JPPIPA 6(2) (2020)

Jurnal Penelitian Pendidikan IPA

Journal of Research in Science Education

http://jppipa.unram.ac.id/index.php/jppipa/index

Antioxidant Activity of Fruit Extract Powder Beans (Phaseolus

vulgaris L.) Using DPPH Method
Rizki Nugrahani1*, Yayuk Andayani2, Aliefman Hakim2

1 Pharmacy Study Program, Faculty of Health Sciences, Nahdlatul Wathan University, Mataram, Indonesia.
2 Sciences Education Study Program, Postgraduate, Mataram University, Mataram, Indonesia

DOI: 10.29303/jppipa.v6i2.409

Article Info Abstract: Free radical effect is one of many factors that can cause coronic desease, heart
Received : March 12th, 2020 coronary desease, diabetes militus. Beans plant (Phaseolus vulgaris L.) countant
Revised : June 25th, 2020 antioxidant compound. Antioxidants could inhibit oxidation reaction, that is cousing
Accepted: July 1th, 2020 free radical compounds. The objective of this research was to determine the potential of
bean extracts as an antioxidant. DPPH method was used to measure antioxidant
activity, absorbances was measured using UV-Vis spectroscopy. Scrining
phytochemical used to identified secondary metabolites in samples. The result showed
IC50 1268.18; 2512; 1698.18 and 4442.75 μg/mL for all variety of stroge (less 1, 1, 2, 3
months) and IC50 control BHT was 1.744 μg/mL. Extract of bean powder sample
classified as very weak antioxidant activity, variety of storage could effect to
antioxidant activity. Phytochemical screening results obtained flavonoids, phenols,
alkaloids, saponins, steroids and terpenoids present in the sample, while tannin are not
included in the sample.

Keywords: antioxidant activity; Phaseolus vulgaris L.; DPPH; IC50

Citation: Nugrahani, R., Andayani, Y., & Hakim, A. (2020). Antioxidant Activity of Fruit Extract Powder Beans (Phaseolus
vulgaris L.) Using DPPH Method. Jurnal Penelitian Pendidikan IPA (JPPIPA), 6(2), 194-198. doi:
https://doi.org/10.29303/jppipa.v6i2.409

Introduction Green beans (Phaseolus vulgaris L) is one type of

plant that can be a source of natural antioxidants
One of the causes of some coronary diseases such (Kurnia, 2013). The parts of the beans are known to
as cancer is due to the effects caused by free radical have great benefits because of their abundant
compounds. Free radicals are molecules with free nutritional content. Phaseolus vulgaris L contains
unpaired electrons and are very reactive (Lobo, et al., variations in anthocyanin and antioxidant activity
2010). One method of preventing free radical formation (Dzomba, 2013).
is by using substances that can act as free radicals According to Lima et al (2014) Phaseolus vulgaris
known as antioxidants. L contains large amounts of bioactive phenolic
Antioxidant compounds have the ability to compounds. Pure flavonoid compounds such as
inhibit oxidation reactions from normal metabolic anthocyanin, quercetin glycosides and condensed
processes in living things that cause free radical tannins found in the seeds show better and significant
compounds. Antioxidants are able to give one electron antioxidant activity when compared with existing
to free radicals so they will produce a stable new radial synthetic antioxidants (Sihombing et al., 2010).
(Sánchez, et al., 2016) (Hamid, et al., 2010)..
___________
Email: [email protected]

Copyright © 2020, Nugrahani et al.

This open access article is distributed under a (CC-BY License)
Jurnal Penelitian Pendidikan IPA (JPPIPA) July 2020, Volume 6, Issue 2, 194-198

Research on antioxidant activity in bean extract produces a white precipitate while the addition of
has been done before (Kurnia, 2013). The research used dragendorff reagents which will cause an orange-red
fresh extracts that were soaking using water solvents, precipitate.
but studies of the antioxidant activity of bean extract in A number of sample powders were dissolved
powder form have never been reported. For more with water then heated to boiling. After that, it is
extensive information about the benefits of green beans filtered and the filtrate is used as a test solution. The
as a medicinal ingredient, it is necessary to study and filtrate is put into a closed test tube then shaken for 10
investigate how the antioxidant activity of bean extract seconds. The presence of saponins is indicated by the
in powder preparations is obtained through a heating formation of stable froth.
process at high temperatures and has been stored for a A number of sample powders were dissolved
certain period of time. with methanol. The solution is filtered, the filter results
are then evaporated on the Waterbath. The evaporated
Method extract was dissolved with chloroform, then added
with anhydrous acetate, then the mixture was dropped
The materials needed in this study were P with concentrated H2SO4 through the test tube wall.
Vulgaris L extract powder obtained from the traditional The presence of triterpenes is indicated by brownish or
medicine industry in the town of Mataram, distilled violet rings at the boundaries of two solvents, while the
water, DPPH merck, BHT, 96% v/v methanol, 50% v/v appearance of green indicates steroids.
methanol, magnesium powder, concentrated A number of samples (0.1 gr) were extracted
hydrochloric acid 37 % v/v, concentrated H2SO4 97% with 20 ml of 70% methanol. The resulting solution was
v/v, FeCl3 1% w/v, FeCl3 5% w/v, ammonia, taken as much as 1 ml and then added 5% FeCl3. A
chloroform, dragendorff reagent, meyer reagent, positive reaction is shown by the formation of green or
acetate anhydride, amyl-alcohol. bluish green.
The tools that will be needed in this research are A number of extract powder dissolved with
analytical balance, volumetric pipette, porcelain cup, water then boiled for 5 minutes and filtered. Part of the
water bath, water bath, incubator, desiccator, Shimadzu filtrate obtained was added with a 1% FeCl3 solution.
UV-1800 spectrophotometer, glassware, GF silica gel Positive results are shown by the formation of a
palate, fortex, TLC chamber, sprayer, filter paper cotton blackish green color.
and aluminum foil. Analysis of Antioxidant compounds Using Thin
The material used in this study is the extract of Layer Chromatography Techniques (TLC). As much as
beans obtained from the immersion process of fresh 0.5 grams of bean extract powder that has been
fruit using water which is then processed to form a dissolved with 25ml methanol solvent, TLC was carried
powder with a heating method. The process of making out using a mixture of methanol and chloroform with a
extract powder is carried out by a traditional drug ratio of 1:39. After elution is complete, the plates are
production house in the city of Mataram. There are four dried and sprayed with a 0.5 mM DPPH solution in
types of samples based on sample age namely 0 methanol. Positive anti-free radical tests produce
months, 1 month 2 months and 3 months calculated yellow spots on a purple background in about 30
from the extract made. Each sample used has a minutes. A qualitative antioxidant activity test was also
different production date, not one batch. carried out on BHT used as a standard antioxidant.
The material used in this study is the extract of Antioxidant Activity Test with DPPH Color
beans obtained from the immersion process of fresh Immersion by the process of making a 0.5 Mm DPPH
fruit using water which is then processed to form a solution, weigh rapidly ± 19.7 grams DPPH, then
powder with a heating method. The process of making dissolve it with methanol p.a until the volume is
extract powder is carried out by a traditional drug exactly 100 mL in a volumetric flask and beaten. The
production house in the city of Mataram. There are four solution is stored in a dark colored bottle.
types of samples based on sample age namely 0 Determination of the maximum wavelength
months, 1 month 2 months and 3 months calculated DPPH solution is by dissolving 0.5 mM DPPH in
from the extract made. Each sample used has a methanol measured wavelengths ranging from
different production date, not one batch. wavelengths of 450 nm - 600 nm. Determination of
A number of samples were dissolved with CHCl3 Antioxidant Activity through the following process: P
(chloroform) and NH4OH then filtered and the filtrate Vulgaris L. extract powder was dissolved in methanol
was put into a closed test tube. CHCl3 extract was then and made in various concentrations of 200, 400, 800,
added 2 M H2SO4, until 2 layers were formed. The acid 1600, 3200 and 6400 ppm respectively 10 ml. Each
layer above the meyer reaction is added which solution is added with 1 ml of 0.5 mM DPPH solution
and soaked for 30 minutes in a dark room so that the
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reaction is more perfect (Wardatun, 2011), then Table 1. Phytochemical Test Results
measured at the maximum wavelength predetermined. Chemical Storage time (month)
As a blank solution methanol was added with a 0.5 mM Compounds <1 1 2 3
DPPH solution. As for positive control BHT used with Flavonoid + + + +
concentrations of 1, 2, 3, 4, 5 and 6 ppm added 1 ml of Alkaloid + + + +
Saponin + + + +
0.5 mM DPPH solution.
Tanin - - - -
Absorption measurement process by means of a
Fenol + + + +
test solution with a concentration variation incubated Steroid/triterpenoid +/+ +/+ +/+ +/+
in a dark room for ± 30 minutes then the absorption
was measured at a maximum wavelength of 517 nm The results of the analysis of antioxidant
using a UV-Vis spectrophotemeter. compounds by TLC technique can be seen that after
The method of calculation is the percentage of spraying with a 0.5 mM DPPH solution. The eluen used
inhibition calculated using the formula: resistance = as the mobile phase is methanol and chloroform with a
{(blank absorption/ sample absorption) x100%}/blank ratio of 1:39, the results obtained are yellow spots with
absorption. IC50 values are calculated by linear a purple background patches have Rf values of 0.36,
regression equations obtained from the inhibitory 0.48 and 0.61 (Figure 1).
percentages and concentration relationship curves

Result and Discussion

The results of phytochemical screening of

samples of powdered bean extract (Phaseolus vulgaris
L) which varied their storage time were based on the
color changes caused when the samples were reacted
with certain reagents. The results obtained are
flavonoids, phenols, alkaloids, saponins, steroids and (a) (b)
triterpenoids (Table 1). Tannin compounds were not Figure 1. Test results for Activity Content with TLC (a) Under
UV Lamp 366 nm (b) After DPPH Spraying.
found in all sample variations. These results are
consistent with the results of previous studies that the
Testing antioxidant activity with DPPH is used to see
extract of beans contains flavonoids and several types
the IC50 value, which is the concentration needed to
of polyphenol compounds (Dzomba, et al., (2013),
inhibit 50% of free radicals. The percentage inhibition
Sihombing, et al., (2010) and Kurnia (2013)). The
and IC50 values for each sample are presented in Table
presence of secondary metabolite content is expected to
2
be an indication of what compounds will act as
antioxidants in bean extract powder.

Table 2. Antioxidant Activity Test Results

% inhibition and concentration (ppm) IC50
Storage time
200 400 800 1600 3200 6400 (μg/mL)
< 1 month 17,97 21,09 52,54 81,45 92,58 90,63 1268,18
1 month -5,27 2,34 28,52 59,38 91,80 91,60 2512
2 months -2,93 26,37 49,41 85,16 91,80 88,67 1698,18
3 months -3,52 0,59 6,45 18,55 39,26 72,66 4442,75
BHT 37,70 51,56 68,95 74,80 83,01 84 18 1,744

From the data in Table 2 below it is known that inhibition seen that fresh samples stored for less than 1
the greater the concentration of the sample used the month when compared to samples stored for 3 months,
greater the percentage of inhibition increases. The the percentage of inhibition has decreased very
percentage of maximum inhibition for samples with a significantly for each variation of concentration, this is
storage life of less than 1 month - 2 months is at a relevant to the results obtained by Eveline, et al., (2014)
concentration of 3200 ppm and has decreased and research Febrianti, et al., (2014) on purple sweet
thereafter. For samples with 3 months storage requires potatoes. The effect of storage on antioxidant activity in
greater concentration to achieve the maximum this study was not shown by data on a significant
percentage of inhibition. The storage period of the decrease in antioxidant activity for each storage time
sample has an influence on the value of percent
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given to this sample because the sample used had a Table 2 shows that all samples have an IC50
different date or production date. value> 500 μg/mL, this value is much greater than the
The antioxidant activity test of bean extract IC50 BHT value used as a positive control of 1,744
powder was carried out by immersion reaction using μg/mL. From these data it can be categorized that the
the DPPH radical (2,2-diphenyl-1-picrylhydrazyl). The antioxidant compounds contained in the bean powder
reaction between DPPH and antioxidants is illustrated samples are classified as very weak antioxidants
in Figure 2. (Ciptaningsih, 2012). This is probably caused by
heating at high temperatures given during the process
of making ketal extract into powder preparations.
Besides the extract used is not derived from extracts
that have been purified so that there are many types of
compounds that are still contained therein. The
classification of antioxidant power is based on IC 50
values, the smaller the IC50 value the stronger
antioxidant activity (Molyneux, 2004).

Conclusion
Figure 2. DPPH and Antioxidant Reactions
Beans extract powder samples showed
The working principle of free radical inhibition antioxidant activity with IC50 values for samples with
by antioxidants is to convert DPPH radicals which are storage less than 1, 1, 2, 3 months respectively 1268.18,
reactive and unstable compounds into stable 2512, 1698.18 μg/ml and 4442.75 μg/ml. Phytochemical
compounds by means of antioxidant compounds that screening shows samples containing chemical
will donate one H atom or one electron to an unpaired compounds flavonoids, alkaloids, saponins, phenols,
DPPH compound. Reduction reaction occurs between steroids and triterpenoids. The data obtained showed
DPPH and antioxidants are marked by a color that the strength of the antioxidant activity of bean
change that occurs, the color of the purple DPPH extract powder samples was classified as very weak
solution will turn yellow after the addition of compared to BHT which had a smaller IC50 value.
antioxidant compounds. The antioxidant activity test
using the DPPH method is very easy to do because the Acknowledgements
reaction can be directly observed both using the TLC
method and with simple spectrophotometry (Kedara Thank you to Dr. Yayuk Andayani, M.Si for the
and Singh, 2011). direction and financial assistance given as well as Dr.
The amount of immersion activity is expressed as Aliefman Hakim for guidance during the course of the
IC50 value, which is the concentration of the substrate study.
(in this case the concentration of antioxidant
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