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Introduction: A reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous analysis of two drugs, levamisole hydrochloride (LH) and oxyclozanide (OX), in co-formulation for veterinary use. Materials and Methods: The new HPLC method was validated as per the ICH and other guidelines. A C18 column was used with a gradient program; eluent A was an equal mixture of methanol and acetonitrile, and eluent B included a 25 mM phosphate buffer at pH 7.0 containing 30 mM sodium decanesulfonate and triethylamine (50:50:1 v/v) with pH adjusted to 7.0 using H3PO4 [51:49 v/v] .The detection wavelength was set at 220 nm. For the final gradient program, the retention times were 8.2(for LH) and 13.6(for OX) minutes, respectively, at a flow rate of 1 ml/min over 20 minutes run time. Results: The method was precise, specific and robust. The correlation coefficient, R2 was 0.9998 and 0.9999 for LH and OX, respectively in the range of 5 – 280 μg / mL. The percent y-intercepts and percent residual standard deviations were 1.6%/0.4% and 1.4%/1.0% for LH and OX, respectively. The LOD and LOQ of the method were 0.21 μg / mL and 0.62 μg / mL for LH and 0.06 μg / mL and 0.18 μg / mL for OX. The method has an average accuracy of 100.5% for LH and 101.1% for OX when tested on veterinary bolus formulations, and the samples could be stored under typical lab conditions for about 7 days without significant degradation. Conclusion: This HPLC method is suitable for assaying levamisole hydrochloride and oxyclozanide simultaneously from veterinary formulations.

Animal trypanosomosis is one of the major disease problems affecting agricultural productivity in Ethiopia. The impact of the disease is believed to vary with season and agro-ecologies in line with fly vector distribution. A cross-sectional study on bovine trypanosomosis was conducted from November 2015 to June 2016, in seven selected villages of Gidami district, Oromia Regional State, western Ethiopia. A total of 930 blood samples were collected and subjected to parasitological and hematological analysis. The overall prevalence of bovine trypanosomosis was 14.1%. The seasonal prevalence shows 9.06% in early dry and 18.4% in early rainy seasons. Three trypanosome species, Trypanosoma congolense, Trypanosoma vivaxand Trypanosoma bruceiwere identified in the examined animals. T. congolensefollowed by T. vivaxwere the predominant species (respectively 59.0 and 35.9% in early dry season and 62.0 and 22.8% in early rainy season). The prevalence of T. vivaxremained similar in both early dry and early rainy seasons in both lowland and midland agroecologies whereas T. congolensewas more dominant in the lowland area in both seasons compared to mid land study sites. The disease was more prevalent in lowland (23.9%) compared to midland (11.1%) during early rainy season (P < 0.001) whereas no significant difference was observed between the two agroecologies during early dry season (P = 0.165). Packed cell volume (PCV) was much lower in parasitemic animals than in aparasitemic cattle whereas the mean PCV value for parasitemic animals (20.36%; 95% CI 19.56 to 21.16) in early dry season was similar to values in early rainy season (20.46%, 95% CI 18.84 to 21.08%). A similar situation was noticed for animals in both low land and mid land study sites. Overall, the detection of trypanosomes in blood was significantly affected by agro-ecology, season and body condition of the animals. Special emphasis should be given to integrated trypanosomosis management in early rainy months where fly population is believed to start increasing.

Glycosylceramides (GlyCers) are precursors of ceramides (Cers) that are major components of the outer layer of human skin, the stratum corneum. A Cer deficiency is associated with skin diseases such as psoriasis and atopic dermatitis and can be treated with Cer-containing semisolid formulations. Plants may serve as alternative sources for expensive semisynthetic Cer production. Since the GlyCer contents of plants vary widely, there is a need to develop a rapid, simple, selective, and precise method for GlyCer quanti-fcation in plants. In the present study, an efective and validated automated multiple development—high-performance thin-layer chromatography (AMD—HPTLC) method has been developed for GlyCer quantification in 9 d iferent plant materials. An 18 -step gradient elution program (n-hexane, chloroform, ethyl acetate, meth-anol) led to a clear separation of bands from complex matrices and allowed densitometric analysis for quantification purposes. Apple pomace and wheat germs yielded 26.8 and 39.5 mg of GlyCer per 100 g plant material, respectively, while the yields of cofee grounds were below the limit of quantification. The GlyCer contents of the seeds of six Fabaceaespecies, namely, Albizia grandibracteata, Albi-zia gummifera, Albizia lebbeck, Albizia schimperiana, Acacia etba-ica, and Robinia pseudoacacia, ranged from 9.4 to 23.1 mg per 100 g plant material. GlyCers were separated by preparative thin-layer chromatography (TLC) and identified by offline high-performance liquid chromatography—mass spectrometry (HPLC—MS). Intact GlyCers were detected in the Fabaceae species for the first time. A simple AMD—HPTLC screening and quantification technique for GlyCers was developed, which may serve as a tool in searching plant GlyCers for a possible “phyto”-Cer production.

AbstractEssential oils from the aerial part of Salvia officinalisL., grown in Addis Ababa and Wondo genet regions of Ethiopia, were isolated by hydrodistillation to yield 0.99 and 1.2 mL/100 g on dry weight, respectively. Fifty-one compounds representing 96.36 and 91.47 % of the oils, respectively, were identified by GC-FID and GC-MS. The major constituents in both samples were trans-thujone (19.98-26.42 %), 1,8-cineole (16.63-21.65 %) and camphor (12.98-17.58 %). The oils were assessed for their in vitroantimicrobial activities against 21 bacterial and 4 fungal strains using the disk diffusion method. Escherichia colistrains were most susceptible (MIC = 10-25 mg/mL) with viable cells dropping to zero CFU/mL after 13.5-14 hours of exposure to 200 mg/mL of the oils. Furthermore, the oils demonstrated potent radical scavenging potential (IC50= 4.47-4.65 μg/mL) in 2,2-diphenyl-1-picyryhydrazil (DPPH) assay. In light of the above results, the essential oil from S. officinalisgrown in Ethiopia may have the potential to serve as natural preservative.

Traditionally Plectranthus cylindraceusHochst. ex. Benth (Lamiaceae) is used as a disinfectant and for the treatment of various illnesses. Hydrodistillation of the fresh aerial part of P. cylindraceuscollected in Ethiopia yielded 0.39 % (v/w) colourless oil with a strong but pleasant smell. Analysis of the oil by gas chromatography-mass spectrometry (GC-MS) afforded twenty-nine compounds which accounted for 95.26 % of the oil with two of the components yet to be identified. The oil was found to be rich in oxygenated monoterpenes (90.00 %), the major constituents being camphor (40.93 %) and 1,8-cineol (25.38 %). In vitroantimicrobial activity of the oil investigated by disc diffusion technique indicated that it was active against both Gram-positive and Gram-negative bacteria as well as some fungal strains. The activity of the oil was more prominent against several Gram-negative bacterial strains including Escherichia coli, Shigellaspp and Vibro choleraewith a minimum inhibitory concentration (MIC) value of 25 μg/ml. Among the bacterial strains tested Shigella flexneriType 6 was the most susceptible against which the oil displayed an MIC value of 10 μg/ml. The oil did also demonstrate a significant fungal inhibitory potency against Aspergillus nigerand Candida albicanswith an MIC value of 400 μg/ml. At similar concentrations, the oil and the standard antimicrobials used in this study exhibited comparable activities against the tested bacterial and fungal strains. Moreover, the mode of action of the oil was proved to be bactericidal and fungicidal. The study provides evidence for the broad-spectrum antimicrobial effect of P. cylindraceusessential oil, a possible justification for the traditional use of the plant in the treatment of skin, digestive, respiratory and inflammatory diseases which may be related to microbial infections.

AbstractEssential oils extracted by steam distillation from Cymbopogon citratus(DC.) Stapf, Cymbopogon nardus(L.) Rendle and Eucalyptus citriodoraHook were evaluated for their repellent effects against Anopheles arabiensismosquitoes under laboratory conditions. Blended oils, ointments and cream formulations of the oil of C. nardusin different bases were also evaluated. At 10 % and 20 % concentrations, all the oils showed a minimum of 90 % and 95 % relative protection, respectively, soon after application. These were not significantly different in efficacy from NN-diethyl-meta-toluamide (DEET), P<0.05. Afterwards however, only C. nardusoil produced more than 70 % protection for 3 h and 4 h at a concentration of 10 % and 20 %, respectively. Combinations of 10 % or 20 % of each of the oils of C. citratusand C. nardusgave better protection than their individual effects. In the presence of the oil of E. citriodora, the effects of C. citratusand C. narduswere significantly reduced at both concentrations. The oil of C. nardusin oleaginous ointment (i.e. Vaseline) provided above 80 % relative protection for as long as 4 h which was better than the effect of the 20 % oil, P<0.05. Similarly, the oil in hydrophilic ointment, 1:1 w/w blend of PEG 4000 and PEG 600, gave better repellent effect than the 20 % oil (P<0.05). However, the effect of the o/w cream was not statistically different from the 20 % oil, P>0.05. The blends of C. citratusand C. nardusoils and the ointment formulations of the oil of C. nardusshowed improvement on the percentage of protection of the oil of C. nardus. However, both the blending and the formulations did not improve the duration of protection. In addition, both the percentage and duration of protection were not as good as DEET, P<0.05. This study demonstrated the potential of essential oil of C. nardusas topical repellent against An. arabiensis.

As part of ongoing explorations of the structural diversity of cyclotides, the cyclotide content of a native violet of the East African highlands, Viola abyssinica(which grows at altitudes up to 3400 m), was studied. Six new cyclotides, vaby A−E (1−5) and varv E (6), were isolated and characterized by employing HPLC and MS techniques and quantitative amino acid analysis. Cyclotides 1−5were found to have new sequences, and 1−3have a further novel feature in their sequences, an alanine moiety in loop 2. Two of the cyclotides (1and 4) also exhibited cytotoxic properties in a flourometric microculture cytotoxicity assay. The findings corroborate the hypothesis that investigating the cyclotide contents of violets growing in diverse environments is a promising approach for extending our knowledge of both the structural and biological diversity of cyclotides.

AbstractThe essential oils of fresh aerial parts of Satureja abyssinica(Benth.) Briq. ssp. abyssinicaand Satureja paradoxa(Vatke) Engl. collected in Ethiopia were analyzed by gas chromatography (GC/FID) and gas chromatography-mass spectrometry (GC/MS). The results of the analyses indicated that the components of the two oils are similar qualitatively with significant quantitative differences. Twenty-three compounds comprising 96.9% of the total peak area were identified in S. abyssinicassp. abyssinicaoil with the monoterpene ketones pulegone (43.5%) and isomenthone (40.7%) as major components. The oil of S. paradoxawas found to contain twenty-six compounds comprising 92.0% of the total oil with the major components being cis-piperitone oxide (45.3%), pulegone (9.3%) and piperitenone oxide (8.1%). The in vitroantimicrobial activity of the oils was studied against some medically important pathogens including Gram positive and Gram negative bacteria as well as some fungal strains using standard agar-well diffusion technique. Both oils exhibited significant activity against most of the bacteria and fungi used in the study with minimum inhibitory concentration (MIC) values ranging from 0.50 to 4.00 μL/mL, against the bacteria, and 0.19 to 3.00 μL/mL, against the fungi. Comparison of their activity with standard antibacterial and antifungal agents revealed that both oils were more active against Streptococcus pyogenesthan the standard antibiotic amoxicillin. Moreover, the oil of S. paradoxadisplayed a powerful activity against Candida albicansand Trichophyton mentagraphyteswith growth inhibition zone greater than that of the standard antifungal agent ketoconazole. The oils were also tested for their antioxidant activity in diphenylpicrylhydrazyl (DPPH) assay and showed a dose dependant free radical scavenging activity with IC50 values of 1.8 μL/mL (S. abyssinicasubsp. abyssinica) and 1.6 μL/mL (S. paradoxa).

13 Alkaloids were identified in leaves and twigs of Platycelphium vöense (a monotypic genus in the tribe Sophoreae) by capillary GLC and GLC-MS. Cytisine, N-methylcytisine, anagyrine and 5,6-dehydrolupanine figured as major and N-formylcytisine, N-acetylcytisine, baptifoline, thermopsine, isolupanine, and rhombifoline as minor alkaloids in both leaves and twigs. Lupanine occurred in considerable amount in the leaves and as a minor component in the twigs. The rare alkaloid, 6ß-hydroxylupanine, was detected only in the leaves while N-ethylcytisine was shown to be a minor constituent of the twigs only.

Good quality drugs fulfilling the regulatory parameters and produced per the current good manufacturing (CGMP) standards are very critical for best therapeutic outcome in patient therapy. Hence, this study assesses quality as well as physicochemical bioequivalence of five brands of glibenclamide tablets marketed in Addis Ababa using in vitro and in vivo methods. Friability, disintegration, dissolution, and assay for the content of active ingredients were evaluated using the methods described in the British Pharmacopeia (2009) and United States Pharmacopeia (2007). All the brands of glibenclamide tablets complied with the official specification for hardness, friability, disintegration, and assay. Difference factor (f1) values were less than 15 and similarity factor (f2) values were greater than 50 for all products of glibenclamide. The hypoglycemic effect of different products of glibenclamide tablets was evaluated on normoglycemic mice. The in vivo studies indicated that there is no significant difference in percent reduction of blood glucose level between the brands of glibenclamide and the innovator product (p > 0.05). Hence, based on the in vivo results and in vitro dissolution studies, the brands might be substituted with the innovator product in clinical practice.

Phytochemical investigation of the leaf latex of Aloe trichosantha by preparative TLC gave two closely related anthrones, aloin A/B (1) and aloin-6'-O-acetate A/B (2). The identity of the compounds was established from HRESI-MS, 1H, 13C, DEPT, HMQC and HMBC spectral and chemical data. Whilst aloin A/B occurs in several Aloe species, aloin-6'-O-acetate A/B was isolated for the first time. The isolated compounds inhibited growth of several bacterial and fungal pathogens with minimum inhibitory concentration (MIC) from 10 to 400 μg/mL and 800 to 1000 μg/mL, respectively.

Essential oil from the aerial part of Plectranthus marrubatusJ. K. Morton (Lamiaceae), obtained by hydrodistillation was analyzed by gas chromatography/mass spectrometry (GC/MS) and evaluated for antimicrobial and free radical scavenging activities. Twenty-four compounds representing 99% of the total oil were identified. The major constituents were thymol, p-cymene and γ-terpinene. The oil was tested against 21 bacterial and 4 fungal strains using the disc diffusion method and found to be active against a broad spectrum of pathogens including Gram-positive and Gram-negative bacteria as well as some fungal strains. The minimum inhibitory concentrations (MICs) of the oil against the bacterial strains tested ranged from 10 to 800 μg/mL, and from 400 to 800 μg/mL against the fungal strains employed. The in vitroantioxidant activity was assessed using 2,2-diphenyl-1-picrylhydrazil (DPPH) and showed a low EC50value of 0.15 μl/mL. The study provides evidence for the broad-spectrum antimicrobial and antioxidant effect of Plectranthus marrubatusessential oil, and a possible explanation for its traditional use in the treatment of cold, fever, stomach disorder, diarrhea and as a skin cleaner.

Free radical scavenging activity-guided investigation of the acetone extract of Plectranthus punuctatus(L'Her) (Lamiaceae) resulted in the isolation of a novel diterpenoid 3,11,12,14,19-pentahydroxy-5,8,11,13-abietatraen-7-pyran[4,6]-16-dione whose structure was elucidated based on spectral evidence. The isolated compound showed a significant antioxidant activity in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and deoxyribose degradation assays with IC50values of 9.65 μg/mL and 5.25 μg/mL, respectively.

Phytochemical investigation of the latex of Euphorbia abyssinicaGmel (Euphorbiaceae) afforded a new hydroxy unsaturated fatty acid, 8(R)-hydroxy-dec-3(E)-en-oic acid (1), in addition to the four known compounds lupeol (2), β-sitosterol (3), oleanolic acid (4) and β-sitosterol-3-O-glucoside (5). The in vitroantibacterial and antifungal activities of the isolated compounds, as well as the total methanol extract, were studied against different micro-organisms; compound 1displayed reasonable antifungal activities towards the tested fungi.

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