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Research Article

ANTIOXIDANT ACTIVITY OF TAUCO ETHANOL EXTRACT AND ITS FRACTIONS

RESMI MUSTARICHIE1*, SANDRA MEGANTARA1, WIWIEK INDRIYATI1, ADE ZUCHROTUN2

1
Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty of Pharmacy, Universitas Padjadjaran, Indonesia. 2Department
of Biological, Faculty of Pharmacy, Universitas Padjadjaran, Indonesia. Email: [email protected]
Received: 21 October 2016, Revised and Accepted: 30 January 2017

ABSTRACT

Objective: The objective of this study is to investigate antioxidant activity and phytochemical screening of ethanol extract, fractions of water, ethyl
acetate, and n-hexane from tauco.

Methods: Two types of tauco were extracted using soxhletation methods, followed by fractionation using liquid-liquid extraction methods, and
phytochemical screening. Antioxidant activity test was carried out using 1,1-diphenyl-2-pikrilhiradzil with ascorbic acid (vitamin C) as a reference.
Modified method of Farnsworth was applied for phytochemical screening.

Results: It was found that extracts of ethanol and ethyl acetate fraction containing flavonoids, monoterpenoid, and sesquiterpenoids whereas the
water fraction and a fraction of n-hexane only contain monoterpenes and sesquiterpenoids. The inhibition concentration 50 (IC50) value for the
ethanol extract, water, ethyl acetate, and n-hexane fractions of two taucos in a row were 1192.71 ppm, 1746.01 ppm, 722.38 ppm, 1845.45 ppm and
1190.15 ppm, 1740.30, 710.46, for tauco A and B, respectively.

Conclusion: It was unexpected that tauco ethanol extract and fractions showed much weaker antioxidant activity than vitamin C, which had the IC50
value of 4.41 ppm.

Keywords: Antioxidant, Flavonoid, Free radicals, 1,1-diphenyl-2-pikrilhiradzil, Tauco, Ascorbic acid.

© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.
org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2017.v10s2.19488

INTRODUCTION METHODS

Tauco is one of the traditional food items that come from Indonesia and Collecting materials and pre-treatment sample
are found especially in the area of West Java. Tauco paste form, can be Two well-known brands tauco sample used in this study were obtained
found, in the form tauco dry and wet. Tauco is processed soy products from a supermarket in the area of Bandung, West Java. Tauco dried
obtained from fermentation techniques. This fermentation tauco aims to using an oven at 60-70°C until constant weight.
improve taste, but it also can improve the nutritional content contained
in soy. Tauco society generally used as food flavoring agent [1]. Tauco Extraction and fractionation tauco
generally made of yellow soybeans. Soybean crops including a group The extraction method used in this research was the method
of natural flavonoid that isoflavone group. Soybeans are known to have soxhletation using ethanol 96%. The extraction followed standard
biological activity as an antioxidant, estrogenic effects, antiosteoporosis, soxhletation method [12-14]. It was done approximately 4 hrs or until
and anticancer [2]. Isoflavones are antioxidants that are needed by almost colorless solvent droplets. Viscous liquid extract obtained was
the body to stop the reaction of free radical formation [3]. Isoflavone then evaporated to obtain a thick extract with constant.
glycosides are a major component in soybean and non-fermented soy
products, while the soybean fermented product is found free isoflavones Extract weight
(aglycone) in large quantities [4]. Based on research that has been % Yield = ×100%
Sample weight
done Hutriadi [5], tauco compounds containing daidzein and genistein.
Genistein and daidzein are included in the free isoflavone compounds
formed from the fermentation process soybeans [6]. Genistein is the Water content of the obtained extract was determined to make sure
most powerful antioxidant in soy, followed by daidzein [7]. fulfill Farmakope Herbal requirement, in which the water content in the
extract should not be more than 10% to avoid the rapid growth of fungi
Antioxidants are compounds that have the ability to protect cells and and microorganisms in the extract [15].
tissues from damage caused by the threat of the presence of free radicals
that are reactive [8]. Antioxidants react with free radicals to form non- Hydrolysis of isoflavones
radical compounds are relatively stable [9]. Whether or not strong Hydrolysis aims to separate the intermediate compounds with glycosides
antioxidant activity can be seen from the inhibition concentration 50 aglycone. 10 g tauco extract dissolved in a mixture of methanol and 2N
(IC50) value. IC50 is a number that indicates the concentration of extract hydrochloric acid in the ratio 1:1, refluxed in water bath for 2 hrs at a
that can inhibit the oxidation of 50%. The smaller the IC50 value, the temperature of 40°C. After refluxing, the mixture was evaporated with a
higher the antioxidant activity [10]. Research into the antioxidant rotary evaporator at a temperature of 40°C to remove methanol.
activity in soybean has been conducted, but research on the antioxidant
activity in soybean fermented product has not been done, particularly Fractionation
in tauco. Therefore, it, in this study, will be conducted research on Fractionation performed using liquid-liquid extraction. Extracts tauco
the antioxidant activity assay method tauco with 1,1-diphenyl-2- hydrolyzed then fractionated using a solvent of water, ethyl acetate, and
pikrilhiradzil (DPPH) with ascorbic acid as a reference. Part of this n-hexane. The results of the water fraction, fraction of ethyl acetate and
study has been published in Indonesia [11]. n-hexane fraction were then evaporated with a rotary evaporator.

II-Indonesian Conference on Clinical Pharmacy

 Mustarichie et al.
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Phytochemical screening dried to remove the water content contained therein. Two taucos as
Phytochemical screening was conducted on a sample tauco to much as 2.25 kg sample dried using an oven at a temperature of 70°C
determine the content of secondary metabolites contained therein until constant weight was obtained, which was 832.55 and 830.00 g.
based on Farnsworth method [16] which includes: The water content contained in tauco 37%.
a. Identification of alkaloids
b. Identification of compounds polifenolat Extraction and fractionation
c. Identification tanin Tauco dried samples were extracted using methods soxhletation.
d. Identification flavonoids The soxhletation method will occur repeatedly so that the extraction
e. Identification monoterpenoid and sesquiterpenoids process will be more perfect. Soxhletation method was done because
f. Identification of Steroids and triterpenoid it was known that the active compounds contained in tauco were
g. Identification quinone thermostable. In addition, the soxhletation method’s number of
h. Identification of saponin. samples and solvents needed just a little so that the extraction process
will be more efficient.
Thin layer chromatography (TLC)
TLC plates (stationary phase) used were silica gel GF254. TLC plates A total of 300 g of dried tauco obtained viscous extract of 88.45 and 90 g
prepared with a size of 10×4 cm. Based on research conducted by for Tauco A and B, respectively. We found that the water content in both
Irianti et al. [17], the developer used is toluene:ethyl acetate:acetic acid tauco samples fulfilled Farmakope Herbal Indonesia requirement that
at a ratio of 5:4:1. Subsequently observed in ultraviolet (UV) 254 nm was below 10% [15].
and 366 nm. Spotting observed marked for the calculated value of Rf. To
test qualitatively antioxidants which had been spotted TLC plate tauco The viscous extract obtained was then hydrolyzed to break down
into its component isoflavone glycosides and isoflavone glycosides
extracts and fractions sprayed with DPPH. DPPH solution was prepared
free (aglycone). A total of 10 g of viscous extract hydrolyzed using
by dissolving reagent DPPH as much as 4 mg in 50 ml of 96% ethanol.
HCl and methanol at a ratio of 1:1. Hydrolysis process is carried out
Furthermore, the observed color staining formed.
for 2 hrs at a temperature of 40°C with the aim of accelerating the
Antioxidant activity test termination reaction force of the component isoflavone glycosides.
Hydrolysis was then evaporated using a rotary evaporator to remove
Test of antioxidant activity with DPPH based on Molyneux
the methanol solvent. The residue was then fractionated to attract the
method [18,19] with ascorbic acid as a reference. The test includes:
active compound contained in the extract based on the level of polarity.
1. Preparation of the test solution.
Fractionation was conducted using liquid-liquid extraction using a
2. Determination of the wavelength (λ) maximum DPPH
solvent of water, ethyl acetate, and n-hexane.
DPPH solution of 3 ml of ethanol was added 2 ml, homogenized, and
observed absorbance at a wavelength of 450-650 nm. The maximum
Phytochemicals screening results
wavelength of absorption characterized by the greatest. Blank of
Phytochemical screening was conducted to determine the content of
ethanol was used.
secondary metabolites contained in the extract and the fractions tauco.
3. Determination of operating time DPPH solution in ethanol.
The phytochemical screening results are shown in Table 1.
4. Determination of the sample incubation time.
5. The determination of IC50 values of samples. Table 1 showed that the tauco samples, extracts of ethanol and
ethyl acetate fraction containing flavonoids, monoterpenoid and
A total of 2 ml of test solution in various concentrations added about sesquiterpenoids, and saponin. While the water fraction and a fraction
3 ml of DPPH solution, homogenized, and then allowed to stand. of n-hexane only contain monoterpenes and sesquiterpenoids. This
Absorbance was read at maximum wavelength was 517 nm. Used as result comparable to photo screening result of soybean (Glycine soja
a blank test solution. For testing the reference solution, as many as Sieb. and Zucc.) [20], except for tannin. In this result, we did not find
2 ml of vitamin C in various concentrations added about 3 ml of DPPH tannin content in our samples. It might be due to soybean used and or
solution, homogenized, and then allowed to stand. Absorbance is read mixed soybean and other beans were used in making the tauco.
at maximum wavelength is 517 nm. As a form of ascorbic acid solution
was used in various concentrations as much as 2 ml and 3 ml of ethanol Results of TLC
as much. IC50 values were calculated from a linear regression curve TLC was carried out using the developer toluene:ethyl acetate:acetic
between the % inhibition of uptake with various concentrations of acid (5:4:1). Extracts and fractions tauco observed in visible light, UV
extracts of the test solution with the following formula: 254 nm, 366 nm UV, and spotting DPPH solution. Some TLC results are
shown in Fig. 1 and Table 2.
  A 
% Inhibition = 1 −  test
  × 100%
  Acontrol  

Where
Atest = absorbance of DPPH solution in the sample
Acontrol = absorbance of DPPH solution in ethanol (3 ml DPPH and 2 ml
of ethanol).
Data were obtained as percent inhibition values that were plotted
against the concentration of test solution. IC50 describe extract
concentration required to inhibit 50% of DPPH radical activity. IC50
value obtained from the graph the concentration of the extract of the
percent inhibition of DPPH.
a b c d
RESULTS AND DISCUSSION
Fig. 1: Thin layer chromatography by the developer toluene:ethyl
Collection results materials and pre-treatment samples acetate:acetic acid (5:4:1) of Tauco A. 1 - Ethanol extract, 2 - Water
Two samples tauco soybeans, namely, Tauco A and B, from two well- fraction, 3 - Ethyl acetate fraction, 4 - N-hexane fraction. a - visible
known commercially brands obtained from Bandung, West Java. The light, b - ultraviolet 254 nm, c - ultraviolet 366 nm, d - DPP
water content in tauco was high enough so that samples need to be solution

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Table 1: Phytochemical screening results

Secondary metabolites Tauco samples Ethanol extract Water fraction Ethyl acetate n‑heksana fraction
fraction
Tauco A Tauco B Tauco A Tauco B Tauco A Tauco B Tauco A Tauco B Tauco A Tauco B
Alkaloids ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑
Polyphenols ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑
Tannin ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑
Flavonoids + + + + ‑ ‑ + + ‑ ‑
Monoterpenoid and + + + + + + + + + +
sesquiterpenoid
Steroids ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑
Triterpenoid ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑
Quinone ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑
Saponin + + + + + + + + ‑ ‑
+: Detected, ‑: Not detected

Table 2: TLC of ethanol extract and its fractions of Tauco A and B

Sample Rf Visible light UV 254 nm UV 366 nm Spotting DPPH

Ethanol extract 0.57 ‑ Blue Green Yellow
0.91 ‑ ‑ Green Yellow
Water fraction 0.06 ‑ Blue Yellow Yellow
Ethyl acetate fraction 0.57 ‑ Blue Green Yellow
0.85 ‑ ‑ Green ‑
0.95 ‑ Blue Yellow Yellow
N‑hexane fraction 0.58 ‑ Blue Green Yellow
0.94 ‑ Blue Yellow Yellow
DPPH: 1,1‑diphenyl‑2‑pikrilhiradzil, UV: Ultraviolet, TLC: Thin layer chromatography, ‑: Not detected

TLC carried out as an early detection of antioxidant activity in the

sample. DPPH solution was used as spotting patches to determine
whether or not the compounds that had antioxidant activity in extracts
and fractions of tauco. Their antioxidant activity characterized by the
formation of patches of yellow with a purple background. Both Tauco A
and B found to have this color.

Determination of wavelength maximum DPPH

DPPH solution color change time is reduced by antioxidants can be
measured by using UV-visible spectrophotometry at a wavelength of
515-520 nm (Molyneux, 2004). For this work, it was found the DPPH
solution maximum absorption at a wavelength of 517 nm.
Fig. 2: 1,1-diphenyl-2-pikrilhiradzil operating time
Determination of time operating results DPPH solution in ethanol
DPPH solution is unstable, so it is necessary to determine the operating Determination of IC50 sample
time DPPH. Determining operating time was done to determine the best In this study, each sample weighed 50 mg and dissolved in 50 ml
working time of DPPH solution. Graph DPPH operating time can be seen volumetric flask, so we get the mother liquor with a concentration of
in Fig. 2. 1000 ppm. The mother liquor was then diluted to 100, 200, 300, 400,
and 500 ppm. DPPH solution was prepared by weighing 2 mg DPPH
Fig. 2 showed that the optimum time of DPPH work is in the 35th to dissolved in 50 ml volumetric flask, so we get a DPPH solution with a
50th minutes where at this time there was no significant increase or concentration of 40 ppm. As a reference used ascorbic acid as vitamin C
decrease in absorbance. is known to have very powerful antioxidant activity. Vitamin C can
donate a hydrogen atom to stabilize free radicals and can prevent a
Determination of sample incubation time chain reaction.
Determination of the sample incubation time was needed to see the
time it took the test sample, the ethanol extract, the water fraction, The parameters used in the test the antioxidant activity by DPPH
fraction of ethyl acetate and n-hexane fraction of tauco be reacted method were IC50, i.e., the concentration where the sample was able to
with DPPH, characterized by a steady uptake. The timing of incubation reduce 50% of the initial concentration of DPPH activity. IC50 value of
was also done on samples of the comparison, namely, vitamin C. The the samples obtained using the linear regression equation that states
timing of incubation samples was done in 5 minutes intervals for 2 the relationship between the concentrations on the X axis the percent
hrs at a wavelength of 517 nm. It found that the incubation time the inhibition on the Y axis. Testing the absorbance of each sample at various
ethanol extract tauco, namely, in the 70th minutes to the 80th minutes, concentrations carried out at a wavelength of 517 nm is then calculated
the incubation time the water fraction of tauco, namely, in the percent inhibition values of each sample. The greater the concentration
100th minutes until the 110th minutes, the incubation time ethyl acetate used, the greater the reduction of free radical activity (% inhibition).
fraction of tauco, namely, in the 85th minutes to the 95th minutes, and the Samples which act as antioxidants will donate hydrogen atoms to the
incubation time n-hexane fraction of tauco, namely, in the 75th minutes radical DPPH. DPPH radical reduction will experience characterized
to the 85th minutes. As for ascorbic acid C incubation period was from by a color change from purple to yellow. When the color changes, then
the 30th minutes until the 40th minutes. there will be a decrease in the absorbance of DPPH. The greater the

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concentration of the sample used, the greater the ability of antioxidants bean, Bogor peanut, peanut, (except n-hexane shells extract of soybean)
to reduce DPPH. Measured absorbance decreases with an increase in had IC50 of DPPH scavenging capacities <50 ppm. Most likely, the low
concentration of the sample. Hence, the greater the concentration of the antioxidant activities of this report due to tauco process and that
sample is used, then the greater the percent inhibition of the sample. soybeans used in these two tauco were mixed beans, not soybeans only.

Value percent inhibition of each test solution and reference solution at According to the Jun et al. [21], a compound considered to have a very
various concentrations is used to create the linear regression equation. strong antioxidant activity when have IC50 values of <50 ppm, strong
IC50 value of ethanol extract, the water fraction, fraction of ethyl category if you have IC50 values of 50-100 ppm, when the medium
acetate, and n-hexane fraction of each tauco in a row was 1192.71 ppm, category have 101-250 ppm IC50 value, a category feeble have IC50
1746.01 ppm, 722.38 ppm, 1845.45 ppm and 1190.15 ppm, 1740.30, values 251-500 ppm, and was listed inactive when the IC50 values
710.46, for Taucho A and B, respectively. IC50 value tauco sample is above 500 ppm. Based on test results, extracts and fractions of tauco
much larger than its peers, namely, vitamin C. Comparison of IC50 values have the ability to neutralize DPPH radical, but the antioxidant activity
ethanol extract tauco A and B, tauco water fraction, the fraction tauco of extracts and fractions tauco included into the category of inactive
ethyl acetate, n-hexane fraction tauco, and vitamin C can be seen in because it had IC50 values above 500 ppm.
Fig. 3.
CONCLUSION
Ethyl acetate fraction had the smallest IC50 values between ethanol
extract, the water fraction, and the fraction of n-hexane of tauco so that Based on this research, it is known that extracts and fractions of tauco
it could be said ethyl acetate fraction had good antioxidant activity than have antioxidant activity with the ability to neutralize free radicals
the ethanol extract, the water fraction, and the fraction of n-hexane DPPH. Best antioxidant activity of extracts and fractions tauco is shown
from tauco. Tauco was fermented from soybeans, where soy was known by ethyl acetate fraction with IC50 value of 722.38 ppm (inactive),
to contain secondary metabolites that isoflavone class of flavonoids. followed by ethanol extract with IC50 value of 1192.71 ppm (inactive),
Isoflavones in soy can be found in bound form with sugar (isoflavone the fraction of water with IC50 value of 1746.01 ppm (inactive), and the
glycosides) or in free form (aglycone isoflavones). In the process of fraction of n-hexane with IC50 value of 1845.45 ppm (inactive), while
fermentation of soy, isoflavone aglycone compound to be transformed vitamin C have IC50 values are 4.41 ppm (very strong). These results,
into compounds isoflavone aglycone [6]. Therefore, the product of however, was unexpected that tauco ethanol extract and fractions
fermentation of soy, isoflavone aglycone is found in large quantities [4]. showed much weaker antioxidant activity than vitamin C. Further
The antioxidant activity of ethanol extracts tauco not as good as the research is needed to test the antioxidant activity of tauco using other
antioxidant activity of ethyl acetate fraction tauco. When the ethanol methods. This will strengthen the scientific data about the antioxidant
extract of hydrolyzed tauco, there will be a breakdown of compounds activity of tauco.
isoflavone glycosides into isoflavone aglycone and glikosidanya.
Compounds isoflavone aglycone this hydrolysis results are less polar ACKNOWLEDGMENT
solvent that will dissolve in ethyl acetate which is semi-polar. Based We thank Arina Syifa Hidayati for her technical support.
on research conducted by Jun et al. [21], the aglycone isoflavones
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