Indonesian Journal of Chemical Research

http://ojs3.unpatti.ac.id/index.php/ijcr Indo. J. Chem. Res., 10(3), 143-148, 2023

Antioxidant Capacity Fraction of the Pelawan Stems (Tristaniopsis merguensis Griff)

Robby Gus Mahardika 1*, Khairul Fajri1, Henri2

1
Departement of Chemistry, Faculty of Engineering, Universitas Bangka Belitung Jl. Kampus Peradaban,
Merawang, Bangka, 33172
2
Departement of Biology, Faculty of Engineering, Universitas Bangka Belitung Jl. Kampus Peradaban,
Merawang, Bangka, 33172
*
Corresponding Author: [email protected]

Received: July 2022 Abstract

Received in revised: August 2022
Accepted: August 2022 Free radicals destroy other molecules such as proteins, fats, carbohydrates, or DNA.
Available online: January 2023 The impact of reactions from free radical compounds can cause various health problems
such as inflammation, aging, and cancer. Therefore, to avoid damage caused by free
radicals, the body needs an important substance, antioxidants. Natural antioxidants are
more recommended than synthetic antioxidants because synthetic antioxidants must be
in accordance with the prescribed dose because they can cause side effects that are
harmful to the body. This study aimed to determine the antioxidant activity of the
extract and fraction of the Pelawan (Tristaniopsis merguensis Grifft.) stem extract.
Antioxidant testing was carried out using the DPPH method on extracts and fractions of
Pelawan stem extract (T. merguensis Grifft.). In addition, qualitative phytochemical
testing was also carried out on extracts of the stems of Pelawan (T. merguensis). The
results of the three solvent fractions, n-hexane, ethyl acetate, and methanol, were
positive for flavonoid and phenolic secondary metabolites. Meanwhile, the antioxidant
activity in reducing DPPH radicals in the methanol extract fraction of pelawan stems
had a very strong activity with an IC50 value of 36.33 g/mL, while the ethyl acetate and
n-hexane extract fractions had a very weak antioxidant activity with an IC50 value of
387.43 μg/mL dan 759.88 μg/mL.
Keywords: Pelawan Stems, Antioxidants, Phytochemicals, DPPH, IC50

INTRODUCTION Antioxidants act as inhibitors that inhibit the

oxidation process by reacting with reactive free
Currently, many people use private vehicles to
radicals to form non-reactive free radicals, which are
make their activities easier. Increased use of vehicles
relatively stable so that they can protect cells from the
can result in much pollution generated by vehicle
harmful effects of reactive oxygen free radicals
exhaust. Free radicals are one of the things caused by
(Sofia, Prabowo, & Rijai, 2016). Natural antioxidants
pollution (Septiana, Muchtadi, & Zakaria, 2002). The
are more recommended than synthetic antioxidants
pollution from vehicle fumes can increase free
because synthetic antioxidants must be in accordance
radicals, where excess free radicals can cause various
with the prescribed dose because they can cause side
diseases.
effects that are harmful to the body. Sources of
Free radicals are unstable compounds in which
natural antioxidants come from plants which
the outer orbit of the atom has one or more unpaired
generally contain phenolic secondary metabolites
electrons (Khaira, 2016). Free radicals damage cell-
scattered in plant parts such as fruit, seeds, flowers,
forming macromolecules such as proteins, fats,
leaves, pollen, wood, and roots (Sarastani, Soekarto,
carbohydrates, and DNA. The behavior of these free
Muchtadi, & Fardiaz, 2002). Phenolic compounds and
radicals is destructive to other molecules whose
polyphenols can change or reduce free radicals and
electrons are taken. Taking electrons by free radicals
act as anti-free radicals (Pietta, 2000; Agatonovic-
causes a reaction, forming more free radicals. The
Kustrin, W. Morton, & Ristivojevic, 2017).
effects of reactions from free radical compounds can
Based on previous studies, the major content of
cause various health problems such as inflammation,
secondary metabolites was found in
aging, and cancer. Therefore, to avoid damage caused
the Tristaniopsis genus is tannins and phenolics. The
by free radicals, the body needs important substances,
content of phenolic compounds in the
namely antioxidants, which can protect the body from
genus Tristaniopsis has the uniqueness of
free radical attacks (Khaira, 2016).

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glycosylated phenolics (Verotta, 2001). Pelawan obtain a thick extract. The extract and the three
leaves contain secondary metabolites such as fractions were subjected to FT-IR analysis to
alkaloids, phenol, hydroquinones/tannins, and determine the functional groups in the extract.
flavonoids. Another study found that the total
phenolic content in pelawan leaves was 215.22 mg Antioxidant Testing
GAE/g dry extract and had a strong antioxidant This antioxidant test was carried out based on the
activity with an IC50 value of 22.1454 μg/mL DPPH (1,1-diphenyl-2-picrylhydrazyl) method. First,
(Roanisca, Mahardika, & Sari, 2019; Souhoka, Hattu, a test solution was prepared by weighing each
& Huliselan, 2019)). The research above shows that pelawan stem extract from each solvent as much as
pelawan plants can be a source of antioxidants. 0.005 g dissolved in 50 mL methanol to obtain a
Various studies have been conducted to concentration of 100 ppm. The solution is then
determine plant stems and leaves' antioxidant activity pipetted as much as 0.1; 0.5; 1, 2, and 5 mL and
and toxicity. Based on the results of research on diluted in a 10 mL volumetric flask. Then each of
extracts of karamunting (Rhodomyrtus tomentosa), it these solutions was put into a test tube of 1 mL and
is known that the stems have a lower toxicity value of added 2 mL of methanol, and 1 mL of DPPH (0.005
>500 ppm compared to the leaves of 43.38 ppm. gram in 50 mL of methanol). Then prepare the test
However, for antioxidant activity in stems and leaves tube again for the blank solution, 3 mL of methanol
(Rhodomyrtus tomentosa), both have strong added 1 mL of DPPH. Each solution and blank was
antioxidants with IC50 values of 6-50 ppm (Kusuma, stirred using a vortex for 30 seconds and incubated for
Ainiyati, & Suwinarti, 2016)\. This study showed that 30 minutes at 37oC. After that, the solution and blank
stems have high antioxidant activity with lower were tested using a UV-Vis spectrophotometer at 515
toxicity than leaves. In addition, the choice of solvent nm (Mahardika & Roanisca, 2018; Mahardika,
is important in extracting the active components in Roanisca, & Sari, 2020; Fadiyah, Lestari, &
plants. Therefore this study aims to determine the Mahardika, 2020)).
antioxidant activity of extracts and fractions of
Pelawan stem extract (T. merguensis Grifft.) RESULTS AND DISCUSSION
The Pelawan stems refine using a wood-crushing
METHODOLOGY machine and a blender (Heriani, Sari, & Oktasari,
Materials 2021). In general, extraction is divided into two:
The tools used in this study were measuring single and multilevel. Single extraction can be carried
cups, beaker glasses, Erlenmeyer, dropper pipettes, out using only one solvent, whereas, in multilevel
volume pipettes, test tubes, filter paper, stirring rods, extraction, two or more solvents are used. Stratified
vials, measuring flasks, aluminum foil, vortex, extraction will produce certain compounds extracted
analytical balance, rotary evaporator IKA RV 10 and specifically for each solvent used.
a Shimadzu UV-1800 UV-Vis spectrophotometer, and In contrast, single extraction produces the
an FTIR spectrophotometer. At the same time, the extracted compounds, total extracts that can be
materials used in this study were Pelawan stem extracted with solvents. The solvent used in
Simplicia, technical methanol, technical n-hexane, multilevel extraction uses solvents with different
technical ethyl acetate, distilled water, acetone, 2 N polarity levels. Non-polar solvents can extract
hydrochloric acid, Wagner reagent, Meyer reagent, terpenoid groups, including triterpenoid compounds,
magnesium metal powder, amyl alcohol (a mixture of steroids, and saponins, while polar solvents will
hydrochloric acid 37% and 95% ethanol), ethanol, extract compounds such as alkaloids and phenolic
chloroform, glacial acetic acid, sulfuric acid, 1% groups, including flavonoids and tannins (Gong et al.,
ferric chloride, DPPH (Sigma). 2016). In comparison, semi-polar solvents will attract
compounds such as flavonoids, terpenoids, aglycones,
Extraction and glycosides (Harborne, 1987).
The Pelawan stems fine powder was taken 250 The Pelawan stem maceration using multilevel
grams and then macerated using three solvents,
extraction method. Multilevel extraction was done by
including n-hexane, ethyl acetate, and methanol. 2500 sequentially soaking the sample in different solvents,
mL of each solvent was used, and Pelawan stem
starting from non-polar, semi-polar, and polar
extract from each solvent was macerated for 3 x 24 solvents. The solvents used in the extraction of
hours. Then the filtrate obtained from each solvent Pelawan stems are solvents with increasing polarity,
will then be concentrated using a rotary evaporator to
namely n-hexane (non-polar), ethyl acetate (semi-

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Robby Gus Mahardika et al Indo. J. Chem. Res., 10(3), 143-148, 2023

polar), and methanol (polar). The first maceration was and saponins. The phenolic group, including
done by soaking Pelawan stem simplicial powder in flavonoids and tannins, and the alkaloid group is an
n-hexane solvent for 3x24 hours. The filtrate is alkaline secondary metabolite compound containing
separated by filtering, while the dregs are macerated one or more nitrogen atoms and usually in a
again with ethyl acetate solvent, and then separation is heterocyclic ring.
carried out again. The same treatment is for methanol FT-IR analysis was carried out to determine the
solvent. All the extracts were then concentrated using functional groups in the Pelawan stem extract as
a rotary evaporator to evaporate the solvent to indicated by the presence of absorption at certain
produce a thick extract. The yield of the Pelawan stem wave numbers by giving different absorptions to each
extract with n-hexane, ethyl acetate, and methanol sample. The FTIR results can be seen in Figure 1.
solvents were 4.232; 3.0917; and 9.7145 grams with a Based on the FTIR results of the Pelawan stem
yield percentage of 1.69; 1.56; and 3.88%. These data extract, the results were analyzed for wave numbers
indicate that the highest extract yield is methanol. It is with functional groups, which are presented in Table
because the active compounds contained in pelawan 2.The results of functional group analysis of Pelawan
stems are polar. stem extract using FTIR spectrophotometry found a
Phytochemical identification was carried out broad absorption at wave numbers 3000-3600 cm-1,
qualitatively on Pelawan stem extract. The results of which indicated the presence of O–H groups. The
identifying secondary metabolites of Pelawan stem stretching C-H group is in the range of 2850-2960
extract are shown in Table 1. The results of the cm-1. The C=O stretch ranges from 1700-1780 cm-1.
phytochemical tests showed that Pelawan stem extract the C=C group stretches 1500-1680 cm-1, the C-H
in each solvent was positive, containing phenolic groups bending 1350-1470 cm-1. The C-O group
compounds and flavonoids. At the same time, the ranges from 1060-1300 cm-1. At the same time, C-H
negative results on the test are alkaloids, steroids, and is aromatic was found at wave numbers 675-995 cm-1.
saponins. Tests on pelawan leaves have been carried Based on the results of functional groups that have
out by Enggiwanto, Istiqomah, Daniati, Roanisca, & been analyzed, pelawan stem extract from n-hexane,
Mahardika, (2018), where the Pelawan leaves were ethyl acetate, and solvents have the C=O functional
positive for alkaloid, flavonoid and phenolic group, which indicates that pelawan stem extract
secondary metabolite compounds. Based on the contains flavonoid compounds and is also supported
biosynthesis of secondary metabolites in plants, they by the presence of O-H groups found in wave
are divided into three main groups: the terpenoid numbers 3000-3600 cm-1,
group, including triterpenoid compounds, steroids,

Table 1. Results of identification of secondary metabolites of Pelawan stem extract

Ethanol Extract of Pelawan
Pelawan Stem Extract Leaves (Enggiwanto et al.,
2018) and the fraction of ethyl
Test Test Method
Ethyl acetate extract of methanol
n-hexane Methanol leaves (Samsiar, Mahardika,
Acetate
& Roanisca, 2021)
Alkaloid Mayer - - - +
Wagner - - - +
Flavonoid FeCl3 + + + +
Wilstater
Phenolic + + + +
Sianidin
Steroid Forth - - - -
Liebermann-
Saponin - - - -
Burchard

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Robby Gus Mahardika et al Indo. J. Chem. Res., 10(3), 143-148, 2023

Figure 1. FTIR spectrum of Pelawan stem extract

C=C aromatic at 1500-1680 cm-1, C-O at wave differences in the functional groups found in Pelawan,
number 1060-1300 cm-1 as well as the results of the Pelawan leaves do not have C-H stretch groups which
analysis of pelawan stem extract also contains are in the range of wave numbers 2850-2960 cm-1,
phenolic as indicated by the presence of O-H groups and Pelawan stem extract does not there is a C-H
found at wave numbers 3000-3600 cm-1, C-H at wave group (CH2 (methylene) which is present in wave
numbers 2850-2960 cm-1, C=C aromatic at 1500-1680 number 1450 cm-1 and the aromatic C-H group in the
cm-1, and C-O at wave numbers 1060-1300 cm-1. methanol extract of pelawan stem which is present in
Based on previous research on Pelawan leaves by the absorption wave number between 675-995 cm-1.
Samsiar, Mahardika, & Roanisca, (2021) showed

Table 2. FTIR Spectrum Analysis Results of Pelawan Stem Extract

Wave Numbers (cm-1)

Ethyl Acetate Fraction
Fuctional Groups Band shape Ethyl
n-Hexane Methanol of Pelawan Leaves
Acetate
Extract Fraction (Samsiar, Mahardika,
Fraction
& Roanisca, 2021)
O-H stretch Broad 3364 3384 3379 3320
C-H stretch Sharp 2925 2915 2926 2940
C-H stretch (aliphatic) Sharp 2857 2855 2852 -
C=O stretch Sharp 1735 1729 1720 1710
C=C stretch Sharp 1610 1618 1612 1610
C-H (CH2) Sharp - - - 1450
C-H (CH3) bend Sharp 1374 1376 1362 1320
C-O bend Sharp 1023 1073 1037 1020
C-H (aromatic) Sharp 973 976 - 750

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Antioxidant activity Based on Table 3. The antioxidant activity of

The antioxidant activity test was carried out Pelawan stem methanol extract of pelawan stem has a
quantitatively using a spectrophotometer by small IC50 value compared to another pelawan stem
measuring the concentration inhibition by the extract, namely 36.33 μg/mL. It shows a correlation
sample's ability to scavenge radicals from DPPH. between total phenolic and flavonoid levels with
Antioxidant activity testing was carried out using the antioxidant activity. Based on the total phenolic test,
DPPH (1,1-diphenyl-2-picrylhydrazyl) method. The the methanol extract had the highest total phenolic
reaction mechanism of DPPH with antioxidant content, giving it a strong antioxidant activity.
compounds is radical. DPPH is a radical compound Pelawan stem ethyl acetate extract has antioxidant
that reacts with antioxidant compounds, which can be activity with an IC50 value of 387.43 μg/mL. At the
phenolic compounds or flavonoids. same time, the pelawan stem n-hexane extract had
Antioxidant compounds will reduce DPPH and very weak antioxidant activity giving an IC50 value of
pair with hydrogen from antioxidant compounds to 759.88 μg/mL. The results of the antioxidant activity
form reduced DPPH-H (Lee & Kim, 2022). The test showed that the methanol extract from pelawan
reaction between antioxidant compounds and DPPH stems had the highest antioxidant activity compared
will reduce the intensity of the purple color in DPPH, to the ethyl acetate and n-hexane extracts. It is due to
which can then be measured using a the content of phytochemical compounds, especially
spectrophotometer. The decrease in the intensity of phenolics which have more antioxidant activity
the purple color is due to the interaction of antioxidant extracted in polar solvents. The more phytochemical
compounds that donate electrons to the DPPH compounds that have antioxidant activity are
radicals. The more antioxidant compounds, the extracted, the higher the antioxidant activity. Based
intensity of the purple color will decrease. on the results of the antioxidant activity test of the
Quantitative testing of antioxidant activity was carried pelawan stem methanol extract, this is weaker when
out using a UV–Vis spectrophotometer at a compared to the ethanol extract and acetone of
wavelength of 515 nm to determine IC50. IC50 states pelawan leaves with consecutive IC50 values of
the concentration at which the extract can decompose 18.2772 μg/mL (Enggiwanto et al., 2018) and
50% of free radicals, which in this test is DPPH. The 22.1454 μg/mL (Roanisca et al., 2019). It is due to the
results of the analysis of the antioxidant test can be presence of alkaloids in pelawan leaves, while in
seen in Table 3. pelawan stems, no alkaloids were found.

Table 3. Antioxidant Activity of Pelawan

Antioxidant activity
No. Samples References
(IC50)

1 Pelawan Stem Methanol Extract 36.33 μg/mL This research

2 Pelawan stem ethyl acetate extract 387.43 μg/mL This research

This research
3 Pelawan stem ethyl n-hexane 759.88 μg/mL
Ethanol Extract of Pelawan Enggiwanto et al.,
4 18.2772 μg/mL
Leaves 2018
Acetone Extract of Pelawan Roanisca et al.,
5 22.1454 μg/mL
Leaves 2019

CONCLUSION DPPH radicals in the pelawan stem methanol extract

fraction had a very strong activity with an IC50 value
Based on the results and discussion, it was
of 36.33 µg/mL. At the same time, the ethyl acetate
concluded that qualitatively the phytochemical testing
and n-hexane extract fractions had a weak antioxidant
of Pelawan stem extract (T. merguensis) from the
activity with IC50 values, respectively, 387.43 μg/mL
three solvent fractions, such as n-hexane, ethyl
and 759.88 μg/mL.
acetate, and methanol, was positive for containing
secondary metabolites of flavonoids and phenolics. In
comparison, the antioxidant activity in reducing

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ACKNOWLEDGEMENT Lee, S., & Kim, H. J. (2022). Antioxidant Activities

of Premature and Mature Mandarin (Citrus
We gratefully acknowledge the funding from
unshiu) Peel and Juice Extracts. Food Science
Universitas Bangka Belitung through UBB PDTU
and Biotechnology, 31(5), 627–633.
2021 funding from LPPM UBB which has financed
Mahardika, R. G., Roanisca, O., & Sari, F. I. P.
this research.
(2020). Inhibition of α-Glucosidase Activity and
The Toxicity of Tristaniopsis merguensis Griff.
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