standard operating procedures and identified as per
Atef et al. (2019).
3.3 Preparation of medicinal plant extracts
The extraction process was done according to Jansirani et al. (2014) with modifications. Briefly, the dried
mature plant leaves of the selected plants were ground
into moderately course powder with the help of a pestle and mortar, weighed using a digital balance, and
subjected to aqueous and ethanolic extraction by a
maceration process keeping the material to liquor ratio
(MLR) of 15:150 w/v and 15:150 w/v, respectively.
After the powder was soaked in different solvents
(distilled water and 99% ethanol) for 24 hours at
50
◦ C with occasional stirring, the extract was filtered
through Whatman No.1 filter paper to obtain a clear
first filtrate. The second filtrate was obtained by soaking the used grounded leaves in both solvents (water
and ethanol) while keeping the same temperature and
MLRs for 24 hours. Both filtrates were mixed and
concentrated in a rotavapour in controlled conditions
to 300 mg/mL and stored in an airtight container at
4
◦ C for further studies.
3.4 Preparation of inoculum
Each bacterial strain was sub-cultured in Mueller Hinton agar slats at 37
◦ C overnight. The bacterial isolated
colonies were picked and dissolved in the 1 mL of the
sterile normal saline and their turbidity adjusted to the
equivalent of 0.5 McFarland standard solution (1×10
8
CFU/mL).
3.5 Antibacterial activity of the plant extracts
The plant extracts were tested for their antibacterial
activity by using Disc diffusion method following the
Kirby-Bauer Test Protocol (Hudzicki 2009) that is in
line with recommended standards of Clinical and Laboratory Standards Institute (CLSI) (Jean B. Patel et al.
2015).
3.5.1 Disc diffusion method
The Mueller Hinton agar plates were prepared by pouring 20 mL of molten media on to sterile Petri dishes at
56
◦ C and they were left to solidify. Thereafter, they
were labelled and overnight incubated for sterility.
Using sterile cotton swabs, 0.1% suspension of each
bacterial strain was spread evenly over the Mueller
Hinton agar plates and six depressions were created
in the agar medium at a spacing of 2 cm apart with
non-toxic pipette tips and filled with 0.1 mL (300
mg/mL) of plant extract in each well. Standard agar
plates with Ciproflaxin (30 µg) and Amikacin (30 µg)
were considered as positive control for the bacteria
while distilled water and 99% ethanol was used as the
negative control. All the plates were left to stand for
45 minutes for the diffusion of the extracts into the
medium. Then they were covered and incubated aerobically at 37
◦ C overnight. All the tests were performed
in triplicate. The antibacterial activity of the extracts
was then evaluated basing on the inhibition zones measured using an inhibition zone ruler that was placed
edge to edge across the zone of inhibition over the plate
disc and the mean diameters recorded in millimetres.
3.5.2 Minimum Inhibition Concentration (MIC)
determination of the aqueous and ethanolic
extracts
Minimum Inhibitory Concentration is the lowest concentration that can inhibit any growth of bacteria on
the prepared culture plates (Batra 2012), It was determined for the aqueous and ethanolic extracts using
serial dilutions as described by Fg et al. (2016) with
slight modifications. Serial dilutions of the aqueous
and ethanolic extracts of Datura stramonium (DS),
Ricinus communis (RC), and Galinsonga parviflora
(GP) were prepared at various concentrations (30, 3,
0.3 and 0.03 mg/mL) of extracts and put in 5 cm
test tubes. Using non-toxic pipette tips, depressions
were created in overnight incubated Mueller Hinton
agar plates. Then 0.1 mL of different concentrations
(30, 3, 0.3, and 0.03 mg/mL) of each extract were
added to each depression on the Mueller Hinton agar
plates and incubated overnight at 37
◦ C. The lowest
concentration that inhibited the growth of microorganisms was considered to be the minimum inhibitory
concentration.
4 RESULTS
4.1 Antibacterial efficacy of extracts from selected
medicinal plants
4.1.1 Disc diffusion assay
The results in Table 1 and Figures 1 and 2 show
that the aqueous and ethanolic extracts of selected
medicinal plants exhibited varied antibacterial activity.
Based on the mean diameters of the inhibition zones,
Racinus Communis aqueous dye extracts exhibited
higher antibacterial activity against Staphylococcus
aureus and Pseudomonas spp. bacterial strains compared to ethanolic dye extracts. Then Galinsoga parviflora aqueous extracts also showed better antibacterial
activity as opposed to that demonstrated by ethanolic extracts. The trend was not different for Datura
stramonium where its aqueous dye extracts demonstrated higher activity in comparison to ethanolic
extracts. It was also clearly shown that for all selected
plants, their aqueous and ethanolic dye extracts are
more susceptible to Gram-positive bacteria (Staphylococcus aureus) than to Gram-negative bacteria
(Pseudomonas spp).
4.1.2 Antibacterial activity of the controls
The control experiment demonstrated that there was
no influence in the recorded inhibition zones of both
aqueous and ethanolic extracts of different plants
against used Gram-positive and Gram-negative bacterial strains, as clearly shown in Figure 3 and
Table 2.
97
Atef et al. (2019).
3.3 Preparation of medicinal plant extracts
The extraction process was done according to Jansirani et al. (2014) with modifications. Briefly, the dried
mature plant leaves of the selected plants were ground
into moderately course powder with the help of a pestle and mortar, weighed using a digital balance, and
subjected to aqueous and ethanolic extraction by a
maceration process keeping the material to liquor ratio
(MLR) of 15:150 w/v and 15:150 w/v, respectively.
After the powder was soaked in different solvents
(distilled water and 99% ethanol) for 24 hours at
50
◦ C with occasional stirring, the extract was filtered
through Whatman No.1 filter paper to obtain a clear
first filtrate. The second filtrate was obtained by soaking the used grounded leaves in both solvents (water
and ethanol) while keeping the same temperature and
MLRs for 24 hours. Both filtrates were mixed and
concentrated in a rotavapour in controlled conditions
to 300 mg/mL and stored in an airtight container at
4
◦ C for further studies.
3.4 Preparation of inoculum
Each bacterial strain was sub-cultured in Mueller Hinton agar slats at 37
◦ C overnight. The bacterial isolated
colonies were picked and dissolved in the 1 mL of the
sterile normal saline and their turbidity adjusted to the
equivalent of 0.5 McFarland standard solution (1×10
8
CFU/mL).
3.5 Antibacterial activity of the plant extracts
The plant extracts were tested for their antibacterial
activity by using Disc diffusion method following the
Kirby-Bauer Test Protocol (Hudzicki 2009) that is in
line with recommended standards of Clinical and Laboratory Standards Institute (CLSI) (Jean B. Patel et al.
2015).
3.5.1 Disc diffusion method
The Mueller Hinton agar plates were prepared by pouring 20 mL of molten media on to sterile Petri dishes at
56
◦ C and they were left to solidify. Thereafter, they
were labelled and overnight incubated for sterility.
Using sterile cotton swabs, 0.1% suspension of each
bacterial strain was spread evenly over the Mueller
Hinton agar plates and six depressions were created
in the agar medium at a spacing of 2 cm apart with
non-toxic pipette tips and filled with 0.1 mL (300
mg/mL) of plant extract in each well. Standard agar
plates with Ciproflaxin (30 µg) and Amikacin (30 µg)
were considered as positive control for the bacteria
while distilled water and 99% ethanol was used as the
negative control. All the plates were left to stand for
45 minutes for the diffusion of the extracts into the
medium. Then they were covered and incubated aerobically at 37
◦ C overnight. All the tests were performed
in triplicate. The antibacterial activity of the extracts
was then evaluated basing on the inhibition zones measured using an inhibition zone ruler that was placed
edge to edge across the zone of inhibition over the plate
disc and the mean diameters recorded in millimetres.
3.5.2 Minimum Inhibition Concentration (MIC)
determination of the aqueous and ethanolic
extracts
Minimum Inhibitory Concentration is the lowest concentration that can inhibit any growth of bacteria on
the prepared culture plates (Batra 2012), It was determined for the aqueous and ethanolic extracts using
serial dilutions as described by Fg et al. (2016) with
slight modifications. Serial dilutions of the aqueous
and ethanolic extracts of Datura stramonium (DS),
Ricinus communis (RC), and Galinsonga parviflora
(GP) were prepared at various concentrations (30, 3,
0.3 and 0.03 mg/mL) of extracts and put in 5 cm
test tubes. Using non-toxic pipette tips, depressions
were created in overnight incubated Mueller Hinton
agar plates. Then 0.1 mL of different concentrations
(30, 3, 0.3, and 0.03 mg/mL) of each extract were
added to each depression on the Mueller Hinton agar
plates and incubated overnight at 37
◦ C. The lowest
concentration that inhibited the growth of microorganisms was considered to be the minimum inhibitory
concentration.
4 RESULTS
4.1 Antibacterial efficacy of extracts from selected
medicinal plants
4.1.1 Disc diffusion assay
The results in Table 1 and Figures 1 and 2 show
that the aqueous and ethanolic extracts of selected
medicinal plants exhibited varied antibacterial activity.
Based on the mean diameters of the inhibition zones,
Racinus Communis aqueous dye extracts exhibited
higher antibacterial activity against Staphylococcus
aureus and Pseudomonas spp. bacterial strains compared to ethanolic dye extracts. Then Galinsoga parviflora aqueous extracts also showed better antibacterial
activity as opposed to that demonstrated by ethanolic extracts. The trend was not different for Datura
stramonium where its aqueous dye extracts demonstrated higher activity in comparison to ethanolic
extracts. It was also clearly shown that for all selected
plants, their aqueous and ethanolic dye extracts are
more susceptible to Gram-positive bacteria (Staphylococcus aureus) than to Gram-negative bacteria
(Pseudomonas spp).
4.1.2 Antibacterial activity of the controls
The control experiment demonstrated that there was
no influence in the recorded inhibition zones of both
aqueous and ethanolic extracts of different plants
against used Gram-positive and Gram-negative bacterial strains, as clearly shown in Figure 3 and
Table 2.
97