Selection of Four Lamiaceae Species

Vivi Septya Wati, et al.

Articles

MOLEKUL

eISSN: 2503-0310

https://doi.org/10.20884/1.jm.2025.20.2.13140

Selection of Four Lamiaceae Species (Ocimum americanum L., Ocimum basilicum L.,
Leucas lavandulifolia Sm., and Perilla frutescens (L.) Britton) as Antioxidant Sources
and Metabolite Profile
Vivi Septya Wati1, Irmanida Batubara1,2*, Budi Arifin1, Bambang Pontjo Priosoeryanto3, Yutaka Kuroki4
Department of Chemistry, Faculty of Mathematics and Natural Sciences, IPB University,
Bogor 16680, West Java 16680, Indonesia
2
Tropical Biopharmaca Research Center, IPB University, Bogor 16128, West Java, Indonesia
3
Division of Veterinary Pathology, School of Veterinary Medicine and Biomedical Sciences,
IPB University, Bogor 16680, West Java, Indonesia
4
Delightex Pte Ltd, 230 Victoria Street, #15-01/08, Bugis Junction Towers, 188024, Singapore
1

*Corresponding author email: ime@apps.ipb.ac.id
Received September 21, 2024; Accepted July 09, 2025; Available online July 20, 2025
ABSTRACT. The Lamiaceae family which are widely used in traditional medicine in Indonesia. Plants from this family are known
to have antioxidant bioactivity. Therefore, in this study, an antioxidant test was carried out first as an initial screening. This study
evaluated the antioxidant capacity of four Lamiaceae species: Ocimum americanum L., Ocimum basilicum L., Leucas
lavandulifolia Sm., and Perilla frutescens (L.) Britton. The four Lamiaceae species extracts were evaluated for their antioxidant
capacity using 2,2-diphenyl-1-picrylhydrazyl (DPPH), cupric-reducing antioxidant capacity (CUPRAC), and ferric-reducing
antioxidant power (FRAP). The highest activity extract was fractionated with n-hexane, ethyl acetate, and ethanol-water, and the
results were tested for antioxidant capacity. The most active extract and most active fraction were analyzed for metabolites using
liquid chromatography-mass spectrometry (LC-MS/MS). The results show that the ethanol 50% leaves extracts of O. americanum
L. have the highest antioxidant capacity of about 0.439 (DPPH); 1.517 (CUPRAC); and 1.021 (FRAP) mmol ascorbic acid
equivalent (AAE)/g extract. The highest antioxidant capacity from the partition results was possessed by the ethyl acetate fraction
with a value of 1.109 (DPPH); 1.540 (CUPRAC); and 1.551 (FRAP) mmol AAE/g fraction. Metabolite analysis using LC-MS/MS
succeeded in identifying 18 metabolites consisting of flavonoids, terpenoids, amino acids, phenolic acids, fatty acids, and other
carboxylic acids.
Keywords: Antioxidant, Leucas lavandulifolia, Ocimum americanum, Ocimum basilicum, Perilla frutescens

INTRODUCTION
For generations, Indonesian people have used many
medicinal plants in their daily lives. One of the plant
families that is widely used as traditional medicine in
Indonesia is Lamiaceae. Lamiaceae species are known
to have medicinal properties, have a strong history of
use since ancient times, and are famous for their high
essential oil content (Handayani, 2015). Plants from this
family are widely used by the community as wound
healers, antibacterials, antioxidants, antifungals, antiinflammatories, diuretics, pain relievers, and antidotes
to infections (Venkateshappa and Sreenath 2013). One
of the widely reported bioactivities in this family is as an
antioxidant. Antioxidant activity is important to study
because it can be used as an initial screening for
determining other bioactivities.

Antioxidants are chemical compounds that can
inhibit oxidative stress. Oxidative stress is damage to
lipids, proteins, or DNA due to the presence of excessive
reactive oxygen species (ROS) (Yousuf et al., 2021).
Oxidative stress can cause several chronic diseases such
as diabetes, chronic lung, chronic inflammation, cancer,
coronary heart disease, premature aging, and
neurodegenerative. These diseases can be avoided by
consuming foods rich in antioxidants which can inhibit
or inactivate ROS. Apart from protecting the body from
the effects of excess ROS, antioxidants also play a role
in protecting food and pharmaceutical products against
oxidative damage (Gulcin, 2020). Therefore, in recent
years, the identification of natural and safe sources of
antioxidants, one of which comes from medicinal plants,
has been of interest.

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The types of Lamiaceae plants that are often found
in Indonesia and have the potential to act as
antioxidants are Leucas lavandulifolia Sm. (lenglengan)
(Ramani et al., 2012), Ocimum americanum L.
(kemangi) (Karau et al., 2015), Ocimum basilicum L.
(selasih) (Teofilović et al., 2017), and Perilla frutescens
(L.) Britton (perilla) (Shang et al., 2023). Previous
research has proven the existence of antioxidant activity
in these four plants. However, no one has yet selected
the antioxidant activity of the four, so an antioxidant
activity test is needed to determine the level of
antioxidant activity of these plants.
Lamiaceae
plants
contain
many
phenolic
compounds, polyphenols, and essential oils (Frezza et
al., 2019), but not all of them play an active role as
antioxidants. Therefore, it is important to know the active
metabolites that act as antioxidants. Different plants will
have different activities, because the metabolites they
contain are also different. Several factors also affect the
content of extracted plant metabolites, such as
differences in extraction methods and differences in
extraction solvents (Sayuti, 2023). One extraction
method that is rarely used in research but is widely used
in industry is the autoclave extraction (AE) method. The
AE method has the advantages of higher extraction
yields, increasing the bioactivity of bioactive
compounds, saving time and solvents, being efficient in
extracting macromolecules, and is considered a
promising method for extracting active ingredients (Kim
et al., 2016; Ko et al., 2015). Previous studies showing
active compounds that act as antioxidants among the
four plants extracted using the AE method are still few.
Therefore, it is necessary to identify metabolite profiles
that act as antioxidants in these plants. This research
carried out a selection of antioxidant activity from the
plant extracts of O. americanum L., O. basilicum L., L.
lavandulifolia Sm., and P. frutescens (L.) Britton. as well
as searching for active metabolites that have potential
as antioxidants through compound identification using
liquid chromatography-mass spectrometry (LC-MS /MS).
EXPERIMENTAL SECTION
Material
The sample used in this study was O. americanum L.
(determination result letter No. 7767/IT1.C11.2/
TA.00/2022) and O. basilicum L. (determination result
letter No. 668/IT1.C11.2/TA.00/2022) taken in Bogor
Regency at coordinates -6.54714 LS, 106.71637 BT, P.
frutescens (L.) Britton (determination result letter No.
193/1T1.C11.2/TA.00/2022) taken in Sukabumi at
coordinates -6.85250 LS, 106.95306 BT, and L.
lavandulifolia Sm. (determination result letter No.
668/IT1.C11.2/TA.00/2022) taken in Padang at
coordinates -0.82061 LS, 100.32421 BT. The materials
used in this research were ethanol, ethyl acetate, n-

hexane, chloroform, methanol, copper(II) chloride,
iron(III) chloride, ammonium acetate, dimethyl sulfoxide
(DMSO) (Merck, Darmstadt, Germany), 2,2-diphenyl-1picrylhydrazyl (DPPH), ascorbic acid, neocuproine,
2,4,6-tripiridyl-s-triazine
(TPTZ)
(Sigma-Aldrich,
Steinheim, Germany).
Sample Preparation and Determination of Water
Content
The four samples were determined at the School of
Biological Sciences and Engineering, Bandung Institute
of Technology. The leaves were taken from each
sample, then cleaned and washed with water. Next, the
samples were dried in an oven at 45 °C until dry. After
drying, the samples were ground to a size of ±80 mesh.
Determination of water content refers to AOAC (2000)
with modification. The empty porcelain cup was dried in
an oven at 105 °C for up to 30 minutes, then cooled for
30 minutes in a desiccator, and the empty weight was
weighed. A sample of 3 g was added to the cup, then
dried in an oven at 105 °C for 5 hours. After cooling in
a desiccator for 30 minutes, the cup containing the
sample was weighed. The same thing was done with a
drying time of 1 hour until a constant weight was
obtained. Water content is expressed as the percentage
of sample weight lost due to evaporation.
Sample Extraction and Partitioning
The samples were extracted with hot water and 50%
ethanol using an autoclave process. 20 g of dry powder
was put into a closed container, then 200 mL of solvent
was added and put into an autoclave with a temperature
setting of 100 °C for 10 minutes and an environmental
pressure of 1 atm. After that, the container containing
the sample was kept in a water bath at 60 °C for 1 hour
and left overnight at room temperature to cool naturally.
The extract was filtered using filter paper to remove the
supernatan which was then concentrated using a rotary
evaporator and lyophilized. The yield is shown as a
percentage of the weight of the extract obtained per
initial weight of the simplicia. The obtained extracts were
tested for activity. The most active extract was partitioned
with the solvent n-hexane (nonpolar) until the organic
phase obtained was colorless, followed by the solvent
ethyl acetate (semipolar), and finally ethanol-water
(polar).
Phytochemical Test
The concentrated extracts were tested for
phytochemistry to determine the class of compounds
contained in each extract, including tests for flavonoids,
alkaloids, saponins, triterpenoids, steroids, and tannins.
This test uses standard procedures and procedures from
previous research by Shaikh & Patil (2020) and Mlozi et
al. (2022). (1) Flavonoids: 0.1 g of extract was dissolved
in 1 mL of distilled water, then 2 mL of 2% NaOH
solution was added to the solution. The mixture was

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Vivi Septya Wati, et al.

shaken, then a few drops of dilute HCl were added, and
the color change was observed. Extracts are positive for
containing flavonoids if they change color from dark
yellow after adding NaOH solution to colorless after
adding a few drops of dilute HCl. (2) Alkaloid: 0.1 g of
extract was dissolved in 1 mL of 1% HCl, then the mixture
was stirred and filtered, then 2 mL of
Dragendorff/Mayer reagent was added to the filtrate.
The presence of alkaloids is indicated by the formation
of a reddish-brown precipitate after adding
Dragendorff's reagent or a white/creamy yellow
precipitate after adding Mayer's reagent. (3) Saponin:
0.5 g of extract is shaken using 2 mL of distilled water.
The foam formed is observed. The presence of saponin
is indicated by the formation of foam which can last for
10 minutes. (4) Triterpenoids and Steroids: To a solution
of 0.1 g of extract in 1 mL of chloroform, 1 mL of acetic
anhydride was added, followed by 2 drops of
concentrated H2SO4 added slowly, then the color
change was observed. The presence of triterpenoids is
indicated by a color change from purple to pink to red,
while the presence of steroids is indicated by a color
change from purple to blue or green. (5) Tannin: 0.1 g
of extract is dissolved in 1 ml of distilled water, then the
mixture is stirred and filtered. 10% FeCl solution was
added 3 drops into the filtrate. The formation of a blueblack precipitate indicates the presence of tannins.
Antioxidant Capacity Test
The antioxidant capacity test for each extract and
fraction was performed using three methods: 2,2diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging
Activity, Cupric Ion Reducing Antioxidant Capacity
(CUPRAC), and Ferric Ion Reducing Antioxidant Power
(FRAP). Radical scavenging activity was measured with
the DPPH method refer to Rafi et al. (2018). Each extract
sample is dissolved in ethanol, then 40 µL of the sample
solution is added to 250 µL of 125 µmol/L DPPH
solution in a 96-well plate. Ascorbic acid solutions were
prepared with six different concentrations as positive
controls. The ethanol was prepared as a negative control
(blank). Then, 40 µL of each control is added to 250 µL
of 125 µmol/L DPPH solution in a 96-well plate. Each
sample and control was prepared in triplicate. The
mixture was incubated for 30 minutes in the dark at
room temperature, then the absorbance was measured
at a wavelength of 517 nm using a microplate reader.
The radical scavenging activity was determined using the
following equation: y = 2.6151x – 0.0097, with R2 =
0.9979, and it is expressed in mmol AAE/g sample.
The CUPRAC method of antioxidant capacity test
refers to Badrunanto et al. (2024). Each 40 µL sample
solution was added with 50 µL of 10 mM CuCl2 solution,
50 µL of 7.5 mM neucuproin solution, and 60 µL of 1 M
NH4CH3COO solution (pH 7) into a 96-well plate. The
mixture was incubated for 1 hour at room temperature

and the absorbance was measured at a wavelength of
450 nm using a microplate reader. Ascorbic acid
solutions were prepared with six different concentrations
as positive controls. The antioxidant capacity was
determined using the following equation: y = 1.2808x
+ 0.0012, with R2 = 0.9971. Each sample and control
was prepared in triplicate. Antioxidant capacity is
expressed in mmol ascorbic acid equivalent (AAE)/g
sample.
The FRAP method refers to Rafi et al. (2018), and the
FRAP reagent was prepared following the procedure of
Benzie and Devaki (2017). 30 µL of sample solution is
taken, then 270 µL of FRAP reagent is added to a 96well plate. The mixture was incubated for 30 minutes at
room temperature. Absorbance was measured at a
wavelength of 593 nm using a microplate reader.
Ascorbic acid solutions were prepared with six different
concentrations as positive controls. The antioxidant
capacity was determined using the following equation: y
= 1.7419x – 0.0448, with R2 = 0.9979. Each sample
and control was prepared in triplicate. Antioxidant
power is expressed in mmol AAE/g sample.
Analysis of Metabolite Profiles using LC-MS/MS
LC-MS/MS analysis for metabolite identification
refers to the method of Farag et al. (2016) with mobile
phase modification. The LC-MS/MS used was a
Vanquish
Flex
ultra-high-performance
liquid
chromatography (UHPLC) system coupled to an
Orbitrap high-resolution mass spectrometer (HRMS) (Q
Exactive Plus LC-MS/MS System, Thermo Scientific,
Germany). LC-MS/MS analysis was performed at The
Advanced Research Laboratory of IPB. The column used
was Accucore C18 100 × 2.1 mm, 1.5 µm
(ThermoScientific) with a temperature of 30 °C. The
mass spectrometer was operated with an electrospray
ionization (ESI) source with a Q-Orbitrap mass analyzer.
The scanning range was 100–1500 m/z and the
ionization mode used was negative ion. The sample was
separated using 2 mobile phases, namely 0.1% formic
acid in water (A) and 0.1% formic acid in acetonitrile (B),
with a flow rate of 0.2 mL/minute and an injection
volume of 2 µL. The elution system used is a gradient
with a mobile phase composition of 0–1 minute (5% B),
1–25 minutes (5–95% B), 25–28 minutes (95% B), 28–
33 minutes (5% B). The resulting data was processed
using Compound Discoverer 3.2 software (Thermo
Scientific, Germany) with an online database and an inhouse database. Next, MS-MS was confirmed to predict
the compounds contained in the sample.
Data Analysis
The research data were analyzed descriptively and
presented in the form of pictures, graphs, and tables.
Results are expressed as the mean ± standard deviation
of three replicates. Statistical significance was

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determined using analysis of variance (ANOVA)
followed by Tukey's test where applicable, using Minitab
software version 20 (Minitab, LLC, New York). Analysis
of significant differences was carried out on the yield and
activity of each sample using a confidence level of 95%
(p < 0.05).
RESULTS AND DISCUSSION
Water Content and Yield
The four samples have a water content of <10%,
meaning the samples are in good condition and can be
stored for a long time (BPOM, 2019). The variance test
showed significantly different results (p < 0.05), and the
follow-up test (Tukey) also showed significantly different
perilla samples, with the highest water content. The
water content in a sample is influenced by several
factors, including drying factors, temperature, size, and
thickness of the sample (Gómez-de la Cruz et al., 2015;
Morakinyo & Taiwo, 2016).
The extraction yield of the four samples ranged from
5.98 ± 0.54 to 20.17 ± 1.44%. Analysis of variance
showed that the yields of these extracts were significantly
different with p < 0.05, with the results of further tests
shown in Table 1. The highest yield was possessed by
selasih extract with hot water, namely 20.17 ± 1.44%,
which shows that the metabolites in selasih plants are
more extracted with hot water. Compared with other
extracts, the yield of selasih extract was the highest in
both solvents (hot water and 50% ethanol) (Table 1),
meaning that selasih contained more compounds that
had the same polarity as the two solvents. In addition,
the yield of lenglengan, perilla, and kemangi extracts
with 50% ethanol was higher than with hot water (Table
1). This shows that the three samples contained more
compounds with the same polarity as 50% ethanol. The
four samples were extracted using the autoclave method
because this method has been reported to produce more
extract, increase the bioactivity of compounds, and save
time and solvents, making it more efficient (Kim et al.,
2016; Ko et al., 2015).
The kemangi 50% ethanol extract was further
separated using the liquid-liquid extraction (partition)
method with three solvents of different polarity, namely
n-hexane (nonpolar), ethyl acetate (semipolar), and
50% ethanol (polar), respectively. Partition is carried
out to obtain more specific compounds based on
their polarity. The yield of the fractions obtained is shown
in Table 1, expressed as a percentage of the weight of
the fraction obtained per weight of extract used. The
highest yield was obtained by the 50% ethanol fraction,
which shows that the metabolites in the kemangi 50%
ethanol extract are mostly polar in nature, so they
are more distributed in t he 50% ethanol solvent.
These results are by Vinnata et al. (2018) who also
partitioned the ethanol extract of kemangi using three

solvents (n-hexane, ethyl acetate, and ethanol-water)
and obtained the highest yield in the ethanol-water
fraction (41.33%).
Phytochemical Content
Extracts were tested for phytochemistry as an initial
qualitative screening of the compound content in each
extract. The test results in Table 2 show differences in the
phytochemical content in the extracts of the four plants
with the two solvents. Flavonoid compounds were
contained in each extract, while steroid compounds were
only positive in three extracts (lenglengan, perilla, and
selasih with 50% ethanol). This is because most of the
flavonoid group has the same polarity as the solvent
used, that is, it is polar, while the steroid group is mostly
nonpolar, so it is less extracted. Previous studies have
shown different results with tables such as Ali et al.
(2022) did not showing triterpenoid content in water and
ethanol-water extracts of kemangi. Islam et al. (2017)
also showed different results in methanol extracts of
lenglengan from Bangladesh containing steroid,
alkaloid, tannin, and flavonoid compounds. Nadeem et
al. (2022) also showed different results in water extracts
of selasih containing alkaloids, flavonoids, steroids,
tannins, and terpenoids, while the ethanol extract
contained flavonoids, steroids, saponins, and tannins.
Differences in compound content screening results with
literature can be caused by several factors such as
differences in environmental (water, drought, salinity,
temperature, radiation, chemical, seasonal variation,
and region/location) (Verma & Shukla, 2015), plant
parts, extraction methods, and extraction solvents
(Sayuti, 2017).
The partition results of the 50% ethanol extract of
kemangi were also tested for phytochemistry. The results
(Table 3) are different from the research results of Ali et
al. (2022) which showed that the ethyl acetate fraction
resulting from the partition of the ethanol-water extract
of kemangi positively contained flavonoids, saponins,
tannins, terpenoids, and phytosterols. Based on Table 3,
the 50% ethanol fraction contains the largest group of
compounds; These results are in line with the results
yield.
Antioxidant Capacity
The antioxidant test on each extract and fraction is
intended to determine the potential of each as an
antioxidant by looking at its antioxidant capacity. The
antioxidant capacity is expressed as comparable or
equivalent to the antioxidant capacity of ascorbic
acid in the form of mmol of ascorbic acid equivalents
(AAE) per g of extract or fraction. So the higher the
antioxidant capacity value, the higher its potential as
an antioxidant. The results of the antioxidant test for
each extract using three methods (DPPH, CUPRAC,
and FRAP) are shown in Figure 1.

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Table 1. Yield of extracts and fractions
Extracts
Yield (%)
Lenglengan hot water extract
5.98 ± 0.54e
Lenglengan 50% ethanol extract
13.30 ± 0.70d
Kemangi hot water extract
14.32 ± 0.83cd
Kemangi 50% ethanol extract
16.7 ± 0.29bc
Perilla hot water extract
16.06 ± 1.67bc
Perilla 50% ethanol extract
17.48 ± 0.88ab
Selasih hot water extract
20.17 ± 1.44a
Selasih 50% ethanol extract
18.57 ± 0.27ab
Fractions
Yield (%)
N-hexane fraction
4.39
Ethyl acetate fraction
12.48
50% Ethanol fraction
76.92
Note: Different letters indicate significantly different results (p<0.05) in the Tukey test.
Table 2. Phytochemical content of extracts
LA
LE
KA
KE
PA
PE
SA
SE
Flavonoid
+
+
+
+
+
+
+
+
Saponin
+
+
+
+
+
+
+
Tannin
+
+
+
+
+
+
+
Alkaloid
+
+
+
+
+
(Dragendorff)
Alkaloid (Meyer)
+
+
+
+
+
+
Triterpenoid
+
+
+
+
+
+
+
+
Steroid
+
+
+
Note: + (detected), - (not detected LA (lenglengan hot water extract), LE (lenglengan 50% ethanol
extract), KA (kemangi hot water extract), KE (kemangi 50% ethanol extract), PA (perilla hot water
extract), PE (perilla 50% ethanol extract), SA (selasih hot water extract), SE (selasih 50% ethanol
extract).
Table 3. Phytochemical content of the partitioned fractions
Ethyl acetate
fraction
+
+
+
-

n-hexane fraction
Flavonoid
Saponin
Tannin
Alkaloid (Dragendorff)
Alkaloid (Meyer)
Triterpenoid
+
Steroid
+
Note: + (detected), - (not detected)
The highest average antioxidant capacity for the
DPPH and CUPRAC methods is owned by kemangi
extract with 50% ethanol solvent, while the FRAP method
is owned by selasih 50% ethanol extract, but based on
the results of the Tukey test, it was not significantly
different (p > 0.05) with 50% ethanol extract of
kemangi. Therefore, kemangi extract with 50% ethanol
was concluded to have the highest antioxidant capacity.

50% Ethanol fraction
+
+
+
+
+
-

Tests with several methods (DPPH, CUPRAC, and
FRAP) are used to determine the antioxidant ability of
samples in each method based on the mechanism,
hydrogen atom transfer (HAT) or single electron transfer
(SET) (Munteanu and Apetrei, 2021). The DPPH method
has a HAT and SET mechanism for hydrophilic
antioxidants, the CUPRAC method has a SET mechanism
for lipophilic and hydrophilic antioxidants under
neutral

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conditions, and the FRAP has a SET mechanism for
hydrophilic antioxidants under acidic conditions
(Munteanu and Apetrei, 2021; Shahidi and Zhong,
2015). Based on the mechanism of action of
antioxidants according to Munteanu and Apetrei (2021),
these results show that the 50% ethanol extract of
kemangi contains more lipophilic and hydrophilic
antioxidant compounds which play a synergistic role
than other extracts, with HAT and SET mechanisms.
Figure 1 also shows that the average antioxidant
capacity of the 50% ethanol extract is generally higher
than that of the hot water extract. The results of the
variance test showed that the antioxidant capacity of the
samples using the DPPH method was not significantly
different (p > 0.05), meaning that the antioxidants
contained in the samples played a less active role in the
HAT mechanism in neutralizing DPPH radicals.
Antioxidant tests using three methods (DPPH,
CUPRAC, and FRAP) were also carried out on the
fractions and the results are shown in Figure 2. The
ethyl acetate fraction has the highest antioxidant
capacity in the three methods, meaning that the
fraction contains more compounds that play an active
and synergistic role as antioxidants, in inactivating
reactive species by either SET or HAT mechanisms.
Dibala et al. (2016) who fractionated the ethanol-water
extract of kemangi also showed the highest antioxidant
activity of the FRAP method in the ethyl acetate fraction
compared to the ether, dichloromethane, and n-butanol
fractions.
The antioxidant capacity of the ethyl acetate fraction
(Figure 2) was higher than the kemangi 50% ethanol

extract (Figure 1). Based on these results, further
separation to obtain more specific and pure compounds
such as partitioning can increase the antioxidant
capacity. This can be caused by the mixture of
compounds in the extract having a greater antagonistic
effect than the synergistic effect on antioxidant capacity.
The synergistic effect means that the combined effect of
bioactive compounds is higher than the sum of the
effects of each compound. On the other hand, the
antagonistic effect means that the combined effect of
bioactive compounds is lower than the additive effect
(Chen et al., 2022).
The antioxidants in the DPPH method reduce DPPH
radicals (purple) to DPPH-H (yellow). Absorption was
measured at 517 nm which is the maximum absorption
of DPPH radicals (Gulcin, 2020). Therefore, the higher
the antioxidant activity in the sample, the fainter the
purple color, so the measured absorbance is lower. In
the CUPRAC method, the light blue Cu2+-neocuproin
complex is reduced to a yellow-orange Cu+neocuproine complex with a maximum absorption peak
at 450 nm (Gulcin, 2020). The higher the antioxidant
activity of the sample, the more orange the final color,
and the higher the measured absorbance. Meanwhile,
In the FRAP method, antioxidants reduce the colorless
Fe3+-TPTZ complex to a dark blue Fe2+-TPTZ complex
with a maximum absorption peak at 593 nm. In contrast
to other methods, the FRAP test is carried out under
acidic conditions (pH = 3.6) to maintain iron solubility
(Munteanu and Apetrei, 2021). If the antioxidant activity
of the sample is higher, the color is bluer, and the
measured absorbance is higher.

Figure 1. Average antioxidant capacity based on test methods (DPPH, CUPRAC, FRAP) and solvents from
four plants (Note: different letters indicate significantly different results (p < 0.05) in the Tukey test)
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Vivi Septya Wati, et al.

Figure 2. Average antioxidant capacity of the partitioned fraction of kemangi 50% ethanol
extract based on the DPPH, CUPRAC, and FRAP test methods (Note: different letters indicate
significantly different results (p < 0.05) in the Tukey test)
Metabolite Profile
The kemangi 50% ethanol extract and ethyl acetate
fraction as the highest antioxidant capacity were
analyzed for the metabolite profile using LC-MS/MS. The
chromatogram of the analysis results is displayed in the
form of a base peak in negative ionization mode (Figure
3). Based on Figure 3, the differences in each sample
can be seen, some showing similar peaks with different
intensities. The similarity of the chromatogram peaks of
each sample indicates that there are similarities in the
metabolites contained. The difference in intensity
indicates a difference in the concentration of metabolites
in the sample.
A total of 18 compounds in Table 4 were successfully
identified putatively based on confirmation of precursor

ion values, best match values, references, and MS-MS
fragmentation patterns from the library (ChemSpider
and mzCloud). These metabolites consist of 9
flavonoids, 1 terpenoid, 1 amino acid, 2 phenolic acids,
3 fatty acids, and 2 other carboxylic acids. Several
previous studies also reported the presence of these
metabolites in kemangi extract. Zengin et al. (2019)
carried out LC-MS/MS analysis on ethyl acetate,
methanol, and water extracts from kemangi leaves and
flowers and reported the presence of flavonoids,
carboxylic acids, fatty acids, and other phenolic
metabolites. Karau et al. (2015) also confirmed the
presence of flavonoids, terpenoids, alkaloids,
phytosterols, fatty acids, and phenolic metabolites in
kemangi ethyl acetate extract.

Figure 3. Base peak chromatogram in negative ionization mode of the kemangi 50% ethanol extract
and the ethyl acetate fraction.
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Table 4. Putatively identified metabolites using LC-MS/MS
RT
[min]

Error
[ppm]

Molecular
weight

C27H30O15 9.097

-0.32

594.15828

C21H20O11 9.424

-0.46

Quercetin-3β-DC21H20O12 8.806
glucoside

-0.34

Rutin

C27H30O16 8.477

-0.38

Trifolin

C21H20O11 9.164

-0.46

Apigenin

C15H10O5

12.70
8

-1.64

Glycitein

C16H12O5

15.86
1

-0.78

Luteolin

C15H10O6

Name
Flavonoids
Kaemferol-3rutinoside
Astragalin

Formula

Skrofulein/Cirsim
C17H14O6
aritin
Terpenoid
Asiatic acid

C30H48O5

11.52
8
14.47
7
17.13
2

-0.94
-1.31

MS2

Reference

68, 245, 255, 285, Čulina et
593
al. 2021
67, 227, 255, 285, Abdelaty et
448.10036
447
al. 2021
Farag et al.
464.09532 67, 151, 463
2016
Beltrán151, 179, 255,
610.15315
Noboa et
300, 609
al. 2022
67, 227, 255, 284, Database
448.10036
447
MzCloud
Beltrán68, 117, 151, 225,
270.05238
Noboa et
269
al. 2022
MartínezCruz dan
284.06825 68, 268, 283
ParedesLópez 2014
Zengin et
286.04747 153, 241, 285
al. 2019
67, 133, 151, 161, Dharsono
314.07863
313
et al. 2022

-1.13

488.34962 69, 296, 407, 487

Velamuri et
al. 2020

-1.72

246.10002 74, 203, 245

Database
MzCloud

KE

FEA
























Amino Acid
2-(acetylamino)C13H14N2O
3-(1H-indol-39.084
3
yl)propanoic acid
Phenolic Acids
Caffeic acid

C9H8O4

6.929

-4.71

180.04141 135, 179

BeltránNoboa et
al. 2022

(R)-(+)rosmarinic acid

C18H16O8 9.981

-1.44

360.08400 59, 133, 161, 359

BeltránNoboa et
al. 2022







Fatty Acids
(15Z)-9,12,13Trihydroxy-15octadecenoic
acid

C18H34O5

13.10
5

-1.14

Azelaic acid

C9H16O4

9.717

-4.92

188.10393 57, 125, 143, 187

Database
MzCloud

-0.42

272.23503 68, 225, 271

Database
MzCloud

1624.19
Hydroxyhexadeca C16H32O3
7
noic acid
Other Carboxylic
Acids

330.24025

286

68, 171, 329

Database
MzCloud








Selection of Four Lamiaceae Species
Indole-3-acetic
acid

Vivi Septya Wati, et al.

Database

MzCloud
Shanaida et

Citric acid
C6H8O7
1.454
-3.87 192.02626 87, 111, 191
al. 2017
Note: RT (retention time), error (molecular weight tolerance), MS2 (MS-MS fragmentation), KE (kemangi 50%
ethanol extract), FEA (ethyl acetate fraction)
C10H9NO2 8.545

-4.83

175.06248 67, 130, 174

The most abundant metabolite profile in Table 4 is
the flavonoid group. This is possible because flavonoids
are one of the main groups of natural plant products
with more than 9000 known structures (Pilon et al.,
2019). Flavonoids have many bioactivities, one of the
best known being an antioxidant. The profile is
dominated by the flavonoid group, so the metabolites
that play an active role in increasing antioxidant capacity
are thought to be the flavonoid group. The group of
flavonoids that have been detected and have been
proven to have antioxidant activity includes astragalin
(Choi et al., 2013), rutin (Choi et al., 2021), trifolin,
quercetin-3β-D-glucoside (Zhang et al., 2014),
kaemferol-3-rutinoside (Chen et al., 2013), apigenin,
and luteolin (Tian et al., 2021). In addition, identified
phenolic acid compounds such as rosmarinic acid
(Elansary et al., 2020) as well as fatty acids such as
azelaic acid (Jones, 2009) have also been shown to
have antioxidant activity. The combination of these
compounds is thought to produce a higher synergistic
effect on antioxidant activity compared to their
antagonists. Previous research by Zengin et al. (2019)
also showed the presence of flavonoid compounds in
kemangi leaf and flower extracts such as visenin-2,
eriodictyol-7-O-glucoside, viteksin, luteolin, luteolin-7O-glucoside,
isovitexin,
quercetin-O-glucuronide,
isoquercitrin, kosmosin, eriodictyol, quercetin, sirsiliol,
apigenin, pilosin, sirsimaritin, sirsilineol, xantomirol,
nevadensin, genkwanin, salvigenin, and gardenin B.
CONCLUSIONS
The highest antioxidant capacity was possessed by
kemangi plants (O. americanum L.) with 50% ethanol
solvent with a value of 0.439 (DPPH); 1.517 (CUPRAC);
and 1.021 (FRAP) mmol AAE/g extract. Further
separation showed that the ethyl acetate fraction had the
highest antioxidant capacity in all three methods with a
value of 1.109 (DPPH); 1.540 (CUPRAC); and 1551
(FRAP) mmol AAE/g fraction. Metabolite analysis using
LC-MS/MS succeeded in identifying 18 metabolites
consisting of flavonoids, terpenoids, amino acids,
phenolic acids, fatty acids, and other carboxylic acids.
ACKNOWLEDGMENTS
A collaboration between Delightex Pte Ltd and IPB
University supports this work. The author is grateful to
Delightex Pte Ltd and IPB University for their financial
support in this research and publication.

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