A study of in-vitro antimicrobial effect of phytopreparations was carried out in Debrezeit between November 2007 and May 2008. Debreziet town is located at 47 km south-east of Addis Ababa. The area has an altitude of 1,850 meters above sea level with an average annual rain fall of 866 mm. It has a bimodal rainy seasons; a main rain season extends from the month of June to September and a short rainy season from March to May. The annual average minimum and maximum temperature is 11 ⁰C and 26 ⁰C, respectively. Day length is fairly constant throughout the year (12-13 hrs) with about 6 hours of sunshine during the rainy season and 8 to10 hours for the rest of the year. Humidity is about 50.9 % [7].

An experimental study on in-vitro antimicrobial efficacies on selected plants was conducted between November 2007 and May 2008 in AAU, Faculty of Veterinary Medicine, Debrezeit.

1. Combertum molle (“Agalo, Abalo” in Amharic, “Bika, Dadamata” in Oromiffa) known with common name velvet-leaved combretum (Fig 1). This is a member of the family Combertaceae which is a small deciduous tree growing up to 15 meters high with an often-crooked trunk, commonly branching to the base. The bark is dark brown to black and deeply grooved in squares. The leaves are oppositely arranged, elliptic to lanceolate, large that covered with soft hairs, rounded at the base. The flowers generally appear before the leaves and the fruits yellowish, four-sided with wings. The leaves were used to assess the antimicrobial effect on bacterial isolates from mastitic cases.

2. Commicarpus pedenculosus (“Tihuan tila” in Amharic, “Sara” in Oromiffa) known with common name Chick weed (Fig 2). This is a member of the family Nyctaginaceae which is a prostate or scrambling, glabrescent to pubescent herb with stems up to 3m long. Leaves ovate up to 7cm long, 4cm wide, the base shallowly cordate, truncate, orcuneat, apex acute to obtuse; margins entire to sinuate; petioles up to 2m long. Inflorescence a dense many-flowered head, on a long stout peduncle up to 23cm; flowers sub sessile, but pedicels enlarging in fruit up to 4-10 mm long. The leaves were used to assess the antimicrobial effect on bacteria isolated from mastitic cases.

3. Lagenaria siceraria (“Kil” Amharic) known with common name bottle gourd (Fig 3) is belongs to the family Cucurbitaceous. The leaves were used to assess the antimicrobial effect.

One species of bacterium, Staphylococcus aureus, coming from the clinical mastitis case and representing common bacterial pathogens (from Department of microbiology of Faculty of Veterinary Medicine Debre Zeit) was used in testing.

The plants were chosen based on the results showed by previous workers on the leaf of the plants [3, 5, 6]. Combertum molle was collected from its natural habitat, south Gondar and the other two plants (Commicarpus pedenculosus and Lanegeria siceraria) were collected from Debrezeit. After collection the plants were washed with tap water to remove unnecessary particles. Then dried under shade, chopped by knife and axe in to pieces to facilitate drying and grounded using pestles and wooden mortars. The material was then sieved and weighed before maceration.

50 gm of each of the powdered herb leaves of the three plants were macerated in 80% methanol in a flask separately and mixed using a magnetic stirrer. Then after it is allowed to stand for 3 days at room temperature, each sample was then strained using a tea strainer to remove solids. The resulting filtrate was then further filtered using filter paper to obtain a solution free of solids (Fig 4). The solution was then concentrated in a rotary evaporator to remove the methanol. The plant extracts of the three plants were then taken out and put in evaporating dishes and kept in a dry oven at 40 ⁰C to remove the remaining solvent for 24 hours. The residues left after straining and filtration and were macerated again in the same way. The procedure was repeated in the same way for third time to have sufficient amount of extracts. The resulting concentrated extracts were weighed, transferred and labeled with the respective plant names and stored at + 40 until tested for antimicrobial activity. The flow chart from collection to extraction is shown below.

Six serial dilutions with different concentrations (20 %, 10 %, 5%, 2.5 % and 0.625 %) of each plant extract were prepared using Dimethylsulfoxide (DMSO) as described by Olila et al. [8]. In the first test tube 2ml of DMSO was added and each of the remaining five tubes was filled with 1ml of DMSO. 1ml of 20% solution from the first tube was transferred to a second test tube to prepare 10% .The procedure continues by transferring 1ml of solution from the 10% preparation to a third test tube to get a 5 % concentration, and continued in a similar manner until a 0.625% concentration is reached. Discs of 12mm diameter were impregnated by adding three drops from each reconstituted solution and allowed to dry at 37 ⁰C over night. Dried discs were used to determine antimicrobial effects of the respective plant types. Each disc was gently pressed down to ensure complete contact with the agar and the plates were inverted and incubated at 37 ⁰C for 24 hours. The diameter of zone of inhibition was measured in millimeters.

The antimicrobial test was conducted using agar disc diffusion method. Staphylococcus aureus from clinical mastitis cases were used for this study. The top of 4-5 well isolated colonies of the same morphology were scooped using a wire loop from the nutrient agar and mixed using sterile normal saline (alternative method) and agitated with a vortex mixer [9].

The turbidity of the bacterial suspension was adjusted by comparing with 0.5 McFarland turbidity standards. The standard and the test suspension were placed in a 10ml sized tests tubes and compared against a white back ground with contrasting black lines until the turbidity of the test suspension equates to that of the turbidity standard. Adjustments of the turbidity were made by adding saline or colonies depending on the degree of turbidity. A sterile swab was dipped in to the standardized suspension of the bacteria and excess fluid was expressed by pressing and rotating the swab firmly against the inside of the tube above the fluid levels. The swab was streaked in the three directions over the entire surface of the agar with objective of obtaining uniform inoculations, and a final sweep with the swab was made against the agar around the rim of the Petri dish. The inoculated plates were allowed to stand for not more than 15 minutes and the discs were placed on the agar surface using a sterile forceps. Each disc was gently pressed with the point of the sterile forceps to ensure complete contact with the agar surface [9].

Muller-Hinton agar (38gm) (Biotech UK) medium was used for antimicrobial sensitivity test, and was mixed with 1 litter of distilled water, boiled to dissolve completely and autoclaved at 121⁰c for 15minutes. The medium was later dispensed in to 90mm sterile agar plates and left to set. The agar plates were incubated for 24 hours at 37⁰ C to confirm their sterility. When no growth occurred after 24 hours, the plates were considered sterile and used for antimicrobial sensitivity tests.

Barium solution was used as standard to determine the bacterial concentration was prepared as 1% solution in 1% H₂SO₄ solution. The preparation was kept in dark for the preparation of bacterial suspension. Colonies were picked from the culture under study and placed in 4ml sterile physiological saline and the culture was standardized by comparing with McFarland solution.

The appropriate crude extract impregnated discs and conventional discs were applied at spaces of 24mm apart from center to center and 15mm away from the edge of the plates. This was made no later than 15minutes after the inoculums has been seeded. The plates turned upside down, labeled and incubated at 37⁰C for 24 hours. Diameter of zone of inhibition was measured using a ruler in millimeters and results were recorded as susceptible, intermediate or resistant by comparing with standard values for each conventional antibiotic disc [9].

Effects of Herbal Preparations

Each plant extracts of the three plant species were tested at different concentrations levels (20%, 10%, 5%, 2.5%, 1.5%, 2.5%, 1.25%, and 0.625%) to see their inhibitory effects against Staphylococcus aureus (Fig 6). Of the three candidate plants in this study, two plants showed antibacterial activity against the tested bacteria and the remaining plant did not show any activity after alcoholic extraction (Table 1).

zone of inhibition in mm for each plant extracts at doubling concentrations ranging from 0.625% to 20% (Table 1). The inhibiting zone increased with increasing concentrations of the extracts for the two plant species (Commicarpus pedenculosus and Combertum molle). There are no inhibition zones for bacterial growth at the lower concentrations 1.25 % and 0.625 % of Commicarpus pendunculosus and 0.625 % Combertum molle respectively. A significant difference was observed against Staphylococcus aureus inhibition by the leaf of both Commicarpus pedenculosus and Combertum molle at higher concentrations (Table 1 and Fig 5). The zone of inhibition for the leaf of Commicarpus pedenculosus against Staphylococcus aureus was higher than Combertum molle at concentrations 2.5% to 20 %, whereas Combertum molle has more effect than Commicarpus pedenculosus at lower concentration (1.25%) (Fig 5). The extracts of Lagenaria siceraria, however virtually demonstrated no activity at all concentrations against Staphylococcus aureus.

Figure 1

Fig 1. Combertum molle (velvet-leaved combretum)

Figure 2

Fig 2. Commicarpus pedenculosus ( Chick weed)

Figure 3

Fig 3. Lagenaria siceraria (bottle gourd)

Figure 4

Fig. 4. Flow chart for extraction

Figure 5

Fig. 5. In-vitro efficacy tests of 80% methanol crude extracts of leaf different plants on

Figure 6

Fig. 6. Inhibitory effects of against Table 1. In-vitro efficacy testes of 80% methanol crude extracts of leaf of different plants on .