2.3 Antioxidant Assay
2.3.1 Preparation
The DPPH crystals were weighed 4 mg to be
dissolved in 100 mL of methanol in the measuring
flask for obtaining a DPPH solution with a
concentration of 0.004% or 40 ppm (part per
million) used in the test. The solution is stored in a
tightly sealed place and protected from light.
2.3.2 Concentration Series Determination
The concentration series used was 0.2,4,6,8,10 ppm..
These concentration variations were used in
antioxidant activity testing by DPPH method.
Preparation of stock solution with a concentration of
1000 ppm (10 mg dry extract diluted with methanol
to 10 mL). Furthermore, dilution to obtain the
concentration and Duplo repetition or two
repetitions and a negative control that DPPH
solution and methanol (without the addition of
extract).
2.3.3. Determination of the Maximum
Absorption Wavelength DPPH
2 mL of 0.004% DPPH solution was added with 2
mL of methanol. After being left for 30 minutes in
the dark, Absorption of the solution was measured
by UV-Vis spectrophotometer at 515 -520 nm
wavelength to obtain the maximum wavelength.
2.3.4 Antioxidant Assay
The antioxidant test was carried out through a series
of sample solutions from the methanol extract and
the three fractions with 2 repetitions using a
methanol solvent. Each solution plus 2 mL of DPPH
solution, in order to obtain a solution of a
predetermined concentration, was allowed to stand
for 30 minutes (calculated after addition of DPPH
solution), measured its absorbance at the maximum
wavelength. The absorbance data obtained is used to
determine % inhibition (damping). Through sample
concentration curve versus % inhibition, IC
50
extract
value can be obtained with statistical analysis using
linear regression. There is also a measure of
absorbance of blanks (methanol). The antioxidant
test indicator is the color change of DPPH. The data
in this study is the percentage of DPPH radical
reduction obtained using the formula:
% AA=
𝐴𝑏𝑠. 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠. 𝑆
𝐴𝑏𝑠. 𝐶𝑜𝑛𝑡𝑟𝑜𝑙
× 100%
Data analysis using linear regression equation using
the formula:
y = b (x) + a
Explanation:
y: % AA
x: log concentration
a: intercept
b: slope
3 RESULT AND DISCUSSION
In this study conducted testing of antioxidant
activity of aerial parts of L. microphyllum. Dry
powder aerial parts of L. microphyllum macerated
using methanol to obtain methanol extract. Methanol
extract in fractionation based on polarity level using
n-hexane, ethyl acetate, methanol and water. Each
extract obtained was tested for its antioxidant
activity using DPPH method. This method was
chosen because is simple, easy, fast and sensitive
and requires only a little extract. This method is
often used to detect the anti-radical ability of a
compound because the result proves to be accurate,
reliable, relatively fast and practical. The principle
of this method is the measurement of synthetic free
radical capture in polar organic solvents such as
ethanol or methanol at room temperature by a
compound having antioxidant activity. This process
of radical capture through the mechanism of taking
hydrogen atoms from antioxidant compounds by
free radicals so that free radicals capture an electron
from antioxidants. With the arrest of these radicals,
the diazo double bond in DPPH decreases, resulting
in decreased absorbance. The free radical used is
DPPH (2,2-diphenyl-1-picrylhydrazyl).
DPPH reacts with antioxidant compounds by
taking hydrogen atoms from antioxidant compounds
to obtain electron pairs. The compounds that can
potentially as antioxidants are from the class of
flavonoids, alkaloids, phenols, and tannins. Before
the testing of antioxidant activity, the first
determination of the maximum wavelength of DPPH
used with the wavelength range 515-520 nm. The
maximum wavelength is obtained from the
maximum absorbance of 515 nm. This wavelength
will be used in subsequent antioxidant testing. The
compound that reacts as a radical catcher will reduce
DPPH which can be observed by the color change of
DPPH to yellow from purple when the odd electron
from the DPPH radical has paired with hydrogen.
From the free radical captured compound which will
Antioxidant Activity from Lygodium microphyllum Aerial Parts
251