The effect of solvent concentration

on antioxidant activity of akar kuning

(Fibraurea chloroleuca Miers) extract
Cite as: AIP Conference Proceedings 2049, 030003 (2018); https://doi.org/10.1063/1.5082504
Published Online: 14 December 2018

Mauritz Pandapotan Marpaung, and Desti Wulan Handayani

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© 2018 Author(s).
 The Effect of Solvent Concentration on Antioxidant
Activity of Akar Kuning (Fibraurea chloroleuca Miers)
Extract
Mauritz Pandapotan Marpaung a) and Desti Wulan Handayani

Department of Pharmacy, University of Kader Bangsa

Jl. Mayjen H.M. Ryacudu No. 88, 8 Ulu, Seberang Ulu I, Palembang, South Sumatra 30111, Indonesia

a)
Corresponding author: mauritzchem@gmail.com

Abstract

Akar kuning (Fibraurea chloroleuca Miers) is a traditional medicinal plant that can treat malaria, diabetes, and as an
antioxidant. The antioxidant activity of an extract was determined by many factors, including active compounds in
the extract, the extraction method, the kind and concentration of solvent. This study aimed to determine the effect of
solvent concentration of antioxidant activity akar kuning extract. The solvents used in this study were ethanol with
concentration of 50%, 75% and 96%. Measurement of antioxidant activity using DPPH method (2,2-diphenyl-1-
picrylhydrazyl) with quercetin as a positive control. The antioxidant activity of an extract was determined by IC 50
(Inhibitory Concentration), EC 50 (Efficiency Concentration) and ARP (AntiRadical Power) values. The results of this
study showed antioxidant activity IC 50 value of 50% ethanol extract 9.4078 ± 1.6036 mg/ml, 70% ethanol extract
6.7312 ± 2.911 mg/ml, and 96% ethanol extract 5.2004 ± 3.8368 ug/ml. EC 50 value of 50% ethanol extract 0.0941
ug/g DPPH, 70% ethanol extract 0.0673 ug/g DPPH, and 96% ethanol extract 0.052 ug/g DPPH. ARP value for 50%
ethanol extract 1062.69 g DPPH/g sample, 70% ethanol extract 1485.88 g DPPH/g sample and 96% ethanol extract
1923.08 g DPPH/g sample. Akar kuning extract with ethanol 96% showed higher antioxidant activity than other
extracts. Higher concentration of ethanol then the stronger antioxidant activity. Based on the results of this study
indicated the ethanol solvent concentration in the akar kuning extract had an effect on antioxidant activity.

Keywords: Akar kuning (Fibraurea chloroleuca Miers), Antioxidant, DPPH,

INTRODUCTION
Akar kuning (Fibraurea chloroleuca Miers) was a species of flowering plant family
(Menispermaceae). These herbs include plants lianas (vines) on trees or other plants in the vicinity of up to 40 m
[1]. Based on the distribution areas, akar kuning was spread in Malaysia, Brunei, the Philippines, Sumatra, Java,
North-East Kalimantan, and Sulawesi [2]. In Sulawesi, akar kuning has been used to treat malaria and diabetes
in the decoction [3]. The benefits contained in akar kuning were caused of secondary metabolites. Secondary
metabolites in akar kuning were alkaloids, tannins, saponins and flavonoids [4]. These compounds had many
benefitss in medicine, one of which was a antiradical free [5].
Free radicals are compounds, atoms or molecules are unstable with one or more unpaired electrons.
With the unpaired electrons, these compounds are very reactive in pairs with the surrounding molecular
electrons. If this process occurs in the body, there will be a degenerative disease such as cancer, heart disease,
cataracts, diabetes, liver and arthritis [6]. To prevent these diseases, antiradical compounds are needed. The
chemical compounds that had free antiradical activity were antioxidant compounds. Antioxidants were
compounds that gave one or more hydrogen atoms/electron to unstable free radical compounds. Antioxidants
can prevent oxidative damage and diseases caused by free radical reactions. The antioxidant activity was
determined by the ARP (AntiRadical Power) value. The greater of ARP value, the stronger antioxidant activity.
ARP value could be determined as IC50 (Inhibitory Concentration) and EC 50 (Efficiency Concentration) were
known. IC50 was the sample concentration (mg/mL) to reduce DPPH by 50%. EC50 was a parameter antioxidant
activity obtained after IC50 was known in mg per mg DPPH sample [7].
To maximize the antioxidant compounds dissolved in solvent, the selection of solvents with the right
concentration in extraction were important. The kind of solvent and solvent concentration were factors that
The 3rd International Seminar on Chemistry
AIP Conf. Proc. 2049, 030003-1–030003-5; https://doi.org/10.1063/1.5082504
Published by AIP Publishing. 978-0-7354-1775-5/$30.00

030003-1
influence the quality of an extract [8]. The solvents used in the extraction was ethanol with concentration of
50%, 70% and 96%. Ethanol solvents had properties similar to the properties of antioxidant compounds. Ethanol
that have polar and nonpolar groups were expected to dissolve the antioxidant compounds. With the ethanol
concentration variation was expected to be known optimum antioxidant activity. Therefore, the aim of this study
was to determine the effect of ethanol concentration as solvent on antioxidant activity in akar kuning extract.

MATERIAL AND METHODS

Materials
Materials used in this study were quercetin, DPPH (2,2-diphenyl-1-picrylhydrazyl) ((Sigma-Aldrich),
ethanol p.a (pro analysis) (Merck), distilled water, deionized water.

Instruments
The instruments used in this study were spectrophotometer UV-Vis (Shimadzu UVmini-1240),
analytical balance (Boeco Germany), glass wares (Iwaki), micropipette (Socorex), cup porcelain, pipette, and
rotary evaporator.

Determination of akar kuning

Akar kuning plants were collected from Sengir Village, South Bangka Regency, Bangka Belitung
Islands. Akar kuning was determined at the Herbarium Laboratory, University OF Andalas, Padang.

Extraction of akar kuning

To make simplicia, akar kuning were sorted wet and dry. Then smoothed with a mesh no. 60. 100 g of
simplicia were macerated with ethanol 50% and 96%. While making extracts with 70% ethanol, 300 g of
simplicia were macerated. Maceration performed twice repetition. Results maceration evaporated by using a
rotary evaporator at 40°C to obtain a thick extract. After that, the extracts were weighed and rendemen was
calculated.

Preparation of DPPH solution (100 mg/ml).

1 mg DPPH dissolved in ethanol pa to 10 ml.

Formulation of standard quercetin (100 mg/ml)

To make a standard solution concentration of quercetin 1000 mg/ml, 10 mg of quercetin dissolved in
ethanol p.a until 10 ml. Then diluted by taking 1 ml and added to 10 ml of ethanol p.a.

Formulation of quercetin concentration (1; 5; 10; and 15 mg/ml),

Pipette 0.1; 0.5; 1; and 1.5 ml of quercetin 100 mg/ml each into a 10 ml volumetric flask. Then add
ethanol p.a up to the mark.

Formulation akar kuning extract (100 µg/ml)

To make stock solution of ethanol extract 1000 mg/mL, 10 mg of extract were dissolved in ethanol to
10 ml. Then diluted by taking 1 mL of extract and adding ethanol p.a to 10 ml.

Qualitative test antioxidants

DPPH 100 mg/ml solution was put into test tubes. Then added aqua deionization as a negative control,
quercetin 1000 mg/ml as a positive control and extract respectively of 1 ml. After that, shaken until
homogeneous and incubated for 30 minutes. Ingredients that contain antioxidants will fade dark purple to
yellow.

Determination of the wavelength of maximum absorption of DPPH

2 mL DPPH 100 mg/ml was put into test tubes and added 2 ml of ethanol p.a. After that, shaken until
homogeneous and poured in a cuvette. Then, the maximum wavelength was determined using
spectrophotometer UV-Vis.

Measurement DPPH control solution

2 mL DPPH 100 mg/ml was put into test tubes added 2 ml of ethanol p.a. After that shaken until
homogeneous and incubated for 30 minutes then poured in a cuvette and measured at the maximum wavelength
(521.5 nm).

030003-2
 Measurement of the antioxidant activity of quercetin standard solution,
Quercetin 100 mg/ml was diluted WRFRQFHQWUDWLRQVRI
ȝJPO, 5 mg/ml, 10 mg/ml and 15 mg/ml. 2 ml
of various concentrations were added to 2 ml of DPPH and 2 ml of ethanol p.a. Then shaken until homogeneous
and incubated for 30 min at room temperature and a dark room. After that, the absorbances were measured at a
maximum wavelength (521.5 nm).

Measurement antioxidant activity on akar kuning extract.

Akar kuning extract 100 mg/ml were made in the concentration of 1 mg/ml, 5 mg/ml, 10 mg/ml and 15
mg/ml. 2 ml of various concentrations were added 2 ml of DPPH and 2 ml of ethanol p.a. Then shaken until
homogeneous and incubated for 30 min at room temperature and a dark room. After that, the absorbance of
extracts were measured at maximum wavelength (521.5 nm). Measurements were replicated 3 times. To
determine the IC50 value, the percentage of inhibitions were calculated first. From the percent inhibition
concentration can be determined by the method of linear regression. The formula of the inhibition percentage
was:
ࢇ࢈࢙Ǥࢉିࢇ࢈࢙ǡ࢙
࢖ࢋ࢙࢘ࢋ࢔࢚ࢇࢍࢋ࢕ࢌ࢏࢔ࢎ࢏࢈࢏࢚࢏࢕࢔ ൌ  ࢞૚૙૙Ψ (1)
ࢇ࢈࢙ǡࢉ

Notes:
Abs.c: absorbance of control (DPPH)
Abs.s: absorbance of the sample (extract)

For EC50 values were in g sample/g DPPH IC50 of data obtained from the formula:
ூ஼ହ଴
‫ܥܧ‬ͷͲ ൌ  (2)
௞௢௡௦௘௡௧௥௔௦௜஽௉௉ு

With EC50 value, then the value of ARP can be determined by the equation:
ଵ଴଴
‫ ܴܲܣ‬ൌ  (3)
ா஼ହ଴

RESULTS AND DISCUSSION

To determine the specific spesies of plants, determination must be made. The result of determination
showed the sample was akar kuning. The name species of akar kuning was Fibrauera chloroleuca Miers from
Menispermaceae family. Akar kuning were wet and dry sorted to produce simplicia with mesh no. 60. The
resulting simplicia was extracted using maceration method with ethanol solvent. Maceration was a cool method
by soaking simplicia in solvent without heating. This extraction method had advantages such as the equipment
needed was simple, easy, cheap, and can be used to extract compounds that were not heat resistant [9]. By
maceration method, antioxidant compounds that were not resistant to heat could be maximally absorbed in
ethanol solvent. After maceration, the extracts were evaporated by rotary evaporator at 40°C. Evaporation of the
extracts at 40oC was to prevent the degradation of the active compounds in extracts.
Table 1 showed rendemen of extract with ethanol 96% was higher than extracts with ethanol 50% and
70%. Futhermore, extract with the ethanol 96% had antioxidant activity determined from IC 50, EC50, and ARP
values higher than extracts of ethanol 50% and 70%.

Table 1. Values of Rendemen, IC50, EC50, ARP

ARP (DPPH ug/g

No. Samples Rendemen IC50 (ug/mL) EC50 (g/g DPPH)
sample)
1. Quercetin - 3.7847 .0378 2645.5
2. Extract with ethanol 31.48% 9.4078 ± 1.6036 .0941 1062.69
50%
3. Extract with ethanol 29.26% 6.7312 ± 2.911 .0673 1485.88
70%
4. Extract with ethanol 10.294% 5.2004 ± 3.8368 0.052 1923.08
96%

To determine the specific spesies of plants, determination must be made. The result of determination
showed the sample was akar kuning. The name species of akar kuning was Fibrauera chloroleuca Miers from

030003-3
Menispermaceae family. Akar kuning were wet and dry sorted to produce simplicia with mesh no. 60. The
resulting simplicia was extracted using maceration method with ethanol solvent. Maceration was a cool method
by soaking simplicia in solvent without heating. This extraction method had advantages such as the equipment
needed was simple, easy, cheap, and can be used to extract compounds that were not heat resistant [9]. By
maceration method, antioxidant compounds that were not resistant to heat could be maximally absorbed in
ethanol solvent. After maceration, the extracts were evaporated by rotary evaporator at 40°C. Evaporation of the
extracts at 40oC was to prevent the degradation of the active compounds in extracts.
From the evaporation results obtained thick extracts and calculated the rendemen of extracts. Table 1
showed the extract with ethanol 96% gave the highest rendemen of 10.294%. This showed the ethanol 96% can
extract the active compounds better than ethanol 70% and 50%. Besides that, ethanol 96% had similar properties
with active compounds in the extract. A substance can be dissolved in other substance if it has the same
properties of the substance. The polar substance will dissolve in polar substances and the nonpolar substances
will dissolve in nonpolar substance [10]. The ethanol 96% was less polar than ethanol 70% and 50%. With this
property, ethanol 96% could dissolved the active compounds in polar and nonpolar. From the results of
rendemen showed that the greater solvent concentration, the more extracts obtained. This showed the solvent
concentration is directly proportional to the amount of extract produced [8].
To find out the presence of antioxidant compounds in extract, qualitative test were carried out by
adding DPPH solution to extract. The results of this test gave a yellow color, which means positive akar kuning
extracts contained antioxidant compounds. The color changes to yellow because antioxidant provide hydrogen
atom when reacting to DPPH with unpaired electrons to form DPPH-H pairs [11]. DPPH solution was a
compounds which were free radicals because it had unpaired electrons [12].
Quantitative Test of antioxidant activity was carried out by DPPH method. DPPH method was based
on the purple color changes to yellow. With this color change will gave maximum DPPH wavelength at 521.5
nm when measured by spectrophotometer UV-Vis. This method was often used to determine antioxidant activity
because this method were very easy, cheap, simple and require little material in the testing antioxidant activity
[13].
From absorbance measurement, quercetin standard curve was obtained by linear regression equation y
= 3.1358x + 38.132 with a correlation coefficient (R²) = 0.9351. This showed that the concentration of the
extract affects the inhibtion percentage of akar kuning extract by 93.51% with 6.49% is the external factor of the
extract concentration. Quercetin as a standard solution beccause of quersetin was flavonoid group that had
strong antioxidant activity. Besides that, quercetin had OH (hydroxyl) group at the 3', 4', 3, 5, and 7 positions
which are able to capture free radicals [14].
Indicators to determine the antioxidant activity of the extracts were IC 50 (Inhibitory Concentration),
EC50 (Efficiency Concentration) and ARP (AntiRadical Power). IC50 was an indicator of the antioxidant power
in inhibiting free radicals of DPPH by 50%. If IC 50 < 50 mg/ml classified as very strong antioxidant, IC50 50-
100 mg/mL classified as strong antioxidant, IC50 101-250 pg/ml classified as moderate antioxidant, IC50 251-
500 mg/mL classified as weak antioxidant, and IC50 > 500 mg/ml classified as very weak antioxidant [15].
Based on the classification, akar kuning extract with ethanol 50%, 70% and 96% classified as very strong
antioxidants. In Table 1 showed the highest antioxidant activity which is characterized by the lowest IC 50 value
found in extract with ethanol 96%. This showed that the secondary metabolites in extract that act as antioxidant
activity were more soluble in ethanol 96%. This is evidenced by the highest rendemen value in the extract with
ethanol 96%. Secondary metabolite compounds were acted as antioxidants such as flavonoids, alkaloids and
saponins [16]. EC50 indicator is the quotient of the IC50 value on DPPH concentration in mg/mg DPPH. ARP
indicator showed the antiradical strenght obtained when the value of EC 50 known [7]. IC50 value is directly
proportional to the EC50 value. The smaller of IC50 and EC50 value, the stronger antioxidant activity. Meanwhile,
the greater the value of ARP, the stronger antioxidant activity.
By the provisions of IC50, EC50 and ARP values showed that akar kuning extract with ethanol 96% had
the most stongest antioxidant activity. Table 1 also showed that the greater concentration of ethanol solvent, the
stronger antioxidant activity. From the results of this study showed the effect of ethanol solvent concentration on
antioxidant activity in akar kuning extract.

CONCLUSION
The results showed that the akar kuning extract with ethanol solvent 96% had the highest antioxidant
activity with IC50 
“ ȝJml, EC50 of 0.052 ug/g DPPH and ARP 1923.08 ug/g sample. The higher
concentration of ethanol solvent, the stronger the antioxidant activity in akar kuning extract. This showed the
differences of ethanol solvent concentration influenced on antioxidant activity of akar kuning extract.

030003-4
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