Collection and authentication of the plant material
The leaves of Rhamnus prinoides was collected from the cultivated garden of a family in October 2014, around Debre Markos Town, East Gojjam Zone, Amhara Region, 300 km away from Addis Ababa, North West Ethiopia and identified by a taxonomist at the National Herbarium, Department of Biology, College of Natural and Computational Sciences, Addis Ababa University, and the specimen (number YM002) was deposited for future references.
Preparation of extracts
One hundred fifty gram of coarsely powdered leaves was macerated in 80 % methanol for a period of 3 days with occasional shaking using a shaker (Bibby scientific limited stone Staffo Reshire, United Kingdom). The extract was filtered with Whatman No 1 filter paper (Schleicher and Schuell Microscience Gmbh, Germany). The residue was remacerated for the second and third times with fresh solvent. The extracted solution was combined and concentrated by Rota vapor (Buchi Rota-vapor R-200, Switzerland). Then, it was dried in a lyophilizer (Operan, Korea vacuum limited, Korea).
The solvent fractionation was conducted by using both Soxhlet and maceration techniques. Soxhlet extraction was carried out by sequential extraction of the powdered leaves in solvents of increasing polarity viz. chloroform and methanol as described by a study . The powdered leaf was extracted by chloroform at a temperature not exceeding 40 °C. This extraction process was continued exhaustively until clear solution in the timble was siphoned into the solvent flask. Then, the chloroform fraction was filtered with Whatman No. 1 filter paper and concentrated using rotary evaporator. The concentrated solution was dried in oven at a temperature of 40 °C.
The dried marc of the chloroform fraction was extracted using absolute methanol following the same procedure described for chloroform fraction. Finally, the dried marc of methanol fraction was macerated with distilled water and dried in the lyophilizer to get the aqueous fraction.
The percentage yield of the crude extract, aqueous fraction, methanol fraction, and chloroform fraction were 17.5 %, 4.32 %, 8.74 % and 6.21 %, respectively. Each dried crude extract and solvent fractions was reconstituted in sterile 20 % DMSO (for crude extract and the methanol fraction), 70 % DMSO (for the chloroform fraction) or sterilized distilled water (for aqueous fraction) to prepare the tested concentrations of the plant material.
The qualitative phytochemical investigations of the crude extract, and chloroform, methanol and aqueous fractions of leaves of Rhamnus prinoides were carried out using standard tests [20–22]
Inoculum preparation and standardization
Standard and clinical isolates of different bacterial strains including Escherichia coli (E. coli) (American Type Culture Collection (ATCC) 25922), E. coli (clinical isolate), Pseudomonas aeruginosa (P. aeruginosa) (ATCC 27853), P. aeruginosa (Clinical isolate), Staphylococcus aureus (S. aureus) (ATCC 25923), S. aureus (clinical isolate), Shigella flexneri (S. fleneri) (ATCC 12022), S. fleneri (clinical isolates), Streptococcus pneumoniae (S.pneumoniae) (ATCC 49619), S. pneumoniae (clinical isolate), Streptococcus pyogen (S. pyogen) (ATCC 19615), S.pyogen (clinical isolate) and Salmonella typhi (S. typhi) (ATCC 13062) were obtained from Ethiopian Public Health Institution (EPHI). Then, each bacterial strain was inoculated and spread on pre-labeled nutrient agar and 5 % sheep blood agar (for streptococcus species) aseptically in a Safety Cabinet (Bioair instruments, Eurolone® Company, Italy) and incubated for 24 h at 37 °C. The bacterial turbidity of each bacterium was prepared by growth method and standardized by following the guideline of Clinical and Laboratory Standard Institute . The turbidity of the inoculum tube was adjusted visually by either adding bacterial colonies or by adding sterile normal saline solution to that of the already prepared 0.5 McFarland standard.
Antibacterial activity assay
Agar well diffusion
The antibacterial agar well diffusion assay was conducted by following the methods described previously . The standardized bacterial broth culture prepared in section 2.4 were streaked evenly on sterile MHA plates or MHA with 5 % sheep blood (for streptococcus species) with cotton swab. After thirty minutes, on each plate, four equidistant wells were made with a 6 mm diameter sterilized cork borer. The labeled wells were filled with 100 μl of 780 mg/ml, 390 mg/ml, and 195 mg/ml of the crude or each of the solvent fractions making the final concentration of 78 mg/well, 39 mg/well and 19.5 mg/well respectively. In addition, the commercial antibiotic discs of ampicillin 0.01 mg/disc (for streptococcus species), cefoxitin 0.03 mg/disc (for E. coli and S. aureus species) and ciprofloxacin 0.005 mg/disc (for other bacterial strains) were used as a positive control. The solvents of the crude and each fraction were used as negative controls. Then, the plates were left undisturbed for about 2 h at room temperature. After incubation at 37 °C for 24 h, the zone of inhibition was measured using a ruler. The experiment was performed in three independent tests for each bacterial strains and the mean of zones of inhibition was calculated for each extract.
Determination of the Minimum Inhibitory Concentration (MIC)
The crude extract and solvent fractions that showed antibacterial activity by agar well diffusion method were subjected to serial resazurin based microtitre dilution technique on 96 well plates (Greiner Bio-One, Germany) as described by previous studies [25, 26]. The first column of microtiter plate was filled with 100 μl stock solution (390 mg/ml) of the crude or each active solvent fraction of test material except the last well in which equal amount of the respective solvent was added. Then, all the wells of microtitre plates were filled with sterilized 100 μl of MHB  or BHI (for streptococcus species) . Two fold serial dilution of the crude extract or solvent fraction (throughout the row) was carried out until the 10th column. The 11th and 12th column were used as the growth control for labeled bacterium in which the 100 μl of the solvents of the extract or that of the solvent fraction was added. Then, 30 μl of 0.01 % w/v resazurin solution was added and mixed in each well.
Briefly, within 15 min of standardization to approximately 5 × 106 CFU/mL with the respective broth, 20 μl of bacterial suspension was added to each well except 7th and 8th row which were reserved as extract color contrast control and sterility control, respectively. Subsequently, the serial dilution procedures gave rise to a final concentration of the plant material ranging from 130 mg/ml to 0.26 mg/ml.
Finally, after wrapping each plate loosely with parafilm, the plates were incubated at 37 °C for 24 h. In the experiment, each plate had a set of three controls: (a) a column with all solutions with the exception of the test extract or fraction, (b) a row with all solutions except bacterial suspension and the extract or fraction was used as sterility control, (c) a row with all solutions except the bacterial inoculums was used as color contrast control.
After incubation, any color change observed from purple to pink or colorless was taken as positive for growth of bacteria. The lowest concentration of plant leaf extract at which no color change occurred was recorded as the MIC value. All the experiments were performed in triplicates for each bacterium. The average value was taken for the MIC of test plant material.
Determination of Minimum Bactericidal Concentration (MBC)
MBC was determined by a method described in different studies . In this technique, the contents of all wells containing a concentration of test material above the MIC value from each triplicate, in the MIC determination test, was streaked on MHA or MHA supplemented with 5 % sheep blood (for streptococcus species) with wire loop aseptically and incubated at 37 °C for 24 h. The lowest concentration of the extract which showed no bacterial growth after incubation was observed for each triplicate and noted as the MBC. The average value was taken for the MBC of test material against each bacterium.
The experimental data are expressed as mean ± Standard Error of the Mean (SEM). Data are analyzed using the Statistical Package for the Social Sciences (SPSS), version 16.0 software. The statistical differences of the mean zone of inhibition of crude extract and solvent fractions for individual bacterium was carried out by employing one way analysis of variance (ANOVA) followed by Tukey Post Hoc Multiple Comparison test at a significance level of P < 0.05.