Material and Methods
Standardized ciprofloxacin 50 ?g/mL was diluted in Dimethyl sulphoxide (DMSO) used as positive controls for the antibacterial susceptibility test; where as DMSO serves as a negative control), p-Iodonitrotetrazolium chloride (INT), Muller-Hinton agar was used as a microbial growth indicator.
3.2. Plant materials
The root of C. ficifolius was collected from Estie town which is located South Gondar, Amhara region, in January 2018 Ethiopia, 165 km far from Gondar. Then the Botanical identification and authentication of the plant materials were performed and voucher specimens (No……….) has been deposited in Herbarium Biology Department, Faculty of Natural and Computational Science, University of Gondar.
The test was car¬ried out against three Gram positive bacterial strains: Staphylococcus aureus (ATCC 25923), Enterococcus faecalis ATCC 1912/R and Streptococcus pyogenes (ATCC 19615) and three Gram negative bacterial strains: Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 2706) and Klebsiella pneumonia (ATCC 700603). All the standard bacterial strains were obtained from Department of Microbiology, University of Gondar, and Amhara Regional Laboratory, Bahr Dar.
3.4. Preparation of plant extract
The roots were washed thoroughly under tap water and shad dried at room temperature for three weeks. There after, the dried roots were grinded into a coarse powder and 600 g of powder was macerated in 80% methanol for the period of 72 h with occasional shaking and stirring. This was repeated for three times and the extracts thus obtained were filtered through filter paper (Whatman Fitter Paper No. 1). Then, the filtrate was allowed to evaporate using oven at temperature 40 °C. Finally, a highly concentrated methanol extracts were obtained and kept in refrigerator for further experiment.
3.5. Determination of antibacterial activity
Bacterial broth culture was prepared to a density of 108 cells ml-1 of 0.5 Mc Farland standards and on each plate; equidistant wells were prepared with a 6 mm diameter sterilized cork borer then randomly labelled. The aliquot was spread evenly onto Muller Hinton agar using sterilized cotton swab. Next, 100 ?L of each extract at concentrations of 100, 200 and 400 ug/mL, by using dimethyl sulphoxide (DMSO) as solvent, were applied aseptically into a respective agar wells. Ciprofloxacin 50 µg/mL, dissolved by s dimethyl sulphoxide (DMSO), was used as positive control and DMSO (100 ?L) was included as negative control. The agar plates were allowed to stand on bench for 30 minutes at room temperature for pre-diffusion and then incubated at 37oC for 24 hours. Antibacterial activity was determined by measuring the Zone of inhibition (ZI) around each well (excluding the diameter of the well by subtracting 6 mm from ZI results). For each concentration, three replicate trials were conducted against six organisms(51).
3.6 .Preliminary Phytochemical screening
The crude extract of cucumis ficifolius was screened for the presence or absence of secondary metabolites such as alkaloids, steroidal compounds, terpenoids, phenolic compounds, flavonoids, tannins, phlobatanins, and saponins by using the methods as described by standard procedures.edeoga (52)and jones and kinghorn(53)
Terpenoids (Salkowski test)
One hundred milligram of the extract was mixed with 2 ml chloroform and 3 ml of sulfuric
acid was added to form a layer. A reddish-brown coloration of the interface was an indication of terpenoids.
A half gram of the dried powdered plant sample was boiled in 20 ml of water in a test tube and filtered. Few drops of 0.1% ferric chloride were then added to the filtrate and the formation of brownish green or a blue-black coloration was regarded as indicator of the presence of tannins.
Detection of alkaloids
Mayer’s test: To a few ml of filtrate, 2-3drops of Mayer’s reagent (Potassium mercuric iodide solution) was added along the slides of the test tube. Formation of white or creamy precipitate indicates presence of alkaloids.
(ii)Wagner’s test: To a few ml of filtrate, 2-3drops of Wagner’s reagent (Potassium iodide solution) was added along the sides of the test tube. Formation of reddish brown precipitate indicates presence of alkaloids.
Detection of flavonoids
NaOH test: 50mg of extract was dissolved in 2ml of alcohol and to the extract, increasing amount of NaOH was added. It shows yellow coloration which decolorizes after addition of an acid.
Detection of saponins
Foam test: About 50mg of extract was dissolved in 2ml of alcohol, diluted with20ml of distilled water and shaken for 15min in a graduated cylinder. A layer of stable foam indicates presence of saponin glycosides.
Detection of phenolics
FeCl3 test: About 50mg of extract was dissolved in 2ml of distilled water, add 2drops of neutral 5% FeCl3 solution and observed for colouration. Formation of blue, green and violet colour indicates the presence of phenolic compounds
Detection of steroids:
Salkowski test: To about 50mg of extract, 2ml of chloroform and 2mlof concentratedH2SO4were added and shaken well. Then observed for coloration of CHCl3 and acid layer .appearance of Chloroform layer in red color and acid layer as greenish yellow fluorescence indicates the presence of steroids.
Detection of anthraquinone
Boil 200 mg of extract with 6 ml of 1% HCl and filtered. Shake the filtrate with 5 ml of benzene, filtered and add 2 ml of 10% ammonia solution to the filtrate. Then shake the mixture and the presence of a pink, violet or red colour in the ammoniacal phase indicated the presence of free hydroxyl anthraquinones