Samuel Sahile

Isolation and characterization of potential antibiotic producing actinomycetes from water and sediments of Lake Tana, Ethiopia

OBJECTIVE To isolate, evaluate and characterize potential antibiotic producing actinomycetes from water and sediments of Lake Tana, Ethiopia. METHODS A total of 31 strains of actinomycetes were isolated and tested against Gram positive and Gram negative bacterial strains by primary screening. In the primary screening, 11 promising isolates were identified and subjected to solid state and submerged state fermentation methods to produce crude extracts. The fermented biomass was extracted by organic solvent extraction method and tested against bacterial strains by disc and agar well diffusion methods. The isolates were characterized by using morphological, physiological and biochemical methods. RESULTS The result obtained from agar well diffusion method was better than disc diffusion method. The crude extract showed higher inhibition zone against Gram positive bacteria than Gram negative bacteria. One-way analysis of variance confirmed most of the crude extracts were statistically significant at 95% confidence interval. The minimum inhibitory concentration and minimum bactericidal concentration of crude extracts were 1.65 mg/mL and 3.30 mg/mL against Staphylococcus aureus, and 1.84 mg/mL and 3.80 mg/mL against Escherichia coli respectively. The growth of aerial and substrate mycelium varied in different culture media used. Most of the isolates were able to hydrolysis starch and urea; able to survive at 5% concentration of sodium chloride; optimum temperature for their growth was 30 °C. CONCLUSIONS The results of the present study revealed that freshwater actinomycetes of Lake Tana appear to have immense potential as a source of antibacterial compounds.

In vitro antibacterial activity of leaf extracts of Zehneria scabra and Ricinus communis against Escherichia coli and methicillin resistance Staphylococcus aureus

ABSTRACT Objective To evaluate the antibacterial activities of the crude leaves extracts of Zehneria scabra (Z. scabra) and Ricinus communis (R. communis) against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and methicillin resistance S. aureus. Methods The crude powdered leaves of Z. scabra and R. communis were extracted successively by organic solvents in increasing polarity [benzene, chloroform:acetone (1:1), 70% alcohol and distilled water]. The antibacterial susceptibility of the crude leaves extracts of were tested against standard strains of E. coli (ATCC 25922) and S. aureus (ATCC 2923) and clinical isolates of E. coli, S. aureus and methicillin resistance S. aureus using agar well diffusion method. Results In Z. scabra and R. communis leaf extracts, the most sensitive standard strain was S. aureus with an inhibition zone of (14.00±1.20) mm and (15.90±2.13) mm, respectively. The minimum inhibitory concentration (MIC) values of Z. scabra extracts against test organisms ranged from 1.95 mg/mL for extract 3 in clinical and standard strains of S. aureus to 250 mg/mL for extract 1 and 4 in clinical and standard strains of E. coli. The MIC values of R. communis extracts against test organisms ranged from 1.95 mg/mL for extract 2 and 3 standard strains of S. aureus to 250 mg/mL for extract 1 in clinical isolate of E. coli. Most of the minimum bactericidal concentration and MIC values of plant extracts were almost similar particularly in R. communis, or minimum bactericidal concentration equal to one dilution factor less than MIC value of the extracts mainly in Z. scabra. Conclusions The potency of plant extracts against test organisms were depend on different organic solvents used. Clinical isolate of bacterial pathogens showed less zones of diameter compared to the standard strains. Gram-positive had wide inhibition zones than Gram-negative bacteria. Further studies should be carried out to isolate the pure compounds and standardization of the methods of plant extracts for an in vitro testing.

Antimicrobial activity of crude and semi-purified fractions of Warburgia ugandensis against some pathogens

T annual incidence of urolithiasis in industrialized regions is considered to be 1,500-2,000 cases per millions with reoccurrence rate of 75% in 20 years. There is no effective management therapy for renal calculi. Allopathic and herbal therapies havetheir inherent limitations and side-effects. Bergenia ligulata has been used since ancient time in many herbal compositions and it is major component of Cystone® (Himalaya, herbal healthcare) for treating kidney stones. The present work has been designed to study the anti-lithiatic potential of B. ligulata, isolation of the potent metabolite(s) and its mechanism of action. Commercially available dried rhizomes of B. ligulata were powdered and subjected to activity guided fractionation using in vitro calcium oxalate crystal growth inhibition assay. Further, rat hyperoxaluric model was used to assess anti-lithiatic ability in vivo. The isolated fractions showed anti-calcifying activity in vitro. The sequential isolation of the potent fraction led to the purification of the most active molecule. The metabolite was eventually characterized as bergenin employing LC-MS, NMR, FTIR and UV spectroscopy. Bergenin was found to be effective in reducing oxidative stressmarkers like malondialdehyde (187% in diseased vs. 20% in treated) and elevating reduced glutathione levels (-46% in diseased vs. -15% in treated). It exhibited anti-lithiatic activityas assessed by measuring the activity of lactate dehydrogenase and alkaline phosphatase in serum samples. The creatinine clearance was also normalized with bergenin treatment in rat hyperoxaluric model. The present study provides significant evidence in the effectiveness of bergenin in treating and managing renal calculi.D mellitus is a metabolic disorder constituting a major health concern today whose prevalence has continuously increased in the world. The aim of this study is to evaluate the anti-diabetic potential of methanolic extract of Hyoscyamus albus (HAMeOH) in diabetic rats. Hyoscyamus albus (Solanaceae) is an herbal medicine traditionally applied as a parasympatholytic and nervous sedative. The oral glucose tolerance test (OGTT) was carried out by administering glucose (2 g/kg, b.w), to non-diabetic rats treated with HAMeOH at oral doses 100 and 200 mg/kg, b.w and glibenclamide 5 mg/kg. Also, Streptozotocin-induced diabetic rats, these diabetic rats were administered (100 and 200 mg/Kg b.w ) and standard drugs glibenclamide was given to rats for 30 days. The oral administration of both doses of HAMeOH significantly reduced the levels of blood glucose and glycosylated hemoglobin in diabetic rats. Determination of plasma insulin levels revealed the insulin stimulating action of the leaves extract. It is concluded that HAMeOH have significant anti-diabetic activity.Grewia nervosa (Lour) Panighrahi, belonging to the family Malvaceae s.l. is widely distributed along the Western Ghats of India. Although it has been commonly used in traditional medicine, the medicinal properties have not been scientifically evaluated. Phytochemical analysis established the presence of phenolic compounds, tannins, alkaloids and saponins in leaves. The aqueous and methanol extracts from leaves and bark of G. nervosa were investigated for medicinal properties using in vitro assays. The methanol extract of leaves demonstrated 97.5% inhibition of α-amylase activity. Additionally, the methanol extract of leaves also demonstrated antioxidant activity (5.41± 0.23 mmol/g, dw) that was higher compared to aqueous extract (3.32±0.45 mmol/g, dw). Further the methanol extract of bark exhibited anti-lipoxygenase activity indicative of its potential to control inflammatory activity. These results suggest that Grewia nervosa would be a potential source for treatment of diabetes and its associated complications such as oxidative stress and inflammationC herbs (Asteraceae) are extensively used as food additives and in folk medicine. Anti-cancer, anti-human immunodeficiency virus type 1 (HIV-1), anti-inflammatory, antinociceptive and antiproliferative activities as well as antioxidant effects have been reported for Chrysanthemum species. We report the isolation and identification of flavonoids and new and known terpenoids from the endemic species, C. macrocarpum and C. deserticolum “guertoufa”, used in Algerian Sahara as tea drinks and in “couscous” and soups “Chorba”. Structures of the isolated compounds were established by 1-D and 2-D homo and hetero-nuclear NMR (1H, 13C, COSY, HSQC, HMBC, and NOESY), mass spectrometry, UV and comparison with literature data. C. deserticolum extracts were tested by four methods to identify the antioxidant activity namely, ABTS•+, DPPH• scavenging, CUPRAC and ferrous-ions chelating activity methods. The in vitro anticholinesterase activity was achieved by the use of the basic enzymes that occur in causing Alzheimer’s disease: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Anti-inflammatory, antinociceptive, antiproliferative and antioxidant activities of C. macrocarpum extracts and isolated compounds are also reported here.T practice of traditional medicine for the control of fertility in most parts of India is based on the uses of plant medicines for many years. The aim of the present study was to evaluate the post coital antifertility activity of different varieties of Curcuma longa and underlying mechanism thereof. The effects of n-hexane, chloroform and acetone fraction of hydroalcoholic extract of three different varieties i.e. Salem, Krishna and Rajapore of Curcuma longa rhizomes were studied at three different doses to evaluate their antifertility, early abortifacient, antiovulatory activities and underlying mechanism thereof. Acute toxicity and thin layer chromatographic studies of same were also carried out. All varieties found to have significant antifertility activity (p<0.01). The n-hexane fraction of all varieties showed anti-implantation activity at the dose of 150 mg/kg weight whereas chloroform and acetone fraction of all varieties exhibited 100% reduction in pregnancy at the dose of 300 mg/kg body weight. Antifertility activity of test drugs was found through its antizygotic mechanism whereas all test drugs devoid of any antiovulatory and early abortifacient activity at all doses.M charantia (MC) fruits have previously been reported to reduce blood glucose in laboratory animals and human subjects with diabetes. Increase in insulin secretion is one of the chief mechanisms of antidiabetic action of MC extracts or their purified molecules. In present study the effect of aqueous extracts of MC (AEMC) was studied on insulin secretion in isolated pancreatic islets from normal Wistar rats with an attempt to evaluate the mechanism of action. Islets were incubated in HBBS buffer containing 3.3 or 16.7mM glucose, and AEMC, diazoxide, nimodipine and calphostin C, alone and in combinations. Release of insulin in external media was measured by ELISA. Cytotoxicity studies, to assess the integrity of the islets cells, were carried out by trypan blue uptake and LDH release assay. Trypan blue gained access to 9.6 ± 1.2% cells and 8.3 ± 1.1% dead islet cells were observed in LDH release assay on AEMC exposure, suggesting that the extract was non-toxic at tested concentration. AEMC stimulated insulin secretion from the isolated islets at 3.3 and 16.7 mM glucose. The effect of AEMC was dose dependent. As loss of cell integrity was not observed on AEMC exposure, hence, alteration of membrane integrity as the possible mechanism of insulin release is ruled out by this study. Addition of dizoxide and nimodipine completely diminished glucose induced insulin secretion. AEMC induced insulin secretion at 16.7mM glucose was partially inhibited by dizoxide and nimodipine, however no reduction was observed at 3.3mM of glucose. No change in insulin secretion at basal level of 3.3 mM of glucose suggests that the phytochemicals of AEMC may not be binding to either KATP or Ca channels. Calphostin C significantly (p<0.01) reduced AEMC induced insulin production both at 3.3 mM and 16.7 mM. The finding suggests that PKC inducing activity of AEMC phytochemical/s may be responsible for its insulin secretagogues potential.Bioassay-guided fractionation of the CH2Cl2/MeOH extract of the Thai marine sponge Acanthodendrilla sp. resulted in the isolation of six bromotyrosine-derived alkaloids; aerothionin (1), homoaerothionin (2), 2-hydroxy-3,5-dibromo,4methoxyphenylacetamide (3), 2,4-cyclohexadiene-1-acetamide-3,5-dibromo-1,6-dihydroxy-4-methoxy (4), 11-oxoaerothionin (5), and 11,19-dideoxyfistularin (6) . The structures of the isolated compounds were identified on the basis of detailed spectroscopic analysis. The compounds were tested for the acetylcholinesterase-inhibiting activity, and 3 showed the best acetylcholinesterase-inhibiting activity (92.0% at 0.1 mg/mL).T hepatoprotective activity of methanolic extract of bark of Ficus bengalensis against paracetamol and CCl4 induced liver damage was investigated. Treatment of rats with paracetamol and CCl4 produced a significant increase in the levels of serum glutamate pyruvate transaminase SGPT, serum glutamate oxaloacetate transaminase SGOT, alkaline phosphatase ALP, total and direct bilirubin. Rats pretreated with methanolic extract of barks of F. bengalensis 100 and 250 mg/kg body weight p.o. exhibited rise in the levels of these enzymes but it was significantly less as compared to those treated with paracetamol or CCl4 alone. The results of methanolic extract of F. bengalensis were comparable with the standard hepatoprotective agent silymarin 100 mg/kg. Maximum hepatoprotective effect was found to be at the dose of 250 mg/kg body weight in case of CCl4 induced hepatic damage while 500 mg/kg body weight in case of paracetamol induced hepatic damage. Obtained data suggest that methanolic extract of F. bengalensis bark possesses a potential antihepatotoxic activity.The medicinal quality of plants has been known and exploited by man for centuries. A large number of modern drugs have been isolated from traditional herbal plants[1]. Numerous secondary metabolites obtained from plants, with previously unknown pharmacological activities, have been extensively investigated as a source of medicinal agents[1,2]. The acceptance of traditional medicine as an alternative form of health care and the development of microbial resistance to the available antibiotics led to investigation on the antimicrobial activity of medicinal plants. The increasing failure of chemotherapeutics and antibiotic resistance exhibited by pathogenic agents has led to the screening of several PEER REVIEW ABSTRACTT genus of Phoebe of family Lauraceae is found the most abundance in Borneo and the Malaysian Peninsular. Phoebe tavoyana is locally known as ‘medang rungkoi.’ The woods of Phoebe species have the commercial values usually for housebuilding. As a wood of a good type soft to moderately hard, light, slightly colored than the hardwood used for carving and sculpture, paneling for doors altars wardrobes, carriages and ceiling. Phytochemical study on the leaves of Phoebe tavoyana (Meissn.) H.K.F. from Chebar Besar Reserved Forest, Kuala Kangsar, Perak, Malaysia has resulted the isolation of four known aporphines; laurolitsine (1), roemerine (2), laetanine (3), boldine (4) and one morphinandienone type, sebiferine (5). The structures of alkaloids were determined by spectroscopic analysis. This paper reports the antiplasmodial activity of three alkaloids from the leaves of Phoebe tavoyana (Lauraceae). The results showed that (1), (2) and (5) have shown potent inhibitory activity against the growth of Plasmodium falciparum 3D7 clone, with IC50 1.49, 0.89 and 2.76μg/mL respectively. No previous phytochemical investigation has been performed on this plant.Natural polysaccharides have been widely used because of their biocompatibility and biodegradabilityproperties. An attempt has been made to explore tamarind seed xyloglucan (TSX), a glucosaminoglycan polysaccharide extracted from the kernels of seeds of Tamarindus indica Linn., family Fabaceae for bimodal(immediate and controlled)drug release of multilayer tablet. Chemically TSX powder is highly branched carbohydrate polymer. High drug holding capacity of this polysaccharide was investigated for bimodal release.An in-house extracted TSX polysaccharide was characterized for swelling index, flow property, viscosity and compatibility with drug. Multilayer tablet was comprised of immediate release layer of tramadol hydrochloride (an analgesic agent), followed by tri-layer. This tri-layer consisted of upper and lower barrier layers of TSX and middle layer of drug granular matrix. Multilayer tablets were compressed based on 3 2 factorial design consideringconcentrations of matrix and barrier TSX layers as independent variables. Immediate release layer released the drug within 90 min in acidic media, revealing the retarded action showed bypolysaccharide layer attached to this layer. Granules of matrix layer were prepared by wet granulation technology.Multiplayer tablet of TSX was evaluated for hardness, thickness and drug content. Dissolution test in presence of rat caecal content was found to control the drug release rate for more than 9h. Stability studies confirmed the stable formulation. Thus, this study suggested that inexpensiveand abundantly available natural TSX can act as a potential polymer for bimodal releaseof a multilayer tablet.

Assessment of potential antagonists for anthracnose (Colletotrichum gloeosporioides) disease of mango (Mangifera indica L.) in North Western Ethiopia (Pawe)

Mango (Mangifera indica L.) is considered as one of the most popular fruits among millions of people in the tropical area and increasingly in the developed countries. Anthracnose, caused by the fungus Colletotrichum gloeosporioides, is the most important pre- and post-harvest disease of mango. The objective of this research was to evaluate the prevalence of different promising antagonistic Trichoderma and Bacillus spp. on phyloplane of mango in Ethiopia and to evaluate their antagonistic potential against the pathogen. A total of 19 mango fields were surveyed and anthracnose affected all fields. Culture studies on potato dextrose agar for evaluation of antibiosis activity of Trichoderma spp. and Bacillus spp. revealed that they have inhibitory and lytic effect on C. gloeosporioides, which is an indication of their potential biocontrol agent for management of mango anthracnose as an alternative to chemical control. Significant differences (p < 0.05) were observed among Bacillus isolates in causing lysis of pathogen mycelium, when inoculated on actively growing colony of C. gloeosporioides. Maximum reduction in growth rate of pathogen was observed with Bacillus spp. (B50), which restricted the growth to 2.7 mm compared to 8.3 mm in the control with 67.5% efficacies. There were similar effects (p < 0.05) among Trichoderma spp. in formation of inhibition zones and lysis by varying degrees up to 59.7% efficacies in reducing linear growth of the pathogen in dual culture.

Pathogenic and genetic diversity of Botrytis fabae Sand. isolates from faba bean fields in different agro-ecological zones of Northern Ethiopia

The identity and genetic diversity of the causal agent of chocolate spot (Botrytis cinerea or Botrytis fabae) in Ethiopia is still poorly defined and this hinders efforts aimed at integrated management of the disease, especially breeding for resistance. Chocolate spot disease epidemics are more severe in the moist, humid western districts of northern Ethiopia (NW) compared to the sub-humid eastern districts (NE) but knowledge of pathogen identity and genetic diversity is needed to facilitate epidemiological studies. Of a total of 120 Botrytis isolates examined, all were found to be B. fabae and none fitted the morphological description of B.cinerea. In pathogenicity tests, representative isolates caused typical chocolate spot symptoms and were re-isolated from infected leaves, indicating that B. fabae is the causal agent of chocolate spot in Ethiopia. Marked differences were found among the isolates in colony morphology and growth rate but the morphology of isolates was unrelated to their pathogenicity. A preliminary virulence (=severity of disease) test with 76 isolates revealed differences in virulence of the B. fabae isolates, with NE isolates being generally more virulent than the NW isolates. Genetic analysis based on 129 amplified fragment length polymorphism (AFLP) markers showed that genetic differentiation among pathogen populations was low (GST = 0.02 to 0.03; θ = 0.02, p = 0.05), indicating limited geographic delimitation and significant gene flow. Total gene diversity (HT = 0.22) was mostly attributable to diversity within populations (HS = 0.21). Thus, only 4.5% of the total variability was attributable to frequency differences between agroecological zones. Genotypic diversity (GD), defined as the probability that two individuals taken at random had different genotypes, was high for populations from NW and NE, and from the moist and subhumid agro-ecological zones (GD = 0.99). However, cluster analysis showed high similarity among many isolates (>75% similarity index), suggesting that such isolates have a familial structure or are clonally related. The phenetic tree revealed groups with low bootstrap values that did not reflect the grouping of isolates based on virulence or agro-ecological zone. The implications of these findings for chocolate spot resistance breeding in Ethiopia are discussed.

Effect of Temperature on Growth and Sporulation of Botrytis fabae, and Resistance Reactions of Faba Bean against the Pathogen

Chocolate spot (Botrytis fabae) is a devastating disease of faba bean and reduces its production and productivity. Three controlled condition experiments were conducted to assess the effect of temperature on growth and sporulation of B. fabae, and faba bean resistance reaction against the pathogen using a single B. fabae isolate and Degaga and Bulga-70 faba bean varieties. For cultural experiment, a circular block of actively growing B. fabae mycelia was placed on faba bean dextrose agar medium and arranged in a completely randomized design (CRD) with four replications. For resistance reaction evaluation, fresh culture of isolate suspension was prepared (2 × 105 spores ml-1) and inoculated on to three weeks-old faba bean seedling detached leaves and the whole plant. Inoculated leaflets and seedlings were factorial arranged in a CRD with four replications. Both sets were incubated at 20, 22, 24 and 26°C. The maximum (84.00 mm) radial growth on 5 days after inoculation (DAI); average conidial size (24.86 × 16.32 μm), sporulation (2.48 × 103 conidia ml-1) on 12DAI and growth rate (1.058 mm day-1) were recorded at 22°C. The least values of these parameters and nil sporulation were obtained from 26°C. The highest average lesion size (17.67 mm in Degaga and 22.83 mm in Bulga-70), AUDPC for lesion sizes (30.92 mm in Degaga and 42.08 mm in Bulga-70) and severity (2.13 score) values were recorded at 22°C on 5DAI in detached leaf test. Infection and disease development was reduced at 26°C. The trend was similar in the whole plant test. Such parameters were linearly increased with temperature to maximum and declined progressively in both reaction evaluation tests. The two evaluation experiments indicated that the optimum temperature for B. fabae growth, sporulation, infection and disease development was at 22°C.

Effect of Integrated Climate Change Resilient Cultural Practices on Faba Bean Rust (Uromyces viciae-fabae) Epidemics in Hararghe Highlands, Ethiopia

Climate variability due to increasing temperature and erratic precipitation could affect faba bean rust disease epidemics and the crop productivity. Rust caused by Uromyces viciae-fabae is one of the serious foliar diseases of faba bean in Ethiopia. Field studies were conducted at Haramaya and Arbarakate during 2012 and 2013 to assess effects of integrated climate change resilient cultural practices on rust epidemics in the Hararghe highlands of Ethiopia. Three climate change resilient cultural practices: intercropping, compost application and furrow planting alone and in integration were evaluated using Degaga and Bulga-70 faba bean varieties and Melkassa-IV maize variety. Treatments were factorial arranged in a randomized complete block design with three replications. Faba bean-maize row intercropping and intercropping integrated treatments significantly reduced disease severity, AUDPC and disease progress rate. These treatments reduced rust mean severity by up to 36.5% (2012) and 27.4% (2013) at Haramaya, and up to 27% in 2013 at Arbarakate on both varieties as compared to sole planting. Compost fertilization also led to slow epidemic progression of rust and significantly reduced disease parameters when integrated with maize row intercropping. Compost fertilization in row intercropping recorded the lowest (23.1%) final mean disease severity and the highest (36.5%) percentage reduction in mean disease severity compared to sole cropping at Haramaya in 2012. The trend was similar in 2013 at both locations. Degaga had the lowest rust disease parameters studied compared to Bulga-70 at both locations over years. The overall results indicated that integrated climate change resilient cultural practices were effective to slow the epidemics of rust and to increase faba bean productivity. Hence, integrated climate change resilient cultural practices along with other crop management systems are recommended in the study areas.