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Characterization, Phytochemical Screenings and Antioxidant Activity

Test of Kratom Leaf Ethanol Extract (Mitragyna speciosa Korth) Using
DPPH Method
To cite this article: R Yuniarti et al 2020 J. Phys.: Conf. Ser. 1462 012026

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The 6th Annual International Seminar on Trends in Science and Science Education IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 1462 (2020) 012026 doi:10.1088/1742-6596/1462/1/012026

Characterization, Phytochemical Screenings and Antioxidant

Activity Test of Kratom Leaf Ethanol Extract (Mitragyna
speciosa Korth) Using DPPH Method

R Yuniarti1*, S Nadia1, A Alamanda1 , M Zubir2, R A Syahputra2, M Nizam2

* [email protected]
1
Study program of Pharmacy, Faculty of Pharmacy, Universitas Muslim Nusantara Al
Washliyah, Medan, North Sumatera, Indonesia
2
Study program of Chemistry, Faculty of Mathematics and Natural Science,
Universitas Negeri Medan, Jl. Willem Iskandar, Pasar V, Medan, 20221, North
Sumatera, Indonesia

Abstract. Antioxidants are compounds that have an important role in health because they can
be used as anti-toxic molecules in the body which are the cause of various diseases. One of the
plants that have antioxidant content is kratom (MitragynaspeciosaKorth). The purpose of this
study was to determine the antioxidant activity of kratom leaf ethanol extract by using the
DPPH trapping method. Exploration of kratom leaf samples was carried out by maceration
using ethanol 96%, macerate was evaporated with a rotary evaporator, phytochemical
screening of kratom leaf ethanol extract and antioxidant testing of DPPH as Free radical.
Result of Simplisia Characterization of kratom leaves containing water, air soluble extract
contents, ethanol-soluble extract levels, total ash content, and acid insoluble ash content
sequentially as follows: 6.65; 18.01; 9.45; 7.14; and 1.06%. Phytochemical Screening results
containing kratom leaf ethanol extract containing chemical composition: alkaloids, flavonoids,
triterpenoids/steroids, saponins, and tannins. The results of antioxidant activity testing showed
that ethanol extract had an IC50 value of 38.56 μg / ml. The results showed that the ethanol
extract of kratom leaves had antioxidant activity in a very strong category.

1. Introduction
Antioxidants are defined as compounds that inhibit oxidation by way of reacting with reactive free
radicals that form reactive free radicals are unstable. In the chemical sense antioxidants are electron-
giving compounds, but in a broader biological sense, that is, all compounds that can reduce the
negative effects of oxidants, including enzymes and protein-binding proteins. Antioxidants are
compounds that can inhibit reactive oxygen species and also free radicals so that antioxidants can
prevent diseases that are associated with free radicals such as carcinogenesis, cardiovascular, and
aging [1].
Antioxidants can be obtained in synthetic and natural forms. Natural antioxidants come from the
extraction of natural ingredients that have the potential to capture free radicals, while synthetic
antioxidants are obtained from synthetic chemicals. However, concerns about the side effects of using
synthetic antioxidants have led to many studies on the potential of natural antioxidants derived from

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The 6th Annual International Seminar on Trends in Science and Science Education IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 1462 (2020) 012026 doi:10.1088/1742-6596/1462/1/012026

plants [2] Some plants are known to have high antioxidant content such as kratom, carrots, cabbage,
tomato, rosella, basil, mangosteen, and others.
Kratom ( Mitragynaspeciosa Korth.) can be used to treat various diseases. Kratom leaves contain
alkaloids, glycosides, terpenoids, flavonoids, and saponins. Alkaloids are the main group of
compounds found in kratom leaves [3]. Several studies on the pharmacological effects of kratom leaf
have also been investigated such as analgesic, stimulant, antidepressant, anti-inflammatory,
antinociceptive, antioxidant, and antibacterial activities [4].
Antioxidant activity can be tested by several methods such as the method of capturing radical
attenuation 2, 2 -diphenyl-1-picrylhydrazyl (DPPH), reducing power method, oxygen radical
absorption capacity test (ORAC) method, FRAP method and CUPRAC method [5]. Of all the methods
available DPPH method is the method most often used for testing antioxidant activity because the
DPPH method is a method that is simple, fast, easy, requires little sample and is sensitive to evaluate
the antioxidant activity of natural compounds [6].
Based on the background described above, the authors are interested in examining the antioxidant
activity of Kratom leaves. The kratom leaf antioxidant activity test was carried out using the DPPH
method.
2. Methodology
2.1 Material
Plant material used is kratom leaf (Mitragyna speciosa Korth) chemicals used, namely : ethanol 96%,
toluene, 2 N hydrochloric acid, iron (III) chloride, chloroform, aquadest, Dragendroff reagent,
Lieberman-Burchard reagent, magnesium powder, 1% ammonia solution, 1% gelatin solution, distilled
water, 2,2- diphenyl-1- picrylhydrazyl (DPPH), vitamin C, methanol and ash-free filter paper.
2.2 Tools
The equipment used consists of glassware (beaker cups, funnels, measuring cups, water flasks, stirring
rods, test tubes). Suction pipettes, vaporizer cups, water baths (water bath), rough balance, electric
balance, a set of the rotary evaporator (Stuart) and UV- Visible Spectrophotometer (Shimadzu UV-
1700).

2.3 Research procedure

2.3.1 Making Simplisia
The kratom leaves that have been collected are washed under running water until they are
clean, drained, then weighed as wet weight, then dried in a drying cupboard to dry. Kratom leaves are
said to be dry if easily destroyed, then weighed as dry weight. Dry simplisia is then made into powder
using a blender, extracted by maceration method.

2.3.2 Simplisia Characterization Examination

Simplisia characterization examination conducted by determination of water content, levels of water
soluble extract, content of ethanol soluble extract, total ash, and acid insoluble ash content .
2.3.3 Ethanol Extraction of Kratom Leaves
Samples of kratom leaves that have been pollinated weighed as much as 500 g put into maceration
container, then added with 75 parts of ethanol solvent (3750 ml). The maceration container is closed
and kept for 5 days in a place protected from direct sunlight while stirring occasionally. Then filtered,
separated between pulp and filtrate 1. The pulp is extracted again with the rest of ethanol (1250 ml)
and allowed to stand for 2 days (filtrate 2). Filtrate 1 and filtrate 2 were mixed and evaporated using a
rotary evaporator with a temperature of 50 ºC until a thick ethanol extract of kratom leaves was
obtained.
2.3.4 Phytochemical Screening of Kratom Leaf Ethanol Extract
Phytochemical screening of kratom leaf ethanol extract was carried out by examination of alkaloids,
examination of flavonoids, examination of triterpenoids and steroids, examination of saponin, and
examination of tannins.

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The 6th Annual International Seminar on Trends in Science and Science Education IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 1462 (2020) 012026 doi:10.1088/1742-6596/1462/1/012026

2.4 Antioxidant Activity Testing

2.4.1DPPH free radical trapping principle
The ability of the test sample to dampen the DPPH oxidation process (2, 2 -diphenyl-1-picrylhydrazyl
) as a free radical in a methanol solution (resulting in a reduction in DPPH purple colour) with IC50
(the concentration of the test sample capable of reducing free radicals by 50%) was used as a
parameter determining the activity of the test sample [7] .
2.4.2 Determination of Operating Time
The standard mother kratom leaf solution is pipetted as much as 5 ml, put in a 25 ml flask and then the
volume is sufficient with methanol up to the mark line (concentration of 200 g/ ml). Then pipette as
much as 2 ml, put into a 10 ml flask, added 1 ml DPPH solution (concentration of 200 g/ml) and
sufficient volume with methanol to the mark line (concentration of 40 g / ml), measured its absorption
to determine the operating time of the test solution until the 60th minute at the maximum absorption
wavelength that has been obtained. Then determine the operating time with vitamin C samples
(comparison).
2.4.3 Kratom Leaf and Vitamin C Antioxidant Activity Test Procedure
The standard kratom leaf solution is pipetted as much as 5 ml, put in a 25 ml flask and then the volume
is sufficient with methanol up to the mark line (concentration of 200 g / ml). Then pipette as much
as 1, 2, 3, 4, and 5 ml into a 10 ml flask to get the concentration of the test solution 20, 40, 60, 80, and
100 μg / ml. Each test solution was added 1 ml of DPPH solution (concentration of 200μg/ml) and
then the volume was sufficient with methanol to mark the line, and then allowed to stand for 10
minutes, then the absorption was measured using a UV-Visible spectrophotometer. Furthermore, the
same way is done for vitamin C.
2.4.4 IC50 Determination
The calculation used in determining radical capture activity is IC 50 (Inhibitory Concentration 50%).
This value indicates the concentration of the test compound that can reduce the DPPH oxidation
process by 50%. A value of 0% means it does not have antioxidant activity, while a value of 100%
means total reduction and testing needs to be continued with dilution of the test solution to see the
concentration limit of its activity. The calculation results are entered into the regression equation with
the extract concentration ( g / ml) as abscissa (x axis) and the value of% damping (antioxidants) as the
ordinate (y axis). The formula determines the IC50 value :
Y = ax + b
Note: y = dependent variable (IC 50)
x = free variable (kratom leaf)
a = slope
b = intersept
Specifically, a compound is said to be a very strong antioxidant if the IC 50 value is less than 50 μg/ml,
strong for IC 50 isworth 50-100 μg / ml, while if IC50 isworth 100-150 μg/ ml and weak if IC 50
isworth 151-200 μg / ml [ 8 ] .
3. Result And Discussion
3.1 Simplisia Characterization Results
The results of the simplisia characterization of kr atomic leaves can be seen in Table 1.
Table 1 .The results of the simisia characterization of kratom leaf powder
No. Examination Earnings Rate (%)
1 Water content 6.65
2 The essence of water soluble essence 18.01
3 Soluble essence in ethanol 9.45
4 Total ash content 7.14
5 Acid insoluble ash content 1.06

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The 6th Annual International Seminar on Trends in Science and Science Education IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 1462 (2020) 012026 doi:10.1088/1742-6596/1462/1/012026

Based on data from Table 1 shows that the water content obtained 6.65% has met the general
requirements, which is no more than 10%. Water content checks are carried out to provide maximum
limits or ranges of the amount of water content in simplisia. The maximum allowable value is related
to purity and contamination. If the water content contained in simplisia is more than 10%, the
simplisia will be easily overgrown with fungi at the time of storage so that the quality of the simplisia
will decrease. Water soluble extract concentrations obtained by 18 , 01 % and soluble extract
concentrations in ethanol obtained by 9.45%. Water soluble extract and ethanol soluble extract are
indicators of the amount of efficacy substances that can be found by both water and ethanol solvents.
Total ash content was 7, 14 % and acid insoluble ash was 1.06%. Determination of total ash content
and acid insoluble ash content was carried out aimed at providing an overview of internal and external
mineral content from the initial process until the formation of the extract and to evaluate the extract
against contamination of silica-containing materials, such as soil and sand.
3.2 Phytochemical Screening Results
Phytochemical screening tests were carried out to determine the class of chemical compounds
contained in kratom leaf extract. The results of screening of Simplisia extract of kr atomic leaves can
be seen in Table 2 .
Table 2 .Phytochemical screening results of kratom leaf extract
No. Examination Results
1 Alkaloids +
Dragendroff reagents +
Wagner reagent +
2 Flavonoids +
3 Triterpenoids / Steroids +
4 Saponin +
5 Tannin +

Description: (+) positive: Contains the compound being examined

(-) negative: Does not contain the compound being inspected
Based on the results of phytochemical screening tests of ethanol extract of kratom leaves containing
Alkaloids, Flavonoids, Triterpenoids / Steroids, S. aponin and Ta ninextracts . The screening results in
Table 2 show that the kratom leaf simplisia extract contains a class of chemical compounds that have
potential as antioxidants, namely flavonoid compounds.
Alkaloid testing with Dragendorff and Wagner reagents obtained positive results with the formation of
orange deposits and brown deposits. In reactions using Dragendorff and Wagner reagents, the K +
metal ion forms a covalent bond in coordination with the alkaloid nitrogen atom to form a
precipitating potassium-alkaloid complex.
3.3 Results antioxidant activity
The results of antioxidant activity tests of ethanol extract of kratom leaves and Vitamin C with
concentrations of 20, 40, 60, 80 and 100μg / ml compared with DPPH solution can be seen in Table 3
and Table 4.

Table 3 .The results of the antioxidant activity test of kratom leaf ethanol extract
Sample Absorbance measurement to
I II III
DPPH (blank) 0.979 0.979 0.979
20 ppm 0.549 0.547 0.549
40 ppm 0.310 0.309 0.313
60 ppm 0.228 0.234 0.240
80 ppm 0.207 0.201 0.203
100 ppm 0.142 0.143 0.142

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The 6th Annual International Seminar on Trends in Science and Science Education IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 1462 (2020) 012026 doi:10.1088/1742-6596/1462/1/012026

Table 4 .Vitamin C antioxidant activity test results

Sample Absorbance measurement to
I II III
DPPH (blank) 0.925 0.925 0.925
20 ppm 0.178 0.178 0.177
40 ppm 0.167 0.167 0.166
60 ppm 0.127 0.127 0.127
80 ppm 0.093 0.093 0.093
100 ppm 0.085 0.084 0.084

From Table 3 and Table 4, it can be seen that the absorbance value of the test sample decreases with
increasing concentration. This can occur because of the reduction of DPPH radicals by antioxidants,
where the higher the concentration of the test sample, the more antioxidant compounds contained will
increase the greater antioxidant activity and cause its absorbance to decrease. In principle, the
absorbance measured is the absorbance of DPPH solution that does not react with the test material or
DPPH that is still left in the solution, so that with increasing concentration of free radicals in the
solution, the absorbance of the solution will decrease. This means that the activity of capturing free
radicals is increasing. So from these results it can be said that the test sample has antioxidant activity.
In this test, a comparison solution is used namely Vitamin C (ascorbic acid) because Vitamin C is a
very powerful antioxidant and is most often used as a comparison.
3.4 Results of Free Radical Damping Percentage (DPPH) by Samples and Vitamin C
The relationship between concentration and percent reduction of DPPH free radicals by kratom leaf
ethanol extract and Vitamin C can be seen in Figure 1 and Figure 2.

120
y = 0,7725x + 20,21
100
% Damping

80
60 Daun Kratom

40
Linear (Daun
20 Kratom)
0
0 50 100 150

( g/ml)

Figure 1.Free Radical Damping Percentage (DPPH) by sample

120 y = 0,6945x + 36,919
100
% Damping

80
60
40 Vitamin C
20 Linear (Vitamin C)
0
0 50 100 150

( g/ml)
Figure 2 .Free Radical Damping Percentage (DPPH) by Vitamin C

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The 6th Annual International Seminar on Trends in Science and Science Education IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 1462 (2020) 012026 doi:10.1088/1742-6596/1462/1/012026

Both Figure 1 and Figure 2 show that the increase in concentration is directly proportional to the
increase in the reduction of DPPH free radical scavenging. This shows the antioxidant activity of
kratom leaf ethanol extracts and vitamin C (as a comparison). The interaction of antioxidants with
DPPH either electron transfer or hydrogen radicals to DPPH, will neutralize DPPH free radicals. Free
radical scavenging causes electrons to be paired which then causes a colour loss that is proportional to
the number of electrons taken. The reduction in colour intensity of the DPPH solution is produced by
the reaction of the DPPH radical molecule with one hydrogen atom released by the components of the
test sample to form a yellow compound of 2,2 diphenyl-1-picrylhydrazine.
3.5 Results of Inhibitory Concentration Value 50% (IC50 )
The antioxidant activity of the DPPH method is expressed by IC 50 (Inhibitory Concentration) where
IC 50 is a number that shows a sample concentration (ppm) that can reduce free radicals by 50%. The
smaller the IC 50 value indicates the higher the free radical reduction activity. Conversely, if the IC50
value is greater the lower the free radical mitigation activity. Antioxidant activity can be divided into
very strong, strong, medium, weak and very weak categories. Antioxidants are said to be very strong if
they have an IC 50 value of less than 50 μg / ml, antioxidants are categorized as strong if they have an
IC50 value of 50-100 μg / ml, antioxidants are categorized as moderate if they have an IC 50 value of
100-150 μg / ml, antioxidants are categorized as weak if has an IC 50 value of 150-200 μg / ml and an
IC50 value of more than 200 μg / ml is an extremely weak category of antioxidant.
IC50 values were obtained from the linear regression equation which states the relationship between the
concentration of the test sample with DPPH reduction as a parameter of antioxidant activity, wherein
the concentration of the test solution (ppm) as abscissa (x axis) and% damping value as ordinate (y
axis). The results of the analysis of the IC 50 value of the antioxidant activity test of ethanol extract of
kratom leaves and vitamin C can be seen in Table 5.
Table 5. Results of linear regression equations, IC50 values of kraom leaf extract and vitamin C

No Test Solution Regression equation IC 50(μg / ml ) Category

1 Kratom leaf extract Y = 0.7725x + 20.210 38.56 Very strong
2 Vitamin C Y = 0.6945x + 36.919 18.84 Very strong

Based on Table 5 . showed that the antioxidant activity of kratom leaf extract had a very strong
category with IC 50values obtained at 38.56 μg / ml . The measurement of vitamin C as a comparison
has a very strong category of antioxidant activity with IC 50 values obtained at 18.84 μg / ml . From
these results it can be concluded that vitamin C has a stronger antioxidant activity than kratom leaf
extract.

4. Conclusion
Based on the data obtained, it can be concluded as follows:
1. Phytochemical screening results of kratom leaf (MitragynaspeciosaKorth) ethanol extract
showed that kratom leaves contained Alkaloids, Flavonoids, Polyphenols, Triterpenoids /
Steroids, Saponins and Tannins.
2. The results of antioxidant activity testing using UV-Visible spectrophotmeter at a wavelength of
515.5 nm with DPPH method obtained IC50 values of kratom leaf ethanol extract at 38.56 g / ml
and IC50 values of vitamin C as a comparison of 18.84 g / ml. From these results it was
concluded that the test sample and vitamin C as a comparison have very strong antioxidant
activity.

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The 6th Annual International Seminar on Trends in Science and Science Education IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 1462 (2020) 012026 doi:10.1088/1742-6596/1462/1/012026

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