Isolation and characterization of antibacterial compounds from a Garcinia livingstonei (Clusiaceae) leaf extract

Although pharmaceutical industries have produced a number of new antibiotics in the last three decades, resistance to these drugs by infectious microorganisms has increased. For a long period of time, plants have been a valuable source of natural products for maintaining human and animal health. The use of plant compounds for pharmaceutical purposes has gradually increased worldwide. This is because there are many bioactive constituents in plants which hinder the growth or kill microbes. Plants could be considered a potential gold mine for therapeutic compounds for the development of new drugs. In this study, sixteen South African plant species were selected based on their antibacterial activity after a wide screening of leaf extracts of tree species undertaken in the Phytomedicine Programme, University of Pretoria. Literature search excluded eleven plants because of the work already performed on their antibacterial activities, while Pavetta schumaniana was found toxic and thus not included in the screening. The remaining four plants namely; Buxis natalensis, Macaranga capensis, Dracaena mannii and Garcinia livingstonei were screened for antibacterial activity by determining the minimum inhibitory concentrations (MIC) against 4 nosocomial bacterial pathogens Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa, and also by using bioautography. The extracts of Macaranga capensis, Garcinia livingstonei, Diospyros rotundifolia and Dichrostachys cinerea had good antibacterial activity with MIC values of 0.03, 0.04, 0.06 and 0.08 mg/ml against different pathogens. The average MIC values of the plant extracts against all the tested pathogens ranged from 0.23-1.77 mg/ml. S. aureus was the most susceptible bacterial pathogen with average MIC of 0.36 . The extract of Diospyros rotundifolia was the most active with an average MIC against all the organisms of 0.23 mg/ml. The extracts of Buxus natalensis, Dracaena mannii, and Pittosporum viridiflorum, Acacia sieberiana, Erythrina lattissima, Cassine papillosa and Pavetta schumanniana had lower antibacterial activity. G. livingstonei was selected for further work on the basis of its good activity. The bulk acetone extract of Garcinia livingstonei (20g) was subjected to solvent-solvent fractionation which yielded seven fractions. Only the chloroform and ethyl acetate fractions showed good bioactivity in the microdilution assay and bioautography. Column chromatography was used to isolate two bioactive biflavonoids from the ethyl acetate fraction. The structures of the two compounds were elucidated using nuclear magnetic resonance (NMR) spectroscopy, and were identified as amentoflavone (1) and 4′ monomethoxyamentoflavone (2). These two compounds have been previously isolated from plants that belong to the Clusiaceae. The two compounds were isolated in sufficient quantity with a percentage yield of 0.45% for amentoflavone and 0.55% for 4′ monomethoxyamentoflavone from 20 g crude acetone extract. The antibacterial activity was determined against four nosocomial bacterial pathogens (Escherichia coli, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa). The MIC values ranged from 8-100 μg/ml. Except for Staphylococcus aureus which showed resistance to amentoflavone at >100 μg/ml. All the other tested organisms were sensitive to both compounds. It has long been recognized that naturally occurring substances in higher plants have antioxidant activity. Based on this, the antioxidant activities of the two isolated compounds were tested using the Trolox assay. The two flavones had good antioxidant activity. Amentoflavone had a Trolox equivalent antioxidant capacity (TEAC) of 0.9. The second compound 4′ monomethoxyamentoflavone had a TEAC value of 2.2 which is more than double the antioxidant activity of Trolox, a vitamin E analogue. To assess the safety of the two compounds on cell systems, cytotoxicity was determined using a tetrazolium based colorimetric assay (MTT assay) using Vero monkey kidney cells. The compounds indicated little to low toxicity against the cell line with cytotoxic concentration (CC50) of 386 μg/ml and >600 μg/ml for compound 1 and 2 respectively. Berberine (used as the control toxic substance) had a CC50 of 170 μg/ml. The Ames genotoxicity assay is used to assess the mutagenic potential of drugs, extracts and phytocompounds. The compounds isolated in this study were assayed for genotoxicity using the Salmonella typhimurium TA98 strain. Amentoflavone was genotoxic at the concentration of 100 μg/plate, but 4′ monomethoxyamentoflavone was inactive at the highest concentration of 400 μg/plate tested. The results of the antibacterial, antioxidant and cytotoxicity testing were encouraging and indicated the potential usefulness of Garcinia livingstonei in traditional medicine and drug discovery. However, the genotoxicity assay revealed potential mutagenic effects of amentoflavone, a compound isolated from the plant. Therefore, it is suggested that application of Garcinia livingstonei extracts in the treatment of human and animal ailments be done with caution to avoid mutagenic effects on the treated subjects. A relatively small change in the structure of the two compounds by replacing an hydroxyl group with a methoxy group had a major effect in increasing antibacterial and antioxidant activity and in decreasing cellular and genotoxicity. This illustrates the potential value of modifying a molecule before its possible therapeutic use. CopyrightDissertation (MSc (Veterinary Science))--University of Pretoria, 2009.Paraclinical Sciencesunrestricte

Serological Survey of Antibodies against Brucella Organisms in One Humped Camel (Camelus dromedarius) Herds in the Lake Chad Area of Borno State, North Eastern Nigeria

Sero-prevalence of brucellosis in the one-humped camel (Camelus dromedarius) was carried out in the Lake Chad area of Borno state. A total of two hundred and fifty four (254) sera samples collected from adult camels in herds located in the Lake Chad Area of Borno state, North easternNigeria, were tested using Rose Bengal Plate test (RBPT) and Microtitre Serum Agglutination Test (MSAT). Twenty four samples (9.4%) were positive by both RBPT and MSAT, out of which 10 (3.9%) and 14 (5.5%) were males andfemale respectively. There was no statistically significant association between sex and positive serological reaction (P>0.05

Investigations On The Carrier Rate Of Pasteurella Multocida In Black Rats (Rattus Rattus) In A Commercial Quail Farm

The aim was to investigate the level of Pasteurella Multocida infection from two anatomic sites of black rats (Rattus Rattus), popularly referred to as house or roof rats in a commercial quail farmhouse with recurrent fowl cholera outbreaks and also to evaluate the association between the Pasteurella Multocida found in rats co-habiting quail poultry houses and isolates from outbreaks of fowl cholera. Thus 100 pharyngeal and 100 rectum swabs samples taken from rats co-habiting farmhouse were obtained and evaluated bacteriologically for isolation of P. multocida; 54% of pharyngeal swabs and 62% of rectum swabs were positive for P. multocida. Extended phenotypic characterization of the isolates confirmed the presence of subspecies P. multocida multocida. Subspecies Pasteurella Multocida septica and gallicida were not encountered. Ramdom serotyping of 5 isolates each from the two sites confirmed serotypes A:4. Fowl cholera outbreaks were confirmed on the quail houses and carrier rats had the same Pasteurella Multocida subspecies and serotype as the infected quail. The public health significance of the finding is also discussed.African Journal of Clinical and Experimental Microbiology Vol. 10 (1) 2009: pp. 2-

Formulation, stabilisation and encapsulation of bacteriophage for phage therapy

Against a backdrop of global antibiotic resistance and increasing awareness of the importance of the human microbiota, there has been resurgent interest in the potential use of bacteriophages for therapeutic purposes, known as phage therapy. A number of phage therapy phase I and II clinical trials have concluded, and shown phages don’t present significant adverse safety concerns. These clinical trials used simple phage suspensions without any formulation and phage stability was of secondary concern. Phages have a limited stability in solution, and undergo a significant drop in phage titre during processing and storage which is unacceptable if phages are to become regulated pharmaceuticals, where stable dosage and well defined pharmacokinetics and pharmacodynamics are de rigueur. Animal studies have shown that the efficacy of phage therapy outcomes depend on the phage concentration (i.e. the dose) delivered at the site of infection, and their ability to target and kill bacteria, arresting bacterial growth and clearing the infection. In addition, in vitro and animal studies have shown the importance of using phage cocktails rather than single phage preparations to achieve better therapy outcomes. The in vivo reduction of phage concentration due to interactions with host antibodies or other clearance mechanisms may necessitate repeated dosing of phages, or sustained release approaches. Modelling of phage-bacterium population dynamics reinforces these points. Surprisingly little attention has been devoted to the effect of formulation on phage therapy outcomes, given the need for phage cocktails, where each phage within a cocktail may require significantly different formulation to retain a high enough infective dose. This review firstly looks at the clinical needs and challenges (informed through a review of key animal studies evaluating phage therapy) associated with treatment of acute and chronic infections and the drivers for phage encapsulation. An important driver for formulation and encapsulation is shelf life and storage of phage to ensure reproducible dosages. Other drivers include formulation of phage for encapsulation in micro- and nanoparticles for effective delivery, encapsulation in stimuli responsive systems for triggered controlled or sustained release at the targeted site of infection. Encapsulation of phage (e.g. in liposomes) may also be used to increase the circulation time of phage for treating systemic infections, for prophylactic treatment or to treat intracellular infections. We then proceed to document approaches used in the published literature on the formulation and stabilisation of phage for storage and encapsulation of bacteriophage in micro- and nanostructured materials using freeze drying (lyophilization), spray drying, in emulsions e.g. ointments, polymeric microparticles, nanoparticles and liposomes. As phage therapy moves forward towards Phase III clinical trials, the review concludes by looking at promising new approaches for micro- and nanoencapsulation of phages and how these may address gaps in the field

Development of bacteriophage ΦKAZ14 loaded chitosan nanoparticles for biological control of colibacillosis in chickens

Bacteriophage ΦKAZ14 was applied in the biological control of colibacillosis. It had lytic activity on APEC 01: K1:H7 and β lactamase producing Escherichia coli. The icosahedral head of the phage measured 50 nm by 45 nm and long contractile tail of 78 nm by 10 nm. It multiplicity of infection (MOI) was 0.01, the latent period of infection of 15 minutes and burst size of 80 particles per infected cell. The phage survived a pH range of 5.0 to 14, and resistant to a temperature of 65 °C. C-NPs protected the phage from enzymatic degradation in vitro. The spherical C-NPs had average particle size range from 176 ± 3.2 to 188 ± 7.4 nm, a zeta potential of 60.3±0.2 and 50.5±0.4 and polydispersity index (PDI) of 0.506 and 0.472. Fourier transform infrared spectroscopy (FTIR) showed no difference between C-ΦKAZ14 NP and C-NP. Thermal analysis showed the temperature of -20 °C is desirable for storage. C-ΦKAZ14 NPs were not toxic on chorioallantoic membrane. Treatment with C- ΦKAZ14 NPs decrease viable bacterial counts in feces and colonization in the intestine of chickens (p ≤ 0.05). It also significantly decreased mortality rate in E. coli-challenged birds (p ≤ 0.05). C- ΦKAZ14 NPs increased bodyweight of chickens (p ≤ 0.05). Lesions were not observed in major organs. C- ΦKAZ14 NPs therapy is a beneficial control strategy against colibacillosis in chickens

Effects of Sterculia tomentosa stem bark extract on haematological parameters and Trypanosoma brucei infection in rats

Evaluation of antitrypanosomal efficacy of stem bark water extract of Sterculia tomentosa using rats as a model revealed significant suppressive effect on parasitaemia with extended survival time in group D rats treated with extract subcutaneously. The extract did not affect packed cell volume of treated rats. There was no significant difference (

Antibacterial activity of two biflavonoids from Garcinia livingstonei leaves against Mycobacterium smegmatis

Amentoflavone and 4′ monomethoxy amentoflavone were previously isolated from Garcinia livingstonei leaves. These compounds had good activities (MIC 6 and 8 μg/ml) against some nosocomial bacteria. In this study, the activity of these purified compounds were tested against fast-growing non-pathogenic Mycobacterium smegmatis. Amentoflavone was the most active compound, with an MIC of 0.60 ± 0.70 mg/ml. The MIC of 4′ monomethoxy amentoflavone and the positive control isoniazid against Mycobacterium smegmatis were similar 1.40 ± 1.56 and 1.30 ± 1.70 mg/ml respectively. Although, Mycobacterium smegmatis is a non-pathogenic fast growing mycobacterium the activities of these compounds may also be useful in combating infections by pathogenic Mycobacterium spp.http://www.elsevier.com/locate/jethpharmmn201

Avian spirochaetosis associated with colisepticemia in free ranging white breasted guinea fowls (Numida meleagris pallas) in arid region of Nigeria: A case report

An outbreak of avian spirochaetosis in free ranging white-breasted guinea fowls associated with acute colisepticemia was diagnosed and reported in this paper. During investigation of the outbreak, clinical signs observed were sudden death, listlessness, depression, cyanosis of the wattles and combs, increased thirst, anorexia and fever up to 43&#176C. There was sporadic mortality of the affected birds. Ticks were observed on the less feathery parts of healthy and the affected guinea fowls and may have contributed to the transmission of the etiologic agent of spirochaetosis (Borellia anserina) leading to the high mortality in this outbreak. Lack of immunization and prompt case reporting contributed to the severity of infection and losses recorded in the farm. Total collapse in tick control and environmental management were the major factors, which precipitated the outbreak. Keywords: Spirochaetosis; colisepticemia; Numida meleagridis; NigeriaAnimal Production Research Advances Vol. 2 (3) 2006: pp. 161-16

ETHNOVETERINARY MEDICINE AMONG BADE PASTORALISTS IN SEMI ARID ZONE OF NORTHERN NIGERIA: STUDIES ON INDIGENOUS TREATMENTS AND MANAGEMENT PRACTICES.

Ethnoveterinary medicine is the study of indigenous systems of animal health care, the practical aspects of which are relevant to applied development work. The ancient Bade pastoralists posses sophisticated body of ethnoveterinary knowledge about animal diseases, treatments and management practices, which spans from generation to generations till date. To obtain information used for this study, seventy (70) structured questionnaires were designed and distributed to pastoralists aged forty years and above in seven districts of Bade emirate in semi arid zone of northern Nigeria. It was observed that ethno veterinary practices were adopted by 85.7% of the pastoralists. The major reasons for their adoption is that ethnoveterinary medicine is cheap, partly effective, accessible and practicable. Perhaps this practices is not without risk. However, the ingredients used also have potential for investigation. Integrating this concept into orthodox animal health care delivery will bring alternative therapy which is affordable. Key Words:-Ethno veterinary medicine, Bade Emirate, orthodox, Treatment, Pastoralists. African Journal of Livestock Extension Vol.3 2004: 59-6