Title


Science and Technology Indonesia
e-ISSN:2580-4391 p-ISSN:2580-4405
Vol. 8, No. 1, January 2023

Research Paper

Determination of (+)-Catechin and Antioxidant Activity in Faloak (Sterculia quadrifida R.
Br) Stem Bark Infusion
Audrey Gracelia Riwu1, Jusak Nugraha2, Djoko Agus Purwanto3*, Erwin Astha Triyono4
1Doctoral Program of Medical Science, Faculty of Medicine, Universitas Airlangga, Surabaya, 60132 Indonesia2Department of Clinical Pathology, Faculty of Medicine, Universitas Airlangga, Surabaya, 60132, Indonesia3Department of Pharmaceutical Sciences, Faculty of Pharmacy, Universitas Airlangga, Surabaya, 60115, Indonesia4Department of Internal Medicine, Faculty of Medicine, Universitas Airlangga, Surabaya, 60132, Indonesia
*Corresponding author: djokoagus@ff.unair.ac.id

AbstractOxidative stress is a condition that can damage human cells and tissues and has been linked to a number of illnesses, including cancer,cardiovascular disease, autoimmune disorders, and neurological diseases. Oxidative stress conditions can be brought on by pollution,radiation exposure, and an unhealthy lifestyle. Antioxidants are substances that can be used to both prevent and treat oxidativestress. This study aimed to identify and quantify (+)-catechin levels and antioxidant activity of the stem bark of Sterculia quadrifida R.Br extracted by the infusion method, a method similar to traditional medicine processing generally in the community. Determinationof (+)-catechin and antioxidant activity of S. quadrifida were evaluated by HPLC and DPPH assay, respectively. Quantification of (+)catechin content by HPLC system with wavelength 280 nm and antioxidant activity by spectrophotometry method with wavelength517 nm. The results show that the mean value of (+)-catechin level was 7.786% and the IC50 value of the antioxidant activity was51.5`g/mL having a moderate antioxidant activity category. S. quadrifida stem bark infusion can be utilized as a medication candidatefor the prevention or treatment of a variety of disorders caused by oxidative stress.
Keywords
Sterculia quadrifida R. Br, Faloak, (+)-Catechin, Antioxidant Activity, HPLC, DPPH

Received: 30 August 2022, Accepted: 9 December 2022
https://doi.org/10.26554/sti.2023.8.1.59-65

1. INTRODUCTION

Cancer, cardiovascular disease, autoimmune disorder, and car-
diovascular and neurodegenerative disease (i.e Alzheimer’s dis-
ease and Parkinson’s disease) are widely known to have a cor-
relation with oxidative stress. Oxidative stress is a condition
due to an imbalance between the increase of ROS and RNS
secretion that leads to cell and tissue damage. There are many
factors that contribute to this condition like exposure to air
pollution, tobacco smoke, ozone, alcohol, cooking (used oil, fat,
and smoked meat), and another unhealthy lifestyle. These ex-
ogenous substances are metabolized or broken down into free
radicals after entering the body through various mechanisms
(Pizzino et al., 2017). Free radicals are substances (atoms, ions,
or molecules) that contain one or more unpaired electrons that
interfere with or attack nearby electrons in order to form bonds
with them, which causes oxidative stress (Di Meo and Venditti,
2020).

Antioxidants are one strategy for reducing oxidative stress.
By giving electrons to free radicals, antioxidants are able to

neutralize them and inhibit cellular damage potential due to ox-
idative stress. Antioxidants can be found in both manufactured
and natural goods, although generally, natural antioxidants are
preferred because they have less of an impact on the body.
Some sources of natural antioxidants are usually derived from
plant materials, such as vegetables, fruits and herbs (Nimse
and Pal, 2015; Lourenço et al., 2019). (+)-Catechin is one
of the naturally occurring antioxidants, a derivative form of
catechin compound, which is mostlyfound in woodyplants (Ya-
maji and Ichihara, 2012). (+)-Catechin is a avonoid-derived
avan-3-ols compound that is known to have anticarcinogenic,
anti-tumor, antibiotic, and antioxidant activity which makes it
essential in treating various diseases (Lee et al., 2020; Musial
et al., 2020).

Sterculia quadrida R. Br (world’s known name: peanut
tree/kurrajong; local name in Indonesia: Faloak) is one of
the woody plants that belong to the Malvaceae family that can
be used as a source of natural antioxidants because it is found to
have a variety of secondary metabolites, including avonoids,
alkaloids, terpenoids, saponin and phenolic (Tenda et al., 2021;

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https://doi.org/10.26554/sti.2023.8.1.59-65


Riwu et. al. Science and Technology Indonesia, 8 (2023) 59-65

Siswadi et al., 2021). This plant can be found in plentiful sup-
ply from Queensland (Western Australia), Papua New Guinea,
Timor Leste, and Indonesia (Govaerts et al., 2021; Siswadi
etal.,2021). Thestembarkof trees,whichisprocessedthrough
boiling or decoction, is the most widely utilized plant part in
Indonesia (Tenda et al., 2021).

Traditionalmedicinecanbeutilized in avarietyofways and
with dierent types of solvents, but in the community, boiling
with water is the approach that is most commonly applied. The
process of boiling water is thought to be the simplest and safest
one that can be used in regular community life (Keskin, 2018;
Wachtel Galor and Benzie, 2011).

Based on the above background, the aim of this study is
to determine and quantify (+)-catechin using HPLC, followed
by antioxidant activity using DPPH methods in the infusion
of S. quadrida stem bark. For additional. in this study, we
also identify the functional group of compounds with FTIR
spectrophotometry methods. The infusion method is an ex-
traction technique that is similar to the community’s processing
technique, which involves heating water. Therefore, this study
is expected to provide an overview of the (+)-catechin levels
and antioxidant activity found in traditional medicines that are
processed in a common way in the community.

2. EXPERIMENTAL SECTION

2.1 Materials
S. quadrida stem bark was taken from the tree which grows in
the cityof Kupang, East NusaTenggara, Indonesia. Determina-
tion of these plants was carried out at the Indonesian Institute
of Sciences, Biological Research Centre, Cibinong, Bogor. The
determination process was conducted on these plant parts such
as seeds, owers, leaves, fruit, and stem bark. The (+)-catechin
standard was obtained through Sigma Aldrich, which was inter-
preted by the Testing Services Unit of the Faculty of Pharmacy,
Airlangga University, Indonesia.

2.2 Extraction
S. quadrida stem bark was extracted using the infusion method
in order to represent the traditional method of processing by
the community. The stem bark of this plant was gathered,
sorted, crushed, and mashed using a blender in quantities of up
to 500 grams. Aquades solvent was used to dissolve the mashed
simplisia, which was then heated at 90°C for 15 minutes. After
15 minutes, the ltrate is retrieved, gathered, and dried using
the freeze-drying method to produce up to 16 grams of dry
powdered stem bark (Azwanida, 2015).

2.3 Preparation of Samples and Standard Solutions
The powdered S. quadrida stem bark was taken as much as 500
mg and put into a ask with a volume of 50 ml. Then added
ethanol and water with a ratio of 1: 1 until exactly according to
the mark on the ask. The sample then underwent sonication
for 15 minutes and was ltered with a nylon membrane of
0.45 microns into the HPLC vial and analyzed. A raw stock
solution of catechins of 10.4 mg was dissolved in 10 ml of

methanol which is then ltered using 0.2 microns of nylon
(Gottumukkala et al., 2014).

2.4 Calibration
The standard calibration process was carried out by diluting
the catechin stock solution with methanol with a concentra-
tion range of 200-1000`L. The standard curve was obtained
using the peak area of ve concentrations in ve repeat tests
and expressed by a linear-quadratic regression equation Got-
tumukkala et al. (2014).

2.5 FTIR Analysis
Fourier-transform infrared spectroscopy (FTIR) is a technique
used to obtain the infrared spectrum of the absorption or emis-
sion of a solid, liquid, or gas. The FTIR spectrometer simulta-
neously collects high-resolution spectra data over a wide spec-
trum range. FTIR method is commonly used to identify the
groups present in a compound or group of compounds that
describe the identity of the natural product extract under study.
Therefore, FTIR is important to provide an overview of the
characteristics of a particular plant extract (Pharmawati and
Wrasiati, 2020). Approximately 10 mg of water extract of
S. quadrida stem bark was mixed with 100 mg of KBr and
packaged into sample pellets and then read by the FTIR spec-
trophotometer (Perkin Elmer, 1302F6802). The results ob-
tained were analyzed by their functional groups starting from
wave numbers 450 cm−1 to 4000 cm−1 (Pakkirisamy et al.,
2017).

2.6 Determination of (+)-catechin
The (+)-catechin analysis process is carried out using the HPLC
Agilent 1100 system consistingof aquaternarypump, autosam-
pler DAD detector, column compartment, and degasser with
MerckLichrosper100RP-18,250x4mm,0.5 ` column. The
entire analysis process is carried out at a temperature of 25°C
with a mobile phase using a solution of acetonitrile (A) and
methanol (B) with a gradient of 1.2 mL/minute. The mobile
phase is ltered usingnylon 0.2 microns where before using the
gas was removed rst. The sample injected into HPLC was 20
`L with a wavelength (_) of 280 nm. Chromatography peaks
are identied by comparing retention times between analytes
with standards. Quantication was determined by peak integra-
tion using external standard methods (Roman and Solomiia,
2019).

2.7 Antioxidant Activity
The antioxidant activity of the sample was tested using the
DPPH method with ascorbic acid as a control. S. quadrida
stem bark infusion samples and ascorbic acid as standard were
dissolved with ethanol solvents and made in several concentra-
tions. Each solution is picked as much as 1 mL to be added to 3
mL of DPPH solution 40 ppm in ethanol. Then the solution is
shakenrmlyand let standat roomtemperature for30minutes.
Then the absorbance was measured using a spectrophotometer
at a maximum wavelength (_) of 517 nm. These tests were

© 2023 The Authors. Page 60 of 65



Riwu et. al. Science and Technology Indonesia, 8 (2023) 59-65

Figure 1. FTIR Spectrum of S. quadrida Stem Bark

repeated twice. Radical scavenging activity is indicated as the
presentation of free radical inhibitors by a sample calculated
with the formula:

DPPHscavengingactivity(%) =
ABScontrol-ABSsample

ABScontrol
×100% (1)

Where Abscontrol is the absorbance of the DPPH and Ab-
ssample is the absorbance of the sample. The concentration of
the sample and the percent inhibition obtained were plotted on
the x and y axis respectively in the linear regression equation
y=a+bx. This equation is used to determine the IC50 value of
the sample. The IC50 value is the sample concentration that
can reduce DPPH radicals by 50% of the initial concentration.
The IC50 value is obtained from the x value (concentration)
after replacing the y value with 50 (50% inhibition). The in-
terpretation of IC50 results is that the lower the absorbance
of mixed reactions indicates that the higher the activity of free
radicals (Allam et al., 2020; Sinala et al., 2020).

3. RESULT AND DISCUSSION

3.1 FTIR Analysis
The functional groups in S. quadrida stem bark extract was
predicted by interpreting the infrared absorption spectra. The
FTIR spectrum result can be seen in Figure 1, and the inter-
pretation of the functional group can be seen in Table 1.

Based on the results of FTIR analysis, seven compounds
were found in the stem bark infusion of S. quadirifda, namely
alcohol, poly hydroxyl compound, isothiocyanate, aromatic
compound, ketone compound, secondary alcohol, and aro-
matic phosphates.

3.2 Determination of (+)-catechin
Calibration of the (+)-catechin standard used at concentrations
of 200 `L – 1000 `L (208, 416, 624, 832, 1040 ppm) in-
dicates good linearity which means the correlation coecient

Figure 2. (+)-Catechin Standard Calibration Curve

of the equation was close to 1 and the calibrations curves lead
straight lines with a wide range (Gottumukkala et al., 2014;
Pinto et al., 2017). The (+)-catechin content in the S. quadrida
stem bark infusion was calculated using the linear regression
equation obtained by the curve that can be seen in Figure 2.
Chromatograms of the identication of the standard solutions
and infusions can be seen in Figures 3 and 4. The result of the
determination of (+)-catechins levels can be seen in Table 2.

Based on the results of the HPLC test, it is found that
the average result of (+)-catechins found in the infusion of
the S. quadrida stem bark is 7.786 % w/w with an RPD of
0.889% (Table 2). Studies on the quantication of (+)-catechin
in the stem bark of S. quadrida have never been done before in
previous studies. Therefore, the determination of (+)-catechin
levels in this study is also a novelty in the exploration of specic
compounds in the stem bark of S. quadrda.

(+)-Catechin is a derivative form of catechin, polyphenols
avan-3-ols that belong to the avonoid family. This com-
pound is composed of two aromatic rings and several hydroxyl
groups, which are classied into two groups: free catechin and
esteried catechins. This compound has a molecular weight of
290.27 g/mol with the molecular formula C15H14O6 (Figure
5) (Diniyah et al., 2022; Shukla et al., 2018). (+)-Catechin
and other catechin derivatives have been shown to have neuro-
protective eects as well as antioxidant, anti-cancer, and anti-
microbial (Chang and Wu, 2011).

Quantication of (+)-catechin in S. quadrida stem bark
has never been done in previous studies. However, several
studies regarding the quantication of (+)-catechin in other
plants have been carried out. A study conducted by Silva
et al. (2017) stated that the catechin levels found in the spray-
dried extract (ethanol 95%) of Pimenta pseudocaryophyllus was
5.44%. A study conducted by Nurliayana et al. (2016) on the
methanol, dichloromethanen, and hexane extracts of Uncaria

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Riwu et. al. Science and Technology Indonesia, 8 (2023) 59-65

Table 1. TIR Spectrum Values and Functional Groups of S. quadirida Stem Bark

No Wave Number Reference Wave Functional Group Phyto-compounds
(cm−1) Number (cm−1) Assignment Identied

1 3595 3700-3584 OH stretch Alcohol
2 3242 3570-3200 O-H stretch, Hydroxy Poly Hydroxy compound

group, H-bonded
3 2063 2140-1990 N=C=S stretch Isothiocyanate
4 1759 2000-1650 C-H bend Aromatic compound
5 1605 1650-1600 C=O stretching vibration, Ketone compound

ketone group
6 1118 1150-1085 C-O stretch Secondary alcohol
7 899 995-85 P-O-C stretch Aromatic phosphates

Table 2. Determination Results of (+)-Catechin Levels and Antioxidant Activity in S. quadrida Stem Bark Infusion

Catechin Levels Antioxidant Activity (IC50)

Mean (% w/w) SD (%) RPD (%) Mean (`g/mL) SD (%) RPD (%)

7.786 0.049 0.889 51.5 0.29 0.8
Note: SD: Standard Deviation; RPD: relative percentage dierences

Figure 3. HPLC Chromatogram of (+)-Catechin Standard
Solution. Retention Time at 1.812 Min.

Figure 4. Chromatogram of (+)-Catechin in S. quadrida Stem
Bark Infusion

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Riwu et. al. Science and Technology Indonesia, 8 (2023) 59-65

Figure 5. Molecular Structure of (+)-Catechin (Isemura, 2019)

gambir (Hunter) Roxbstembarkwas5.12%,0.92%, respectively,
and no catechins were found in the hexane extract. Based on
the two studies above, it can be said that the dierences in cate-
chin levels found were caused by dierences in plant species
and solvents used in each study.

A study conducted by Koch et al. (2018) showed that the
levels of (+)-catechin in black tea, green tea, and white tea prod-
ucts from Kenyan germplasm were 0.30%, 0.39%, and 0.57%,
respectively. Meanwhile, in green tea products from China
and Japan germplasms were 0.13% and 0.12%. Based on these
results, it was found that (+)-catechin levels in tea products were
found to be lower when compared to (+)-catechin levels in S.
quadrida stem bark infusion. This is a reasonable, because, in
tea, the levelof (+)-catechinhas the lowest levelwhencompared
to other catechins (Koch et al., 2018; Singh et al., 2011). Cate-
chins are the most abundant polyphenols in tea which account
for 70-80% of the total polyphenols. There are four types of
catechins that are most dominant in tea, namely (-)-epicatechin
(EC), (-)-epigallocatechin (EGC), (-)-epicatechin-3-O-gallate
(ECG), and (-)-epigallocatechin -3-O-gallate (EGCG), where
of the four types of catechins, the concentration of EGCGdom-
inates as much as 50-80% of the total catechins (Koch et al.,
2018; Sivanesan et al., 2022). Therefore, further studies are
needed to identify the total catechins in the stem bark of S.
quadrida so that further comparisons can be made.

3.3 Antioxidant Activity
Results from the DPPH assay using the spectrophotometry
method of S. quadrida stem bark infusion can be seen in Table
1. The test also was done by replicating twice because both of
the results were very close to each other. The dierences in
antioxidantactivitybetweenS.quadridastembarkandascorbic

Figure 6. Dierences of Antioxidant Activity Between S.
quadrida Stem Bark Infusion and Ascorbic Acid as a Control

acid can be seen in Figure 6.
Based on the IC50 value, antioxidant activity is divided

into ve categories, namely very strong (<10 `g/mL), strong
(10-50 `g/mL), moderately (50-100 `g/mL), and weak (100-
250 `g/mL), and inactive (> 250 `g/mL) (Roy and Dutta,
2021). This shows that the lowertheIC50value, thehigherthe
antioxidant activity. According to the classication, S. quadrida
stembarkinfusionisclassiedinthemoderatecategorybecause
the IC50 value is 51.5 `g/mL, while ascorbic acid, which
has an IC50 value of 11.6 `g/mL is classied in the strong
category (Figure 6). This is presumably because the stem bark
of S. quadrida contains a variety of compounds compared to
ascorbic acid which is a pure compound.

The antioxidant activity of S. quadrida stem bark has been
reported in several previous studies. Research conducted by
(Saragih and Siswadi, 2019) on ethanol extract found IC50
values from various parts of S. quadrida bark (branch bark,
root bark, old regrown bark, new regrown bark, non-striped
stem bark) are all below 50 `g/mL, and studies conducted
by Dillak et al. (2019) on ethanol extract also obtained IC50
values of 14.17 `g/mL. This is thought to be due to dierent
typesof solvents, where theextractionprocessusingethanol sol-
vents is the best method of knowing antioxidant activity when
compared to water solvents (Hidalgo and Almajano, 2017).

The extraction method also has an impact on antioxidant
activity. High-temperature extraction methods have the poten-
tial to degrade some of the most controllable molecules that
aect antioxidant activity. Infusion methods with high tem-
peratures reaching 90°C can have an impact on the outcome
since, in general, heating induces a quick initiation reaction and
reduces antioxidant activity (Xu et al., 2017; Réblová, 2012).
According to a study done in Réblová (2012), there is a linkage
between rising temperatures and decreased antioxidant activ-

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Riwu et. al. Science and Technology Indonesia, 8 (2023) 59-65

ity. Antioxidant activity of the easily oxidized antioxidants was
decreased at higher temperatures compared to lower temper-
atures caused by a reduction in the ability of antioxidants to
interact with free radicals.

Catechin levels and antioxidant activity are basically re-
lated. Catechins are able to counteract free radicals by giving
an electron from the phenolic OH group which can reduce
free radicals (Bernatoniene and Kopustinskiene, 2018). Cate-
chins can also reduce oxidation by playing a role in chelating
and binding metal ions which can start the oxidation process
(Nain et al., 2022). Catechins and antioxidants have a rela-
tionship with each other, where the higher the concentration
of catechins, the higher the level of antioxidants (Lee et al.,
2014; Musial et al., 2020; Bartoszek et al., 2018). However,
both of them are also inuenced by various factors, such as the
extraction process, the type of solution, temperature, and the
concentration of other compounds in it (Saklar et al., 2015;
Hidalgo and Almajano, 2017).

Due to the high-temperature extraction method, and the
type of solution used in this investigation, it was discovered
that the catechin levels and antioxidant activity were dier-
ent (depending on the type of plant and solvent) than in the
prior study. Nevertheless, extraction with infusion method is
seen as being safer, more practical, and more accessible to the
community.

4. CONCLUSION

(+)-Catechin has been detected and quantied in S. quadrida
stem bark infusion with a level of 7.786%. A moderate level of
antioxidant activity in this plant’s stem bark was also discovered
in this study, with an IC50 value of 51.5 `g/mL. These nd-
ings provide a brief overview of the (+)-catechin content and
antioxidantactivityofS.quadridastembarkwhich is processed
by the infusion method which is common in the community.
Therefore, S. quadrida can be utilized as a medication candi-
date for the prevention or treatment of a variety of disorders
caused by oxidative stress.

5. ACKNOWLEDGMENT

This work is part of the Doctoral Program dissertation of the
Faculty of Medicine, Airlangga University, Indonesia. The au-
thor thanked the Doctoral Program of the Faculty of Medicine
and the Testing Laboratory Unit of the Faculty of Pharmacy,
Airlangga University, Indonesia who had given permission and
provided places and facilities for this research.

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© 2023 The Authors. Page 65 of 65


	INTRODUCTION
	EXPERIMENTAL SECTION
	Materials
	Extraction
	Preparation of Samples and Standard Solutions
	Calibration
	FTIR Analysis
	Determination of (+)-catechin
	Antioxidant Activity

	RESULT AND DISCUSSION
	FTIR Analysis
	Determination of (+)-catechin
	Antioxidant Activity

	CONCLUSION
	ACKNOWLEDGMENT