Advance Sustainable Science, Engineering and Technology (ASSET)
Vol. 2, No.2, October 2020, pp. 0200207-1 ~ 0200207-7
ISSN: 2715-4211 DOI: https://doi.org/10.26877/asset.v2i2.7129

Comparison of Antioxidant Activity of Ethanolic, Methanolic, nHexan, and Aqueous Extract of Parkia speciosa Peel based on
Half -Maximal Inhibitory Concentration Through Free Radical
Inhibition
Fafa Nurdyansyah1*, Dyah Ayu Widyastuti2
1

Food Technology, Universitas PGRI Semarang, Jl. Sidodadi Timur No 24 Semarang,
Central Java, Indonesia

2

Biology Education, Universitas PGRI Semarang, Jl. Sidodadi Timur No 24
Semarang, Central Java, Indonesia

*fafanudyansyah@upgris.ac.id
Abstract. The objectives of this study was to determine the half maximum inhibitory
concentration (IC50) from four types of Parkia speciosa peel extracts (ethanol, methanol, nhexane, and aqueous) through DPPH free radical inhibition. First Parkia’s peel extract made by
drying the Parkia’s peel that has been sorted, then crushed and mashed with a blender. Parkia’s
powder then macerated for 3 replication using each type of solvent and then solvent
evaporation was carried out using a rotary vacuum evaporator. The evaporated extract
produced then tested for antioxidant activity using the IC50 method and phytochemical
screening was performed to analyze the potential content of functional compounds. The results
showed that all types of solvents dissolve alkaloid compounds (except water extract),
flavonoids, saponins, tannins, and phenols. IC50 values produced from the four types of petai
bark extract using methanol, ethanol, water, and n-hexane solvents sequentially were 76.92;
111; 136; and 201 ppm. Methanol extract had the lowest IC50 value of 76.92 ppm which
resulted that the methanol extract of petai skin had a strong (active) antioxidant strength
compared to others.

Keywords: Antioxidant, Exctract, Parkia’s peel, IC50
(Received 15 August 2020, Accepted 10 September 2020, Available Online by 30 September 2020)

1. Introduction
The use of natural materials with bioactive compounds has recently expanded. This is relating to
the increase of various degenerative diseases caused by free radicals such as heart disease,
arteriosclerosis, cancer, and symptoms of aging. Problems related to the function and ability of
antioxidants in the body as inhibitor of cell oxidation chain due to highly reactive free radicals [1][2].
The use of traditional plants as plants that have functional values for health has been widely used and

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researched in Indonesia. The content of compounds such as phenolic compounds, flavonoids, and
terpenes in traditional plants functions as antioxidants, anti-inflammatory, anti-microbial and others.
Antioxidants based on the source are divided into 2 types of natural antioxidants and synthetic
antioxidants [3]. Synthetic antioxidants may have negative effects if consumed for a long time.
meanwhile natural antioxidant is widely used as an in inhibitor to free radicals in the body. The limited
production of endogenous antioxidants has an impact to the body to need additional exogenous
antioxidants to inhibit free radicals. Therefore, exploration of natural sources of antioxidant have been
continuously improved. One of the Indonesian plants source which has the potential to be explored is
petai.
Petai (Parkia speciosa) is one of the abundant plant in Indonesia because it is easy to grow
anywhere. Parkia speciosa peel (Parkia’s peel) is a part of the petai plant which is not used and is
usually thrown away as waste. Parkia’s peel have potential benefit such as antioxidants, antidiabetic,
and antiangiogenic. Potential benefit from Parkia’speel may become from its contain such as phenolic
compounds and flavonoids in large quantities. The antioxidants contained in the extract of petai seeds
and peels after being fractionated with several solvents showed that the ethyl acetate fraction of petai
peel had the greatest antioxidant potential, with an IC50 value of 85.92 ppm. While IC50 from petai
seeds had the greatest activity with IC50 value was 136.29 ppm [4].
The functional compounds found in Parkia’s seed and peel extract influenced by various factors,
one of them is the type of solvent used and the extraction temperature [5]. Types of solvents have
different levels of polarity and solubility for certain types of compounds, while the extraction
temperature determines the optimal temperature of the extraction process so that the maximum
extraction is obtained. Therefore, the use of Parkia’s peel as a source of natural antioxidants is very
promising and it is necessary for further investigation the factors affecting antioxidant extraction
process of Parkia’s peels. Based on the reasons, it is necessary to carry out research on the
comparative test of the antioxidant activity of the ethanol, methanol, aqueous, and n-hexane extracts of
Parkias peel using the inhibitory concentration 50 (IC50) test method.
2. Methods
2.1. Sample preparation
Parkia speciosa peel was separated from the seeds and then sliced to obtain uniform size. The
peel then dried in a cabinet dryer at 50oC, for 48 hours until completely dry. The dried peel was
smoothed with a grinder and sieved with a 70 mesh sieve. The resulting powder was ready for
extraction.
2.2. Extraction
The extraction process was carried out by the maceration method in an Erlenmeyer and wrapped in
aluminum foil. A total of 100 grams of parkiosa peel powder are macerated in Erlenmeyer for 24
hours. After 24 hours of maceration then filtered and obtained macerate, maceration is repeated up to 3
times. After the maceration process ends, the macerate or filtrate were evaporated until a concentrated
sample of each type of solvent is obtained using a rotary vacuum evaporator at 40oC. The concentrated
sample was obtained then analyzed for phytochemical screening and testing for antioxidant DPPH.
2.3. DPPH Antioxidant Assay
Quantitative measurements of radical scavenging assay were carried out according to the method
described by [5]. The quantitative measurement of radical scavenging properties was carried out in
universal bottle. The reaction mixture contained 50 µl of sample at concentration ranging from 0; 20;
40; 60; 80; 100 ppm and 5 ml of a 0.04% (w/v) solution of DPPH in 80% methanol. Gallic acid was
used for comparison or as a positive control. The DPPH solution in the absence of sample was used as
control and the 80% methanol was used as blank. Discolorations were measured at 517 nm by using
spectrophotometer (HITACHI U-1900 spectrophotometer 200V) after incubation for 30 min in the
darkroom. Measurement was performed at least in triplicate. The percentage of the DPPH free radical
was calculated using the following equation:

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DPPH scavenging effect (%) = ((A0- A1)/ A0) x 100
Where A0 was the absorbance of the control and A1 was the absorbance in the presence of the Parkia
speciosa peel extract [6].
2.4. Determination of Half Maximal Inhibitory concentration
The actual decrease in absorption induced by the test was compared with the positive controls. The
IC50 (concentration providing 50% inhibition) values were calculated use the dose inhibition curve in
linear range by plotting the extract concentration versus the corresponding scavenging effect.
2.5. Phytochemical Screening (Qualitative analyses)
- Alkaloid Identification
0.5 grams of sample extract was dissolved in 5 ml hydrochloric acid, then filtered. The filtrate
obtained was used as a sample solution. About 1 ml sample solution then added with 2 drops of
Mayer LP reagent, a positive result showed by the formation of a yellow precipitate.
- Flavonoid Identification
A total of 0.5 grams’ sample extract was added with 2 drops of NaOH solution. The formation
ofan intense yellow color by adding dilute acids indicates the presence of flavonoids.
- Saponin identification
A total of 0.5 grams of sample extract was added with 2 ml of distilled water, then shaked for 10
seconds. Positive results were showed by formation of a stable foam for not less than 10 minutes
- Tanin identification
A total of 0.5 grams of sample extract was added with 2 drops of 1 % gelatin solution in NaCl. the
formation of white pellet indicates the presence of tannin
- Fenolic identification
A total 0.5 grams of sample extract was added with 3 drops of FeCl3 solution. The presence of
Fenolic compound indicated by formation of greenish blue color of the sample solution.
2.6. Statistical Analysis
All the data analysis was replicated and showed as mean ± SD. Analysis of Variance were performed
using one-way analysis of Variance (ANOVA). Significant differences between means were
determined by Duncan’s Test and if P value less than 0.05 were considered statistically significant. All
data were analyzed using SPSS 17 version program.
3. Results and Discussion
The dried Parkiosa’s peel powder was subjected to normal temperature (37oC) condition of maceration
process. as a result, methanolic, ethanoic, n-hexane and aqueous extracts were obtained. The extract
was analyzed for phytochemical screening, antioxidant activity based on half maximum inhibitory
concentration (IC50).
3.1. Yield Extract
The different types of solvents would determine the yield extract. The results of the Parkia speciosa
peel yield extract’s using various types of solvent can be seen in Table 1.
Table 1. Effect of various types of solvents on the yield percentage of the extract
powder weight
final weigt
Yield (%)
Sample
(g)
ekstrak(g)
Methanolic extract
20
4,56
22,80
Ethanolic extract
20
3,27
16,35
Aqueous extract
20
4,99
24,95
n-hexane extract
20
3,05
15,25

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Parkia’s peel dry powder was extracted by maceration for 24 hours using various types of solvents.
The maserate obtained after the extraction process then concentrated with a rotary vaccum evaporator
to remove the solvent, so that a concentrated extract will be obtained. In general, the extract yield
ranged from 15.25 to 24.95%. The extract yield of the four types of extracts had a high enough value,
in which the yield of water extract was higher than methanolic extract. This may be caused by
compounds from Parkia’s peel that dissolve in water more than other solvents. Methanol and ethanol
are also universal solvents capable of binding or dissolving compounds derived from natural materials,
both non-polar, semi-polar and polar [7].
3.2. Phytocemical screening
Phytochemical screening was carried out to determine the potential content of compounds
qualitatively extracted from the material with various types of solvents, so that potential antioxidant
activity can be identified. The results of phytochemical’s screening are as shown in Table 2.
Tabel 2. Results of Phytochemical Screening Parkia’s peel Extract using various types of solvents
Sample extract
Compound group
Metanolic Ethanolic Aqueous n-Heksan
Alkaloid
+
+
+
Flavonoid
+
+
+
+
Saponin
+
+
+
+
Tanin
+
+
+
+
Fenol
+
+
+
+
The results of phytochemical screening of Parkia’s peel extract with various types of solvent showed
that there were compounds derived from the alkaloids (except aqueous extract), flavonoids, saponins,
tannins, and phenols. Parkia’s peel contains secondary metabolite compounds in the form of phenolic
compounds and their derivatives that have the potential as antioxidants [8][9]. Secondary metabolic
compounds that have the potential to act as antioxidants are phenolic compounds, such as phenyl
propanoids, flavonoids, anthocyanins, tannins, melanins, simple monocyclic phenols, and lignins [10].
3.3. Half Maximum inhibitory concentration (IC50)
Quantitative antioxidant activity testing was carried out by measuring DPPH radical activity by
spectrophotometry method at an absorption wavelength of 517 nm. The IC50 (inhibitory concentration
50) value indicates that the ability of the extract or compound to reduce or inhibit free radicals by
50%. Determination of the IC50 activity can be seen in Table 3.

Sample
Concentration
(ppm)
0
20
40
60
80
100

Table 3. Determination of half maximum inhibitory concentration of each sample
% inhibiton of
% inhibiton of
% inhibiton of
%Inhibiton of
IC50
IC50
IC50
methanolic
ethanolic extract
n-hexan extract
aqueous extract
extract
8,388
9,124
9,724
9,681
21,063
16,295
20,186
17,009
76,9
111
201
35,611
21,974
33,677
25,308
43,296
30,581
54,312
35,217
56,002
37,129
68,325
45,748

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IC50

136

50.000

y = 0.7566x - 8.1537
R² = 0.9878

60.000

% inhibition

% inhibition

80.000

40.000
20.000
0.000
0

50

100

y = 0.4517x - 0.5095
R² = 0.9943

40.000
30.000
20.000
10.000
0.000

150

0

Concentration (ppm)

100
50
Konsentrasi (ppm)

(a)
60.000

40.000
% Inhibition

% Inhibition

(b)

y = 0.5873x - 2.3662
R² = 0.9925

50.000

150

40.000
30.000
20.000
10.000

y = 0.3515x + 1.9313
R² = 0.997

30.000
20.000
10.000
0.000

0.000
0

50

100

150

0

50

100

Concentration (ppm)

Concentration (ppm)

(c)
(d)
Figure 1. Regretion curve of DPPH analysis based on Typical solvent (a) methanolic extract, (b)
ethanolic extract, (c) n-hexane extract, (d) aqueous extract
Antioxidant properties of Parkia’s peel extracts was evaluated to find a new natural source of
antioxidant. DPPH radical is a commonly used reagent for evaluation of antioxidant activity because
of its stability in the radical form and simplicity of the assay [11]. The principle of this assay in the
color change of DPPH solution from purple to yellow as the radical is quenched by the antioxidant
[12]. The colour changes can be measured quantitatively by spectrophotometer at 517 nm.
Based on Figure 1 (a), the linear equation value for methanolic extract y = 0.756x-8.153, so the
calculation of the IC50 value for methanoic extract obtained the following values: y = 0.756x-8.153
(for y = 50), then the x value is 76.92 ppm. The IC50 value of the methanol extract can be categories as
active antioxidant power (50-100 ppm) [13]. Furthermore, based on the calculation of the regression
curve In the ethanol extract sample (Figure 1.b), the regression equation y = 0.451x - 0.509 (assuming
the value of y = 50) is obtained, the x value is 111 ppm. The IC50 value of the Parkia’s peel ethanolic
extract showed that the extract had moderate IC50 antioxidant activity (101-250 ppm). The value
obtained is classified as having the ability to reduce moderate radicals, it happened because the
possibility of the extract obtained has low purity or is in the form of crude extract.
The correlation curve between the concentration of aqueous extracts used in reducing free
radical DPPH (Figure 1.d) was calculated as% inhibition against free radicals. So that the regression
equation y = 0.351x + 1.931 (y value = 50) is obtained, then the x value (IC50) is 136 (ppm). The IC50
value ranging from 101-250 ppm had sufficient or moderate antioxidant reducing power [13].
While, the calculation of IC50 from n-hexane extract of Parkia’s peel had a value about 201 ppm. The

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150

result Showed that n-hexane extract has the highest IC50 value compared to the other solvents. so that
in the determination of IC50 n-hexane extract has a low qualification of antioxidant strength (> 200
ppm).
The comparison value of IC50 among four typesof solvent usedin this research, the methanolic
extract exhibit a significant dose dependent inhibition of DPPH activity with 50% of inhibition (IC50)
at concentration of 76.9 ppm (Figure 1.a). Basically, a higher DPPH radical-scavenging activity is
associated with a lower IC50 value. There are studies have been carried out to evaluate the antioxidant
activity of Cassia species using DPPH assay [14] and reported that, particularly C. fistula exhibited
higher antioxidant activity compared to C. spectabilis [15]. Whereby the present study proof that, the
Parkia speciosa peel extract has the potential compound(s) react as antioxidant which is suitable to
develop a drugs for the prevention of human disease related to free radical mechanism.
4. Conclusion
The methanolic extract of Parkia speciosa peel had the highest IC50 activity value compared to the
three types of extracts with a value of 76.92 ppm, while the ethanol extract, aqueous extract, and nhexane extract were 111; 136; and 201 ppm, respectively. The IC50 value in the range between 50-100
has strong antioxidant activity, while the IC50 value in the range 101-250 includes moderate ability.
Acknowledgements
The authors are very grateful to LPPM UPGRIS for providing all facilities for this research.
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