances not assumed -1.475 10 .182 -2.21667 -5.73887 1.130554 Mean zone of inhibition (mm) Sum of Squares df Mean Square F Sig. Between Groups 39.065 2 19.532 4.051 .056 Within Groups 43.398 9 4.822 Total 82.462 11 University of Ghana http://ugspace.ug.edu.gh 74 Post-Hoc Multiple comparisons Mean zone of inhibition (mm) LSD (I) Concentration (J) Concentration Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval Lower Bound Upper Bound 200 mg/ml 100 mg/ml 3.17500 1.55273 .071 -.3375 6.6875 50 mg/ml 4.25000* 1.55273 .023 .7375 7.7625 100 mg/ml 200 mg/ml -3.17500 1.55273 .071 -6.6875 .3375 50 mg/ml 1.07500 1.55273 .506 -2.4375 4.5875 50 mg/ml 200 mg/ml -4.25000* 1.55273 .023 -7.7625 -.7375 100 mg/ml -1.07500 1.55273 .506 -4.5875 2.4375 *. The mean difference is significant at the 0.05 level. University of Ghana http://ugspace.ug.edu.gh 75 APPENDIX 20: ETHICAL CLEARANCE University of Ghana http://ugspace.ug.edu.gh