Materials and methods
Material
Red onion (Allium cepa L.) was purchased from a local market
in Mazandaran Iran. Onion bulbs had a diameter of 70–85 mm.
The outer layers of onions were separated and washed by cold
water. Then, these layers were dried in the shade and open
air. The dried materials were ground to a fine powder and
used as feedstock for the extraction of phenolic and flavonoid
compounds.
Folin Ciocalteu’s reagent, Griess reagent, Gallic acid,
quercetin, sodium carbonate, and ascorbic acid were procured
from Sigma-Aldrich (Buchs, Switzerland). Aluminum chlo-
ride, phosphate buffer, potassium ferrocyanide and trichlo-
roacetic acid were purchased from Fluka (Buchs, Switzer-
land). Other chemicals, including potassium acetate, DPPH
(2, 2-diphenyl-1-picrylhydrazyl), ferrous chloride, sodium
nitroprusside, sodium chloride, ethanol, methanol, n-butanol,
ethyl acetate and BHA were provided from Merck (Darmstadt,
Germany).
The extracts were prepared by the maceration method using
water, methanol, ethanol, n-butanol and ethyl acetate as extrac-
tion solvents. For extraction, 500 mL of solvent was added to
100 g of onion skin powder in an Erlenmeyer flask, and stirred
for 72 h. The obtained extracts were filtered using filter paper
125 mm, No. 3 (Whatman) and concentrated under reduced
pressure in a rotary evaporator (Stuart Co., UK) by removing
the solvent. The extracts were resuspended in distilled water
and sterilized by membrane filtration (45 µm, Whatman) and
stored at 4
°
C until use. The extraction yield was calculated
based on the following formula:
Determination of total phenolic content
Total phenolic content (TPC) of all extracts was determined by
the Folin-Ciocalteu colorimetric method [
17
]. In brief, 0.5 mL
of all dissolved extracts was mixed separately, with 10 mL of
deionized water and 2.5 mL of undiluted Folin Ciocalteu’s
reagent. After 5 min, 2 mL of Na
2
CO
3
(20% w/v) was added
and the volume was adjusted to 25 mL with deionized water.
The mixture was incubated for 60 min and the absorbance was
measured by UV–vis double-beam spectrophotometer (Ana-
lytik Jena, Jena, Germany) at 760 nm against a blank sample
without reactants. Gallic acid was used as a reference stand-
ard and the results were expressed as milligram Gallic acid
equivalents (mg GAE)/g of extract. The assay was performed
in triplicate.
(1)
Yield =
weight of extract
total weight of onion skin powder
× 100
3580
S. Khalili et al.
1 3
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