Stellen Sie sich eine Welt vor, in der traditionelles Wissen und moderne Wissenschaft Hand in Hand gehen, um die drängendsten gesundheitlichen Herausforderungen unserer Zeit zu bewältigen. Diese Studie taucht ein in die faszinierende Welt der äthiopischen Heilpflanzen, insbesondere in die des ressourcenreichen Omo-Tals, um das Potenzial von vier ausgewählten Pflanzenarten – Aloe pirottae, Grewia schweinfurthii, Kosteletzkya begoniifolia und Uvaria leptocladon – im Kampf gegen antibiotikaresistente Bakterien zu erkunden. Angesichts der wachsenden globalen Krise der Antimikrobiellen Resistenz (AMR) und der begrenzten Verfügbarkeit neuer, wirksamer Antibiotika untersucht diese Forschung die antibakterielle Aktivität von Pflanzenextrakten gegen sowohl ATCC-Bakterienstämme als auch multiresistente (MDR) klinische Isolate. Durch die Anwendung von Extraktionsmethoden mit verschiedenen organischen Lösungsmitteln und anschließenden In-vitro-Tests mittels Agar-Well-Diffusionsassays und der Bestimmung der minimalen Hemmkonzentration (MHK) werden vielversprechende Ergebnisse erzielt, die das breite Spektrum an antibakteriellen Eigenschaften dieser Pflanzen offenbaren. Die Ergebnisse deuten darauf hin, dass insbesondere Uvaria leptocladon eine bemerkenswerte Wirksamkeit gegen MDR-Bakterien aufweist, was einen vielversprechenden Ausgangspunkt für die Entwicklung neuartiger antimikrobieller Wirkstoffe darstellt. Diese Arbeit leistet einen wesentlichen Beitrag zur Validierung des traditionellen Wissens über Heilpflanzen und unterstreicht die Bedeutung weiterer Forschung, um die In-vivo-Wirksamkeit, Wirkmechanismen und toxikologischen Profile dieser Pflanzen vollständig zu verstehen. Durch die Kombination von Ethnobotanik, mikrobiologischer Analyse und statistischer Auswertung bietet diese Studie wertvolle Einblicke für Forscher, Pharmaunternehmen und politische Entscheidungsträger, die nach innovativen Lösungen im Kampf gegen AMR suchen und gleichzeitig die Bedeutung des Schutzes der Biodiversität und des traditionellen Wissens hervorheben. Die gewonnenen Erkenntnisse könnten den Weg für die Entwicklung neuer, pflanzlicher Medikamente ebnen und somit einen wichtigen Beitrag zur globalen Gesundheit leisten. Diese Untersuchung dient als Inspiration für zukünftige Studien, die das reiche Potenzial der Pflanzenwelt zur Bekämpfung von Infektionskrankheiten ausschöpfen wollen, und unterstreicht die Notwendigkeit, traditionelles Wissen zu bewahren und durch wissenschaftliche Forschung zu untermauern. Entdecken Sie mit uns, wie die Weisheit der Natur und die Kraft der Wissenschaft zusammenwirken können, um die Herausforderungen der modernen Medizin zu meistern.
Table of Contents
Acknowledgments
Abbreviations and Acronyms
Table of Contents
List of Tables
Abstract
CHAPTER ONE
1. INTRODUCTION
1.1. Background
1.2. Statement of the Problem
1.3. Significance of the Study
CHAPTER TWO
2. REVIEW OF LITERATURES
2.1. Antimicrobials and Antimicrobial Resistance Infections
2.2. Plant Based Antimicrobial Studies in Ethiopia
2.3. Ethnobotanical Data of Medicinal Plants in Omo Valley
2.4. Review on Plants Selected for the Experiment
2.4.1. Aloe pirottae Berger
2.4.2. Grewia schweinfurthii Burret
2.4.3. Kosteletzkyabegoniifolia(Ulber) Ulber
2.4.4. Uvaria leptocladon Oliv
CHAPTER THREE
3. OBJECTIVES
3.1. General Objective
3.2. Specific Objectives
CHAPTER FOUR
4. MATERIALS AND METHODS
4.1. Description of the Study Area
4.2. Study Design and Period
4.3. Selection of Medicinal Plants
4.4. Plant Material Collection and Transportation
4.5. Taxonomic Identification of Plants
4.6. Processing of Medicinal Plants
4.7. Preparation of Plant Extract
4.8. Test Microorganisms
4.9. Antibacterial Assay
4.9.1. Primary Antibacterial Screening Against ATCC strains
4.9.2. Extended Antibacterial Screening Against MDR Clinical Isolates
4.10. Determining Minimum Inhibitory Concentration
4.11. Quality Control
4.12. Statistical Analysis
4.13. Ethical Consideration
4.14. Oprational Definitions
CHAPTER FIVE
5. RESULTS
5.1. Overall Primary Antibacterial Screening Against ATCC Bacteria
5.1.1. Primary Antibacterial Activity of A. pirottae crude extracts against ATCC
5.1.2. Primary Antibacterial Activity of G. schweinfurthii crude extracts against ATCC
5.1.3. Primary Antibacterial Activity of K. begoniifolia crude extracts against ATCC
5.1.4. Primary Antibacterial Activity of U. leptocladon crude extracts against ATCC
5.2. Extended Antibacterial Screening Against MDR Clinical Isolates
5.2.1. Extended Antibacterial Activity of K. begoniifolia Against MDR Clinical Isolates
5.2.2. Extended Antibacterial Activity of U. leptocladon Against MDR Clinical Isolates
5.3. Determination of MIC of U. leptocladon extract against ATCC strains
5.4. Determination of MIC of U. leptocladon extract against MDR clinical isolates
CHAPTER SIX
6. DISCUSSIONS
CHAPTER SEVEN
7. CONCLUSIONS and RECOMMENDATIONS
7.1. CONCLUSIONS
7.2. RECOMMENDATIONS
ANNEXES
Annex I: Ethnobotanical studies in Omo Valley
Annex II: Plant based antimicrobial studies in Ethiopia
Annex III: Overall procedure of plant selection and identification
Annex IV: Semi structured questioner for ethnobotanic information
Annex V: Standard Operating Procedures
Annex VI: Schematic diagram of MDR isolation and identification
Annex VII: Schematic diagram of MDR pathogen verification
Annex VIII- Microbiological verification result (ATCC strains)
Annex IX- Microbiological verification result (MDR clinical isolates)
Annex X- Plates illustrating overall activity
Annex XI: ANOVA Results of plant extracts against bacteria
Acknowledgments
I was always searching the way that I can see something from the plants of Ethiopia that were serving in the health care service of the nation through wise men and women, now I got enlighten, thanks to my advisors Dr. Ermias Leulkal, Dr. Aseer Manilal and Mr. Mohamed Seid. In fact, it will be more difficult not to stress on the jobs of some Ethiopians that strengthened my vision, like Dr. Ermias Leulkal. I have been reading his articles and appreciating his project in Ankober from far, but this paper served as vehicle to see him close for the plants taxonomy and thoughtful comments. Organization for natural medicine and nutrition at Lower Omo, medical laboratory department staffs in Arba Minch University and my friend Mr. Tsegaye Yohannes were in my side and they were backbones for this job through provision of all the needs. Thank you all.
My wife, Anne-Lise Goujon, who has been behind with continuous support, deserves the acknowledgment with our kind baby, Elie.
Difficult to mention all the traditional healers of South Omo (Lower Omo Valley), but I would like to acknowledge them. South Omo health department, Jinka General Hospital, and SNNPR public health inistitute are organizations deserve acknowledgments in general. However, difficult to pass without stating the names of Mr. Mesay Tesfaye and Mr. Getnet Cherinet, who have been helping in laboratory activity.
Abbreviations and Acronyms
AIDS- Acquired immune deficiency syndrome
AMR- Antimicrobial resistance
AMU- Arbaminch University
ANOVA- One-way analysis of variance
ATCC- American type culture collection
DMSO- Dimethyl sulfoxide
ESKAPE- Enterococcus faecium, S. aureus, K. pneumoniae, Acinetobacter baumannii, P. aeruginosa and Enterobacter species
GPS- Global positioning system
HIV- Human immune deficiency virus
MDR- Multi drug resistant
MIC- Minimum inhibitory concentration
MRSA- Methicillin Resistant Staphylococcus aureus
ONM- Organization for Natural Medicine and Nutrition
PHL- Public health laboratory
RTIs- Respiratory tract infections
SNNPR- Southern Nations, Nationalities’ and Peoples’ Region
SPSS- Statistical Package for Social Services
Sp- Species
TDR- Totally drug resistant
UTI- Urinary tract infection
VRE- Vancomycine resistant enterococci
WHO- World health organization
XDR- Extremely drug resistant
List of Tables
Table 1: Ethnobotanical data of medicinal plants selected for antimicrobial screening
Table 2: Group of ATCC strains used for the antibacterial assay
Table 3: Group of clinical MDR bacterial strains and resistance pattern
Table 4: Antibacterial activity index of plant extracts against ATCC bacteria
Table 5: Zones of inhibition from plant extracts in primary antibacterial screening against ATCC bacteria
Table 6: Antibacterial activities of selected plants against MDR isolates
Table 7: Minimum inhibitory concentration of ethyl acetate extract of U. leptocladon against ATCC and MDR bacteria
Abstract
Background: The search for novel antimicrobials has increased due to the scarcity of safe and effective antibiotics. According to the WHO, plants should be the best source to obtain a variety of drugs. In many regions of the world, particularly Ethiopia, the vast majority of traditional medicines are plant based. However, these plants were neglected and scarcely explored. Therefore, screening of plants used in traditional medicine could provide the chance of discovering antimicrobials that fight against infectious diseases.
Objective: The aim of this study was to determine the antibacterial activity of crude extracts of four medicinal plants (A. pirottae, G. schweinfurthii, K. begoniifolia, and U. leptocladon), against ATCC and MDR clinical isolates of bacteria from September, 2018 - May, 2019.
Methods: Based on ethnobotanical data, four plants were collected from different areas of South Omo through several field trips followed by taxonomic identification. Leaves (A. pirottae, K. begoniifolia, and U. leptocladon) and root (G. schweinfurthii) parts of plants specimens were subjected to extraction process using six different organic solvents through maceration and subsequent filtration. The resultant crude extracts were screened for primary in vitro antibacterial activity against ATCC bacterial strains, using agar well diffusion assay. The plants that showed the highest activity indices were further screened against MDR bacterial isolates. MIC was performed on the most active plant extract. Results of antibacterial activities were analyzed using statistical software, SPSS for Windows version 20.
Results: The antibacterial activity significantly varied among the plant species, type of solvents used for the extraction and strains of bacteria tested. Ethyl acetate and ethanol was highly effective for extracting antibacterial principles, irrespective of plant species. The results of primary screening revealed that two plants (K. begoniifolia, and U. leptocladon) were highly active against ATCC strains. The results of the extended screening showed that, among the two plants, ethyl acetate extract of U. leptocladon efficiently inhibited the growth of MDR bacterial isolates. The MIC values of U. leptocladon were varied in inhibiting MDR bacteria tested.
Conclusion & Recommendation: The overall findings of this study demonstrated that all the four plants have antibacterial activities in varying degrees. U. leptocladon showed the widest and highest spectrum of antibacterial activities as per agar well diffusion assay and analysis of MIC. However, further ongoing and in-depth studies are mandatory in order to prove and understand in vivo efficacy, mechanism of action and toxicological profile of these plants.
Key words: Antibacterial activity of plants, South Omo Zone, U. leptocladon, A. pirottae, G. schweinfurthii, K. begoniifolia.
CHAPTER ONE
1. INTRODUCTION
1.1. Background
Archeological evidence showed that, the use of plants in traditional medicinal practice dated to the Middle Paleolithic age, some 60,000 years ago (1). It is reported that morethan 3.5 billion people of the world rely on plants for the treatment of both human and livestock diseases (2). World health organization (WHO) estimated 80% of population in developing countries is dependent on traditional medicine. Specifically people, who are marginalized, can’t afford or access formal health care systems entirely depend on it (3). Despite the widespread use of plant-based treatments, reports of serious adverse effects are rare, and a long history of practice suggests that herbal medicine may be clinically effective. Moreover, plant-originated secondary metabolites have great concern because of their antibiotic activity without conferring resistance (4,5). The reason why plant derived antimicrobial extracts do not develop resistance compared to the synthetic ones is not clear so far. This can be directly linked to the difficulty in understanding the mechanisms of actions as well as active constituents involved. It speculated that plants developed antibiotics as a self-defense mechanism against environmental bacterial pathogens. Besides, that bacteria could have difficulty in developing resistance to unpurified therapeutic botanical extracts due to the presence of multiple and potentially synergistic active compounds (6).
Medicinal plants are considered as the source of diverse secondary metabolites with antimicrobial potentials that contribute to the development of pharmaceutical products (7). In the recent decades, there is a spurt in the researches pertained to plant based natural products. Plants are considered as a good candidate for the development of drugs and drug leads for various diseases. For instance, 135 drugs currently used for the management of human ailments are derived from plants (8). Scientific analysis of plant components follows a logical pathway. Plants can be collected either randomly or by following ethnomedicinal data supplied by local healers in geographical areas where the plants are found. Initial screening of plants for possible antimicrobial activities typically begin by using crude aqueous or alcohol extraction methods and can be followed by various organic extraction methods (9). It is reported that 30 to 50% of drugs are originated from plant secondary metabolites. For instance, emetine, quinine and berberine isolated from plants showed high inhibitory activity against microbial infections. Plants containing protoberberines and related alkaloids, picralima plant type indole alkaloids and garcinia flavonoids, have been found to be active against a wide variety of microbes (1,5,10).
Ethiopian traditional medicine practice is made up of using plants and other products (11). These health care practices serve more than 70% of human and 90% of livestock in the country. Whole plants and plant extracts are accountable to 95% of such medications and cures (12,13). Moreover, Ethiopia is one of the centers of origin where about 60% of the plants are indigenous with their multi-dimensional services; and 12% of the plants diversity is endemic. These plants were backbone to treat all manner of illnesses, from minor cold to life-threatening diseases of tuberculosis and malaria, prior to the introduction of biomedicine (14). According to plant based studies, antimicrobial, anti-inflammatory, acetyl-cholinesterase inhibition, and antioxidant properties exhibited from Ethiopian plants were not only from those known with their medicinal properties but also from the plants used as famine food (15).
In the study of natural antimicrobials, medicinal plants of terrestrial origin have long been a focus of attention. It is experimentally proved that plants are the sustainable source of structurally diverse bioactive metabolites which can provide lead structures for development of novel drugs. During the past few decades, over 500,000 bioactive metabolites have been isolated from terrestrial plants and many of them are utilized to design and develop new therapeutic agents in pharmaceutical industry. It is likely that many of the bioactive compounds from the plants are yet to be discovered (16).
1.2. Statement of the Problem
Infectious diseases are leading health problems with high morbidity and mortality in the developing countries. The development of resistance to multiple drugs is a major problem in the treatment of these infectious diseases caused by pathogenic microorganisms. This multidrug resistance is presently an urgent focus of research and new bioactive compounds are necessary to combat these multidrug resistance pathogens (17). The discovery and production of new novel drug is the mandatory requirement to save mankind from the difficult-to-treat infections and it is speculated that20 novel classes of antibiotics are required to support modern medicine during the next 50 years. However the current situation in the development of new drugs is problematic because big pharmaceutical companies are lacking new candidates in their research pipelines. Hence, it is one debate to a greater degree if natural product research units are responsible for this critical drug research needs (18).
It is indicated that only 5-10% of the 250,000 identified higher terrestrial plants, have been evaluated for their phytochemical and pharmacological activity. So, medicinal components from plants which are not yet screened for their healing power can play an important role to tackle the problem. (7). Therefore, it is believed that continuous antimicrobial screening of plants will provide the chance to discover active candidates with novel bioactive molecules of unique structure and potency that can be utilized to solve the crisis from drug resistance microbial infections and its prevention.
In Ethiopia, the use of plants in traditional medicine as well as in nutrition has been well known (2,15). However, studies pertaining to the antimicrobial screening of these medicinal plants are minute compared to the potential of traditional medications’ documented. Researches on medicinal plants used in traditional medicine practice of Ethiopia have been started only very recently, as this was neglected and considered irrelevant in the past. Among 7600 higher plants in the country, only 144 have been screened for their antimicrobial activity in the last decades (Annex II). Majority of therapeutic claims attributed to Ethiopian herbal medicine have been based on anecdotal accounts handed down from generation to generation, rather than on scientific data derived from laboratory experimentation. Meanwhile, medicinal knowledge and its practitioners are prone to disappear due to lack of support and promotion from scientific community (19).
South Omo Zone is one of the richest areas in Ethiopian plant diversity. Many of the endemic plants in Omo Valley are widely used for the preparation of traditional medicine. The people in Omo Valley heavily relying on these plants based traditional medicine for the treatment of infectious and non-infectious diseases. Literature survey indicated that applications of plants in traditional medicine in this area are documented only by ethnobotanical studies (Annex 1). Until now, no studies have been conducted to validate the antibacterial activity of plants used in traditional medicine. Besides, the plant diversities of Omo Valley are under threat due to climate change, industrialization, and over exploitation.
1.3. Significance of the Study
Prolonged and uneventful uses of traditional medicinal plants serve as indirect testimony to their efficacy. And without the aid of scientific experiments, remedial therapies using these plants are questionable and their uses would remain locally restricted. Therefore, the results of this study
- Will ascertains the value of the four medicinal plants for their possible antibacterial effects.
- May give the scientific community an opportunity to explore more and evaluate the crude extracts of traditionally used medicinal plants in South Omo.
- Will contribute basic data to the on-going efforts towards searching of pharmacologically active agents for the development of new alternative antimicrobial agent to manage infectious diseases.
- May initiate the decision makers to search for advanced technology and empower the economy for improved investigation approaches in plant based antimicrobial development.
- Can be used to guide future research efforts to examine complex plant extracts and individual compounds for in vivo studies to determine efficacy and toxicity matters
- Can be an alarm for ex-situ conservation and in-situ protection of such important biodiversity in the area.
- The different methodologies used in extraction and microbiological investigation of our study plants can be taken as reference.
CHAPTER TWO
2. REVIEW OF LITERATURES
2.1. Antimicrobials and Antimicrobial Resistance Infections
Antimicrobials are products used in eradicating or inhibiting ofmicroorganisms and their growth. Antimicrobials can be classified according to their activity as biocide and/or biostatic (20). Among the first effective antimicrobials against many infections, antibiotics are the one discovered first. It is clear that the use of antimicrobial agents is helping a lot but it has been simultaneously resulted in the emergence of resistant bacteria (21). The pathogenic bacteria that are resistant to drugs are categorized as MDR or XDR. The MDR bacteria are non-susceptible to at least one drug among more than two categories of antibiotics available to treat them; whereas, XDR are bacteria non susceptible to at least one agent in all but susceptible to one or two antibiotic categories (22).
World health organization (WHO) is particularly concerned about resistance among ESKAPE pathogens: Enterococcus faecium, S. aureus, K. pneumoniae, Acinetobacter baumannii, P. aeruginosa and Enterobacter species. These pathogens are among the main reasons for 700,000 people dying every year in the World; if the world fail to arrive with new antibiotics, this figure may probably increase to 10 million by 2050 with overwhelming cumulative cost to World economy by $100 trillion (23). In Ethiopia, studies reported as there is dramatic increase in the emergence and spread of multidrug-resistant bacteria in different parts of the country which causes urinary tract infections, gastrointestinal infection, respiratory tract infections, wound infections, septicemia, etc. and the emergence rate ranges from 60 to 95% (24,25).
The capacity of microorganisms to acquire resistance to antimicrobial agents has exceeded the imagination of researchers (21). The resistance mechanisms to the commonly available antibiotics are diverse and beyond the effort to change the resistant clones with susceptible ones too (26). Irrational use of antibiotics by human for human, animal and plant; inappropriate waste disposal and treatment; heavy metals and fertilizers, going with biocides, pesticides and other aggravating factors immersing in the environment and ground water, are the main neglected factors for the emergence of drug resistance bacterial genes (26,27). AMR coupled with economic crisis of developing countries is a substantial problem. Origins of AMR in developing countries are intricate and may be deep-rooted in the health care delivery system and awareness of the society (28). In Ethiopia, different studies indicated that MDR bacterial pathogens are the threats to the health care service. For instance, enterobacteriaceae, which include bacterial species causing infections in the central nervous system, lower respiratory tract, bloodstream, wound and urinary tract sites, are becoming resistant to the commonly prescribed antibiotics in Ethiopia. Besides, E. coli, P. aeruginosa and Klebsiella species developed drug resistance regardless of the specimen types (24,25,29,30)
WHO and CDC provide a number of recommendations in the management of AMR crisis. The recommendations fall into two main categories. One the prevention of diseases so that the need for antimicrobials will be limited and provide new or more efficient treatments to either supplements or replaces existing antimicrobial therapies. In primary, disease prevention through the use of vector control, vaccination, public education, clinical education, and legislative action is recommended. Prompt and effective disease management through the use of diagnostics for microbial identification, microbial sensitivity testing to existing antimicrobials to determine appropriate therapies need to be advocated. Such management measures should also be expanded in line with further search for effective and safe alternative therapeutic options (31).
2.2. Plant Based Antimicrobial Studies in Ethiopia
Screening of Ethiopian medicinal plants for their antimicrobial activity was inspected by different researchers between 1993 and 2019. These antimicrobial activity screening activities were following ethnomedicinal data supplied by local healers in geographical areas where the medicinal plants found. Literature survey showed that a total of 323 plant samples from 144 medicinal species were processed for the scientifically evaluated for their traditional use in the treatment of human and livestock health problems. The studies conducted on these plants include in vitro antimicrobial investigation against the major bacterial and fungal strains, and in vivo studies for parasitic problems commonly diagnosed in the country.
More than 60% of the results shown significant antimicrobial activities and 18% were based on the crude extracts of different organic solvents such as methanol, water, ethanol, chloroform, petroleum ether and ethyl acetate. However, the antimicrobial activities from the ethyl acetate and alcoholic extracts were recognized as best solvents. Disc diffusion (pour plate and streak plate), agar well diffusion and broth-based turbidometric methods of antimicrobial testing were the microbiological procedures that commonly used. Ethiopian scholars mainly implement disc and agar well diffusion in primary screening. And 50% of these screening activities further evaluated to determine the MIC mainly by broth dilution method (Annex II). However, continuous assessment is crucial to determine if any or more of the aforementioned methods are considered as the most optimal assay to be employed for assessing the potential antimicrobial activity of plant extracts (32).
2.3. Ethnobotanical Data of Medicinal Plants in Omo Valley
Ethnobotanical studies revealed that more than 100 sp. of plants are used for the treatment and management of diverse infectious diseases (Annex I). The four plant species selected for this study were quite rare in the literatures, and two are endemic to South Omo. Perusal of literature indicated that medicinal plants in the study area are not well evaluated for the in vitro antimicrobial activities except the study made on antimalarial activity of A. otalensis (33).
2.4. Review on Plants Selected for the Experiment
2.4.1. Aloe pirottae Berger
Aloe pirottae is one of the fifty six species of Aloe found in Ethiopia. A. pirottae is a member of the group of Aloe with 15–18 spots on a tough skin on the leaves. It has been collected and identified from different localities of Sidamo provinces, of eastern Gamo Gofa provinces, Bale, and Harerge floristic regions also in the lowlands of South Omo (34,35). Literature review of ethnobotanical studies underlines the use of different species of Aloe all over Ethiopia. Recent research review states that more than ten different species used mainly for the treatment of diseases associated with digestive system, wounds, burns and skin (36). Besides, A. pirottae is used during ritual ceremony by Suri people (37). The ethnobotanical studies also indicated that the majority of Aloe species are used for the preparation of traditional medicine. Most of the Ethiopian Aloe species used as traditional medicine have been screened for phytochemicals as well as antimicrobial activity (38,39). However, review of literature showed that antibacterial activity of A. pirottae has never been reported.
2.4.2. Grewia schweinfurthii Burret
It is a tropical African shrub, 4-10m tall with rounded branches, mainly found in an altitude range of 600 to 1700m, on open grassland, acacia woodland, on rocky limestone slopes, between basalt blocks, or sometimes on sandy soils (40). Ethnobotanical studies in Ethiopia showed the medicinal use of different species of Grewia (41,42). Among the different species, only the two species (G. ferruginea & G. occidentalis) have been investigated for antibacterial and antifungal activities (43,44). The leaf of G. schweinfurthii in Ethiopian traditional medicine was used for wound healing (45). In Afar region, the fruit of this shrub is used as food and leaf or stem decoction for malaria treatment (46,47). Some of the Grewia species in the study area is used by Kara and Kwegu people for the treatment of chest pain and cough (48). Survey of literature revealed that neither screening of antimicrobial activity, nor phytochemical analysis of G. schweinfurthii has been done in Ethiopia.
2.4.3. Kosteletzkyabegoniifolia(Ulber) Ulber
Kosteletzkya begoniifolia is one of the seventeen species of genus Kosteletzkya (49). It is a coarse, perennial herb or sub-shrub, 0.4–2.8 m tall, ascending or somewhat scrambling, stems with simple or few-armed stellate yellowish hairs, these characteristics are sometimes accompanied by tiny stellate hair. K. begoniifolia is native to Africa, found in an altitude range comprised between 1000 and 1600 m. This species is only mentioned in the traditional medicine practices of South Western Ethiopia. Antibacterial activity of K. begoniifolia has been already conducted. Results have shown inhibition against S. aureus, P. aeruginosa, E. coli, and S. typhimurium (50) . In the ethnomedicinal literature of Ethiopia, the species more often mentioned is K. adoensis which serves as fodder for cattle and Nyala in Bale Mountain. In South Omo, K. adoensis is used by Maale and Ari people as toothbrush and for the treatment of mouth sore and diarrhea (51–53). Literature survey indicated that antibacterial activity of K. begoniifolia from Ethiopia is scanty (51).
2.4.4. Uvaria leptocladon Oliv
Uvaria leptocladon is one of the hundred and fifty species of the genus Uvaria. Some of the African Uvaria species have been described in traditional medicine as a diuretic for stomach disorders, to treat infections from hookworms, and antidotes for snake bites. U. leptocladon is native to East Africa where it is spreads from Mozambique to Ethiopia, including Madagascar (54,55). Together with U. leptocladon some other species in the Uvaria genus indicated as having different uses and benefits to the people who are utilizing them. For instance, U. kirkii is used for malaria, epilepsy and to relief pain, U. acuminate is used for cough and candidiasis, U. lucida for mental disorder and U. leptocladon for the treatment of epilepsy and candidiasis (56–59). In the Ethiopian ethnomedicinal studies, the use of U. leptocladon is cited only in South Omo, among Maale, Kara and Kwegu people. The root is used by Kara and Kwegu healers to treat chest pain, tuberculosis, cough, bloody diarrhea, stomachache, malaria, and abscess (48). In Maale, traditional practitioners use this plant to heal food poisoning and vomiting (52).
Pharmacological studies on the different species of Uvaria genus indicated that it contains compounds having structural novelty and broad bioactivity (60–63). In 1994, initiative has been taken to analyze some components of U. leptocladon (64). Apart from the above citations, there is no scientific report pertaining to the antimicrobial activities of U. leptocladon.
CHAPTER THREE
3. OBJECTIVES
3.1. General Objective
To determine the antibacterial activity of water, ethanol, methanol, ethyl acetate, chloroform and petroleum ether crude extracts of four medicinal plants (A. pirottae Berger, G. schweinfurthii Burret , K. begoniifolia Ulber and U. leptocladon Oliv) used in traditional medicine practice of South Omo Zone, against different species of human pathogenic bacteria between September, 2018 & May, 2019.
3.2. Specific Objectives
- To determine the antibacterial activity of four plants against the American type culture collection (ATCC) bacterial strains.
- To determine the antibacterial activity of active plant extracts against MDR clinical isolate bacteria.
- To determine the Minimum inhibitory concentration of highly active plant against ATCC and MDR bacteria.
CHAPTER FOUR
4. MATERIALS AND METHODS
4.1. Description of the Study Area
This study was conducted in three localities of South Omo Zone, Southern Nations, Nationalities’ and Peoples’ Region (SNNPR); Ethiopia. The parishes were Beneta, Korcho and Dembayte as located on map. All these parishes/villages belong to two different Woredas (Districts), Hammer and Maale in South Omo Zone.
South Omo Zone is one of the Zones found in SNNPR. Its capital town, Jinka, is located at 750km south of Addis Ababa, Ethiopian capital. The topography of the zone indicates that the area is comprised of environmental condition from lowlands to highlands. The altitude ranges 360 meter at the Southern extreme of the zone near Lake Rudolf and highest altitude at Shengama, 3418 meter, are indications to the wide range of altitude. Climate data showed 0.5 % Dega, 5.1 % Weyna Dega, 60 % Semi-Kola & 34.4 % Kola. This district of the region has average temperature 10.50C - 35.50C and means annual rainfall of 400-1600 mm (Data from Zone finance and economy office).
South Omo is inhabited by a total population of 831805 and 149347 households. The Zone divided in 8 Woredas, 1 town administration and 265 Kebeles. This place is known for diverse cultural and ethnic groups that widely uses plants in traditional medicine.
Fig 1: Map of Study area (This map was deleted by the editors due to copyright issues)
Courtesy: Minalu Adem (A Guide to Omo Valley)
4.2. Study Design and Period
An in vitro experimental study was performed to validate the antibacterial activity of four medicinal plants against the ATCC and MDR bacterial isolates, between September, 2018 and May, 2019.
4.3. Selection of Medicinal Plants
The selection of four medicinal plants for the antibacterial study was exclusively based on their ethnobotanical usage against diverse infections. The ethnobotanical use of selected plants is indicated in Table 1. Detail procedure for selection and identification of plants is described in Annex IV.
4.4. Plant Material Collection and Transportation
Healthy specimens of the four selected plants were collected from the different sites of Omo valley with the help of traditional healers between September and November 2019. The geographical locations of collection sites and plant parts used are indicated on Table 1. The plant samples were collected by the local digging tool as well as using industrial pruning shear (Outils Wolf) in sterile condition. Zip-locked polythene and isothermal bags were utilized for the transportation of specimens.
4.5. Taxonomic Identification of Plants
The taxonomic identification of plants was done with the help of an eminent botanist Dr. Ermias Lulekal, Addis Ababa University. Voucher specimens and photos are deposited at the National Herbarium, Addis Ababa University for future reference.
4.6. Processing of Medicinal Plants
Prior to extraction, healthy plant materials of respected species (the root part of G. schweinfurthii and leaves of A. pirottae, U. leptocladon, and K.begoniifolia) were thoroughly washed and rinsed with distilled water to remove the associated debris. The cleaned samples were then surface dried under shade for one week at room temperature to prevent photolysis and thermal degradation of metabolites. The leaf part of Aloe was shade dried for six weeks. The completely dried material was grounded finely with electrical grinder (Moulinex turbomix 350W). The finely ground plant materials were weighed using digital balance (Ormade DB-300, 300X0.1g- Millano, Italy) and stored in cool dry place until use.
Table 1- Ethnobotanical data of medicinal plants selected for antibacterial screening.
Abbildung in dieser Leseprobe nicht enthalten
MOP- mode of preparation, ROA- rout of administration
4.7. Preparation of Plant Extract
The plant materials were extracted by maceration method described earlier with slight modifications of extraction method and solvents (65,66). Briefly, five grams of each plant materials were weighed and macerated with 50ml of six different extraction solvents of increasing polarity (petroleum ether, chloroform, ethyl acetate, methanol, ethanol and distilled water) separately in 100ml capacity reagent bottle. Standard operating procedures were followed strictly during activity (Annex V). The plant specimens in respective solvent mixture were placed for maceration on a shaker (Labtech RS-12 R) at 120 rpm for 24 hours at room temperature. After the stipulated time period, the resulted extracts were filtered using Whatman’s filter paper No 1. This procedure was repeated thrice. The total extracts were obtained by merging the filtrates from each of the three extractions in conical flask (250ml) following evaporation to dryness in a water-bath (VIGITAL serological) at 40°C. The resulted gummy extract was collected and diluted with dimethyl sulfoxide (DMSO), in order to make a concentration of 1 mg/ml aliquot and kept at 4°C until laboratory analysis.
4.8. Test Microorganisms
The antibacterial activity of plant extracts were determined against a battery of nine ATCC bacterial strains and against six multidrug resistant clinical isolates (Table 2 and 3). The ATCC strains were obtained from the Ethiopian Public Health Institute. MDR clinical isolates were sourced from Jinka Public Health Laboratory. Procedures for the isolation and identification of MDR pathogens presented in schematic form (Annex VI). The verification of each bacterium was confirmed by microbiological tests according to the standard procedure and aseptic precautions (Annex VII-IX). Clinical isolates resistant to one or more antibacterial susceptibility discs among three categories were considered as MDR (22).
Table 2: Group of ATCC strains used for the antibacterial assay
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ATCC- American Type Culture Collection
Table 3: Group of MDR bacteria and antibiogram profile
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4.9. Antibacterial Assay
Diverse organic extracts of four medicinal plants were subjected to primary antibacterial screening against ATCC strains in order to select the active plant extracts. Afterwards, the plant extract which showed the highest and broadest spectrum of activity against ATCC strains were further subjected to extended antibacterial screening against six MDR clinical isolates. Finally, the potent plant extract that showed the highest and broadest activity against ATCC and MDR bacteria was further taken up to elucidate the MIC.
4.9.1. Primary Antibacterial Screening Against ATCC strains
The antibacterial screening of plant extracts was done in vitro by agar well diffusion assay as this method produce consistent results compared to disc diffusion (65,67). The assay was carried out on nutrient agar (Biomark Laboratories, India) as described elsewhere (68,69). All plant extracts were screened against the nine strains of ATCC. Prior to antibacterial assay, all test organisms were sub-cultured to nutrient agar. Agar plates for susceptibility tests were prepared on sterile petridishes as per the manufacturer instructions. Pure colonies of the test organisms were transferred to sterile test tube containing normal saline and the suspension was compared to the commercially prepared 0.5 McFarland test standard. The susceptibility agar plates were swabbed with the prepared bacterial suspension, using sterile cotton applicator swab in order to create a uniform lawn culture. Afterwards, wells of 5mm diameter were made (30 mm apart from each other) on the agar plates by using a sterile cork borer. The resultant wells were filled with 120 μl (1 mg/ml) of the appropriate plant extract and solvent used for dissolution (70). After one hour, plates of susceptibility tests were transferred from biosafety cabinet to incubator and kept at 37 0C for 24 hours. The inhibitory activity was measured by measuring the clear zone including well diameter. The formation of clear inhibition zone with diameter of >10mm considered as having antibacterial activity, with slight modification of zone diameter stated elsewhere (71,72). Activity index of each solvent extract of respective plant species as well as the overall activity index of each plant tested in primary screening were determined using the following modified expressions (73):
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4.9.2. Extended Antibacterial Screening Against MDR Clinical Isolates
Based on the results obtained from primary antibacterial screening and overall activity index, the plant extracts that showed highest and broadest spectrum of activities against the ATCC strains were further subjected to the extended screening against panel of multidrug resistant bacteria comprising of six isolates. The methodology used for primary screening was applied here to.
4.10. Determining Minimum Inhibitory Concentration
Based on the results of the extended screening (agar well diffusion assay), potent candidate plant extract, which showed the broadest and highest activities, was taken up for final analysis. Tube dilution method was used for the procedure (65). Plant extract aliquot (1mg/ml) in DMSO, was used as the test solution. The test solution further diluted serially in buffered saline (pH 7.2) in order to obtain a dose range between 3.9µg/ml to 1000µg/ml in nine sterile Pyrex glass test tubes. Then the plant extract, prepared in different concentration, was dispensed to the test tube which contains nutrient broth prepared in a volume of 1 ml. Afterward, respective tubes were inoculated with 100 µL of a fresh culture of appropriate bacterial suspension (105 CFU/mL) and incubated at 37°C for 24 hours. The MIC value was determined by sub culturing a loop full of suspension from each culture tube to the nutrient agar plates that incubated at 37°C for 24hrs. MICs were described as the lowest concentrations inhibiting visible growth compared with the culture of initial inoculums.
Frequently asked questions
What is the main topic of this document?
This document is a language preview containing the table of contents, objectives and key themes, chapter summaries, and keywords related to a study on the antibacterial activity of certain medicinal plants.
What is the purpose of the study described in this document?
The study aims to determine the antibacterial activity of crude extracts from four medicinal plants (A. pirottae, G. schweinfurthii, K. begoniifolia, and U. leptocladon) against both ATCC (American Type Culture Collection) and MDR (Multidrug-Resistant) clinical isolates of bacteria.
What are the main chapters in the study?
The main chapters include: Introduction, Review of Literature, Objectives, Materials and Methods, Results, Discussions, and Conclusions and Recommendations.
What methodologies are used in the study?
The methodologies include: Ethnobotanical data collection, plant material collection and processing, preparation of plant extracts using different organic solvents, antibacterial assays (primary screening against ATCC strains and extended screening against MDR isolates), determining minimum inhibitory concentration (MIC), and statistical analysis.
What are the key objectives of the study?
The general objective is to determine the antibacterial activity of the plant extracts against human pathogenic bacteria. Specific objectives include: determining antibacterial activity against ATCC strains, determining antibacterial activity against MDR clinical isolates, and determining the MIC of highly active plant extracts.
What is the significance of this study?
The study aims to validate the antibacterial effects of traditionally used medicinal plants, provide data for the development of new antimicrobial agents, guide future research on plant extracts and compounds, and contribute to the conservation of important biodiversity.
What are the MDR bacteria tested against?
The MDR bacteria that were used for the antibacterial assay are: E. coli, K. pneumoniae, A. baumannii, S. aureus, and P. aeruginosa
- Arbeit zitieren
- Jochen Krebs (Autor:in), 2000, Auseinandersetzungen zwischen Königtum,Kaisertum,Papsttum, München, GRIN Verlag, https://www.hausarbeiten.de/document/97869
