Comparative study on the effect of extraction solvent on total phenol, flavonoid content, antioxidant and antimicrobial properties of red onion (Allium cepa)

Disc diffusion method
Several sterile, blank discs (6 mm in diameter) were impreg-
nated with 20 μL of the extract at several dilutions (6.25, 

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Comparative study on the effect of extraction solvent on total phenol, flavonoid content,…
1 3
12.5, 25, 50, 100, 200 and 400 μg/mL). A 5 μL of the 
extract was spotted alternately on both sides of the discs and 
allowed to dry before the next 5 μL was spotted to ensure 
precise impregnation. Distilled water-loaded discs were used 
as negative controls. All discs were fully dried before the 
application on the medium. The positive controls used were 
Gentamycin antibiotic discs (Padtan Teb, Iran) for all bacte-
ria and Miconazole discs (Rosco Diagnostica A/S, Denmark) 
for the fungi. The Petri dishes were incubated at 37 ºC for 
24 h for bacteria and 25–28 ºC for 72 h for fungi. Antibacte-
rial activity was evaluated by measuring the inhibition zone 
diameter around the discs. The antibacterial activity was 
expressed as the mean of inhibition zone diameter (mm) 
produced by the concentrations of each extract. The assay 
was conducted in three replications [
21
].
Determination of minimum inhibitory concentration (MIC)
MIC was determined using the broth microdilution method 
on Mueller Hinton broth in a 96-well round-bottom micro-
titer plate [
22
]. Only the medium and bacterial or fungal 
suspensions were added to the first-row wells as negative 
and positive control, respectively. In the next row, 100 μL of 
Mueller Hinton broth was added to 6 well of the microtiter 
plate, then, 100 μL of the studied concentrations (6.25, 12.5, 
25, 50, 100, 200 and 400 μg/mL) of the aqueous extract was 
added separately into 6 wells. 10 μL of bacterial and fungal 
suspensions were added to each well. The microtiter plate 
was then incubated at 37 ºC for 24 h. The turbidity of the 
wells was first assessed visually and then to determine the 
minimum growth inhibitory concentration of bacteria and 
fungi, 5 mg/mL solution of 2, 3, 5-Triphenyltetrazolium-
chloride (TTC) was used (it is a growth reagent which is 
colorless in oxidized form, but when reduced by microorgan-
isms, it turns red due to the formation of formazan). A 50 
μL of TTC was poured into all wells of the microtiter plate 
and incubated again for 3 h. After 3 h, the microplates were 
checked [
22
].