Nitric oxide scavenging assay
In this assay, nitric oxide scavenging activity was measured
using the protocol described by Ebrahimzadeh et al. [
20
].
First, 40 mg of each dried extract was dissolved in 25 mL of
the solvent (15 mL of methanol + 10 mL of normal saline) to
prepare the extract solution with a concentration of 1600 μg/
mL. For the experiment, 25 mg of sodium nitroprusside
(10 mM), in 100 ml of phosphate-buffered saline, was mixed
with 3 ml of different concentrations of the extract solu-
tion (100, 200, 400, 800 and 1600 μg/mL) and incubated at
room temperature for 150 min. The same reaction mixture,
without extract, but with an equivalent amount of water,
served as control. After the incubation period, 0.5 mL of
Griess reagent was added. The absorbance of the formed
chromophore was read at 546 nm. Quercetin was used as a
positive control.
Antimicrobial activity tests
Microorganisms
Lyophilized vials of Staphylococcus aureus (S. aureus)
(PTCC-1431) as Gram-positive bacteria, Escherichia coli
(E. coli) (PTCC-1395) and Salmonella Typhimurium (S.
Typhi) (PTCC-1609) as Gram-negative bacteria and Asper-
gillus niger (A. niger) (PTCC-5154) and Candida albicans
(C. albicans) (PTCC-5027) as fungi were purchased from
the Microbial and Fungal Collection of Iranian Research
Organization for Science and Technology (IROST). The
tested microorganisms were characterized by standard
microbiology methods. The pure cultures were subcultured
on nutrient agar slants and potato dextrose agar and kept at
4 ºC until ready for the study.
Preparation of inoculum
About 24-h cultured bacteria and fungi were suspended
in sterile nutrient broth. It was standardized according to
National Committee for Clinical Laboratory Standards
(NCCLS, 2002) by gradually adding normal saline to com-
pare their turbidity to McFarland standard of 0.5 which is
approximately 1.0 × 10
6
CFU/mL.
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