Molekul, Vol. 20. No. 2, July 2025: 330 – 338

Articles

MOLEKUL
eISSN: 2503-0310

https://doi.org/10.20884/1.jm.2025.20.2.13792

Antioxidant, Anti-Aging and Antibacterial Activity from Dewa Leaves Ethanolic Extract
(Gynura japonica (Thunb.) Juel)
Umi Fatmawati1*, Anggie Meilinda Zienitha1, Meisi Anggraini1, Lisa Rosita1, Meti Indrowati1, Harlita1,
Slamet Santosa1, Vera Permatasari2, Gian Primahana2, Indri Yati3, Muhammad Eka Prastya2
Biology Education Study Program Faculty of Teacher Training and Education
Universitas Sebelas Maret, Surakarta 57126, Indonesia
2
Research Center for Pharmaceutical Ingredients and Traditional Medicine, National Research, and Innovation
Agency (BRIN), Kawasan Sains dan Teknologi (KST) South Tangerang, Banten 15314, Indonesia
3
Research Center for Chemistry, National Research, and Innovation Agency (BRIN), Kawasan Sains dan
Teknologi (KST) B.J Habibie (PUSPIPTEK) Serpong, South Tangerang, Banten 15314, Indonesia
1

*Corresponding author email: umifatmawati@staff.uns.ac.id
Received December 07, 2024; Accepted June 23, 2025; Available online July 20, 2025

ABSTRACT. Due to their phytochemical constituents, Dewa leaves ( Gynura japonica (Thunb.) Juel.) are often used in
traditional medicinal herbs. However, in vitro and in vivo of antioxidative and anti-aging studies of Dewa leaves on yeast
as a eukaryotic cell model have not been widely carried out. This study aims to determine the antioxidant, anti-aging, and
antibacterial activities derived from G. japonica leaves extract. Extraction was conducted using 70% and 96% ethanol
solvents, total phenolic content (TPC) was assayed using Folin-Ciocalteu method, flavonoid contents (TFC) was assayed
using aluminum chloride method, antioxidant activity was tested using DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS
(2,2-azinobis-3-Ethylbenzothiazoline)-6-sulfonic acid) radicals. Subsequently, an anti-aging activity test was performed on
the yeast Saccharomyces cerevisiae as a model organism following antibacterial activity. The antibacterial activity test was
carried out using well diffusion agar, and the Minimal Inhibitory Concentration (MIC) was determined using the microplate
method. We obtained that G. japonica leaves extracted from 70% and 96% methanol solvents, have TPC of 13.14 and
22.11 mg GAE/gr extract, and TFC of 8.04 and 14.09 mg QE/gr extract, respectively. As for DPPH and ABTS antioxidant
activity, D70 showed the best activity with IC50 values of 1411.36±56.35 µg/mL and 2516.10±18.77 µg/mL,
respectively. The anti-aging test showed that both 70% and 96% ethanol extracts were able to maintain the yeast cell
viability under H2O2 oxidative stress. Further, 70% and 96% ethanol extract also showed antibacterial activity at the best
value against Staphylococcus aureus with a MIC value of 390.62 µg/mL, it means that Gynura leaves extract has a
potency as antibacterial agent. The results of this study indicate that the ethanol extract of G. japonica leaves can be
developed for further investigation as an antioxidant and antibacterial therapeutic agent.
Keywords: Anti-aging, antibacterial, antioxidant, Gynura japonica, Saccharomyces cerevisiae

INTRODUCTION
The use of native Indonesian plants as herbal
preparations that are rich in benefits has started to
become a trend and has continued to increase for
several decades. One such plant is Daun Dewa
(Gynura japonica (Thunb.) Juel) and also known as
Gynura segetum. This plant is one of the Indonesian
medicinal plants which has long been used for
generations to treat various diseases such as cancer,
fever (antipyretic), diabetes, high blood pressure,
and skin diseases (external medicine) (Bari et al.,
2021; Meng et al., 2021; Tan et al., 2016). To
improve the quality, safety, and benefits of G.
japonica plant as Indonesian natural medicine, it is
necessary to standardize the raw materials, both in
the form of simplicial and in the form of extracts or
galenic preparations.

Pharmacological studies show that G. japonica
leaves are proven to have various biological activities
that strengthen the scientific basis for their use as
traditional medicine. G. japonica leaves extract
showed a significantly greater anti-angiogenic effect
in inhibiting the growth and metastasis of tumor
cells (Seow et al., 2011). This extract has also
been shown in experimental animals to reduce blood
cholesterol levels, lower uric acid levels, and be antiinflammatory (Nazri et al., 2019; Tan et al., 2020;
Seow et al., 2014). In vitro tests on G. japonica leaf
extract have been shown to contain phenolic
compounds that can inhibit DPPH free radicals
(Hsieh et al., 2020). This evidence reinforces the
notion that many benefits of G. japonica leaves in
medicine are due to their chemical content which is
efficacious as an antioxidant. The content of phenolic
330

Antioxidant, anti-aging and antibacterial activity
compounds and the antioxidant power of G.
japonica leaves are influenced by some conditions
including the drying and extraction method, and the
solvent used in the extraction process (Rivai et al.,
2012). In addition, G. japonica leaves did not cause
congenital abnormalities or defects in the
organogenesis phase of the experimental rats. Based
on the results of this research, Gynura leaves are
safe to use as medicine during pregnancy
(Kamaruzaman & Mat Noor, 2017).
Of note, G. japonica leaves are reported to
contain some phytochemicals compounds such as
alkaloids, saponins, flavonoids, essential oils, and
tannins (Bari et al., 2021). Interestingly, essential oils
have antibacterial and antifungal properties. Tannins
are ingredients found in medicinal plants and have a
physiological action in inhibiting bacteria. On the
other hand, flavonoids are a class of phenolic
compounds which have bactericidal and fungicidal
activity. G. japonica leaf extract effective in inhibiting
the growth of Staphylococcus aureus, Bacillus

subtilis, Enterobacter aerogenes, Pseudomonas
aeruginosa, Escherichia coli, Proteus mirabili, and
Candida albicans (Seow et al., 2012; Nasiruddin &

Sinha, 2020).
Moreover, cellular manifestations of the aging
process are also influenced by the Reactive
Oxygen Species (ROS) factor produced in cells.
Suppose there is an imbalance between oxidants
and antioxidants in the body due to an increase
in ROS and a decrease in antioxidants from the
body. In that case, it causes cell damage and
affects aging (Prastya et al., 2020). Ethanolic
extract of G. japonica
leaves exhibited strong
antioxidant
activity, because of rich flavonoid
content (Kaewseejan et al., 2015). Antioxidants
from synthetic materials provide side effects that
are quite harmful to health, especially causing
cancer. Therefore, natural sources of antioxidants
that are safer to develop are sought. Chemical
compounds belonging to the group of antioxidants
and can be found in plants, among others, come
from the group of polyphenols, bioflavonoids,
vitamin C, vitamin E, beta carotene, catechins, and
resveratrol (Xu & Zhang, 2017). The results of
previous research have identified the content of
antioxidant compounds in Gynura plants and carried
out in vitro tests (Kim et al., 2021). However, this
research will also test the effect of administering
Gynura leaf extract on the model organism
Saccharomyces cerevisiae, where this organism has
a known genome map, has an mRNA splicing
process that is similar to the metazoan group, and
has a trait inheritance mechanism that is similar to
mammalian cells (Vanderwaeren et al., 2022).
Therefore, it is hoped that the results of this research
can be the basis for developing therapeutic agents
for G. japonica leaves as antioxidants, antiaging
and antibacterial.

Umi Fatmawati, et al.
EXPERIMENTAL SECTION
Material
The materials used in this study were samples of G.
japonica leaf plants, ethanol, Folin-Ciocalteu
reagent, gallic acid, quercetin standard, ammonium
chloride, AlCl3, 2,2-diphenyl-1-picrylhydrazyl (DPPH),
trolox,
ascobic
acid,
2,2-azinobis
(3ethylbenzothiazoline-6-sulfonic
acid)
(ABTS),
potassium persulfate powder (K2S2O8), EAST model
Saccharomyces cerevisiae (InaCC B612), YPD
medium (10 g L-1 yeast extract, 20 g L-1 peptone, 20
g L-1 glucose, and 1 L distilled water). Bacterial
culture for antibiotic assay (Staphylococcus aureus,
Escherichia coli, Pseudomonas aeruginosa, and
Bacillus subtilis).
Plant Extraction
The plant used in this research is Daun Dewa
(Gynura japonica (Thunb.) Juel) taken from Solo
District (GPS location: 7°32’55.9”S 110°50’51.2”E).
The leaves are dried in the oven and then crushed
into powder and then weighed. The sample was then
macerated with ethanol as the solvent. Maceration
was carried out by immersing the sample in a 70%
ethanol solution with a ratio of 1: 10 and 96%
ethanol with a ratio of 1: 5, stored in a dark place at
room temperature for 2-5 days without stirring. The
sample is then separated by filtering and separated
from the solvent by evaporation using a rotary
evaporator at a temperature of 50-60 °C at a speed
of 50-90 rpm to obtain a concentrated extract (Putri
et al., 2024).
The sample extract solution with a test
concentration of 10.000 ppm was prepared by
weighing 10 mg of the sample and dissolved in 1
mL of 96% ethanol then dissolved until
homogeneous. The solution is put in a tube which is
then stored at 4 oC to avoid evaporation. The
positive controls for antioxidant activity were Ascorbic
Acid (AA) and Trolox (Tr) made in a stock
concentration of 1000 µg/mL by weighing 1 mg AA
and Tr and dissolved in 1 mL of 96% ethanol then
dissolved until homogeneous. The solution is put in a
tube covered with aluminum foil and stored at 4 °C
to prevent evaporation (Astuti et al., 2021).
Determination of Total Phenolic Content (TPC)
The total phenol test was carried out using the
standard Folin-Ciocalteu Reagent (FCR) method (Abo
El-Maati et al., 2012)]. 500 µL concentration of
1000 µg/mL sample mixed with 3.5 mL of distilled
water and 250 µL of FCR reagent. The mixture was
homogenized and allowed to stand for 8 minutes.
The solution was then added with 750 µL of 20%
sodium carbonate. The mixture was homogenized
again and then incubated for 2 hours at room
temperature in the dark. Sample absorbance was
measured at a wavelength of 765 nm using an ELISA
reader (Thermoscientific Varioskan-Flash). Gallic
acid stock concentration of 1000 µg/mL was used as
a positive control. Gallic acid standard curves were
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Molekul, Vol. 20. No. 2, July 2025: 330 – 338
made in concentrations of 5, 10, 15, 20, and 25
µg/mL. Total phenol was calculated based on the
equivalent number of mg gallic acid (EAG)/g extract.
Determination of Total Flavonoid Content (TFC)
The total flavonoid test was carried out by the
aluminum chloride method using the quercetin
standard. Samples with a concentration of 1000
µg/mL were used from a stock sample dilution of
10.000 µg/mL and dissolved in 96% ethanol. 500 µL
of sample solution was mixed with 2.45 mL of
distilled water and 150 µL of 5% NaNO2. The
mixture was homogenized and allowed to stand for
2 minutes. The solution was then added with 150 µL
AlCl3 and incubated for 8 minutes. Then 2 mL of
NaOH 1 M was added to the solution. Sample
absorbance was measured at a wavelength of 415
nm using an ELISA reader (Thermoscientific
Varioskan-Flash). Quercetin stock concentration of
1000 µg/mL was used as a positive control.
Quercetin standard curves were made in
concentrations of 10, 20, 30, 50 and 70 µg/mL.
Total phenol was calculated based on the equivalent
number of mg quercetin/g extract (Prastya et al.,
2019).

wavelength of 734 nm. Measurements were carried
out 2 times with Trolox and Ascorbic Acid as positive
controls and treated the same as the samples. The
negative control was 50 µL ethanol added with 150
µL ABTS and the blank was 200 µL ethanol.
Inhibition value of both DPPH and ABTS method
following formula:

Note:
AC = Absorbance Control
AS = Absorbance Sample
The percentage inhibition value is represented by
the IC50 value calculated by the formula above. The
inhibition value was regressed in the linear
regression equation to obtain the 50% Inhibitory
Capacity (IC50) antioxidant capacity of each sample.

Antioxidant Activity Assays with 2,2-Diphenyl-1picrylhydrazyl
(DPPH)
and
2,2-azinobis
(3ethylbenzothiazoline-6-sulfonic acid) (ABTS) Radicals
Both DPPH and ABTS radical’s antioxidant
activity were performed following methods as
described elsewhere (Prastya et al., 2018). For DPPH
activity, several sample solutions were made with the
highest concentration of 10,000 µg/mL then diluted
8 times. Samples were pipetted 100 µL for 3
repetitions and 100 µL 96% ethanol was added to
each well. 100 µL of 2.5 µM DPPH solution was
added to the 96-well plate. The mixture was
incubated for 30 minutes at 37 °C in the dark, then
the absorbance was measured with ELISA microplate
reader at a wavelength of 515 nm. Measurements
were carried out 2 times with Trolox and Ascorbic
Acid as positive controls and treated the same as the
samples. The negative control was 100 µL ethanol
added with 100 µL DPPH and the blank was 200 µL
ethanol. As for ABTS methods, a total of 19.2 mg of
ABTS powder and 3.3 mg of Potassium Persulfate
powder (K2S2O8) were dissolved in 5 mL of ethanol
each. The two solutions were mixed in a 1:1 ratio
and then incubated for 12-16 hours at room
temperature in the dark. ABTS solution was diluted
with 96% ethanol to obtain an absorbance value of
0.6 – 0.7 at a wavelength of 734 nm. Several
sample solutions were made with the highest
concentration of 10.000 ppm and then diluted 8
times. Samples were pipetted 50 µL for 3 repetitions
and 50 µL 96% ethanol was added to each well.
150 µL of ABTS solution was added to the 96-well
plate. The mixture was incubated for 30 minutes at
37 °C in the dark, then the absorbance was
measured with an ELISA microplate reader at a

Anti-aging Activity Assays with Streak Method
Anti-aging activity was conducted following
method as described (Astuti et al., 2021). Briefly,
100,000 µg/mL sample extract solution dissolved in
10% DMSO, and then filtered using a Millipore
membrane 0.22 µm (Sartorius). Inoculation of 375
µL (OD600 0f 0.05) Saccharomyces cerevisiae (InaCC
B612) was carried out in 3 mL of liquid YPD medium
supplemented with 200 uL of 2% glucose sterile
solution and added 30 μL of plant extract with a
concentration of 500 ppm.
The test tubes were
tightly closed and then incubated for 24 hours in a
shaker at 200 rpm at room temperature (30 °C).
Specifically for the control (-) only glucose and yeast
were added, while the DMSO control was replaced
with 10% DMSO of 30 µL. After that the tubes were
incubated for 4 days (96 hours) in a shaker at 200
rpm at room temperature (30 °C). The media was
poured into a petri dish and waited for it to solidify.
Subsequently, streak the results of the inoculum on
the test tube, onto YPD media containing 6 mM
H2O2, 3 mM H2O2 and media without H2O2. After
etching, the petri dishes were tightly closed and then
incubated for 2 days (48 hours) at room
temperature. The lines were observed for yeast
growth and documented. The results showed that
yeast only grew on solid YPD media treated with 6
mM H2O2. Samples showing anti-aging activity were
observed.
Determination of Antibacterial Activity Using
Minimum Inhibitory Concentration (MIC) and
Minimum Bacterial Concentration (MBC) Method
The initial antibacterial activity test conducted
with an antibacterial screening on Gynura leaf
ethanolic extract (70% and 96%) using the well
diffusion agar method. The test bacteria used
included E. coli, P. aeruginosa, S. aureus, and B.
subtilis, Each of bacteria was grown on Mueller
Hinton Agar medium. Subsequently, the test bacteria
were adjusted to a 0.5 McFarland standard
(equivalent to 1.5 x 108 CFU mL-1) and then

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Antioxidant, anti-aging and antibacterial activity
inoculated. Using a micropipette, the test bacteria
were extracted and then used the spread plate
technique (spread method) to inoculate onto solid
media. There was a waiting interval following
inoculation to give the bacteria time to absorb into
the solid media. To make it easier to insert the test
samples, the negative control (DMSO solution in
distilled
water),
and
the
positive
control
(chloramphenicol antibiotics), holes were made in
the solid media. After that, the test medium's
bacterial culture was concentrated for 24 hours at
37°C in an incubator. The last phase involved
measuring and observing the bacterial inhibition
zone, which was shown by the clear zone, carried
out in triplicate.
The stock sample solution was prepared in
100,000 µg/mL at 10% DMSO. The control used
was 2000 ppm tetracycline. Bacterial inoculation was
carried out using MHB liquid medium. The MIC test
was carried out using the dilution method. Beginning
by filling all wells with 100 μL MHB media. Then
added with 100 μL of two-fold diluted sample with
two repetitions as well for the control. The extracted
sample was replaced with 2000 ppm tetracycline
and 10% DMSO 100 μL, then diluted. Next, add 100
μL of the bacteria which is set to Mc Farland
standard of 0.5 which is equivalent to 1 × 108
CFU/mL. Place the cap on the microplate and
incubate for one day (24 hours) in a shaker at 200
rpm at room temperature (30 °C). After 24 hours of
incubation, it was observed whether there was
bacterial growth. If there is bacterial growth, the
concentration is expressed as the MIC value.
Samples at concentrations where there was no
bacterial growth were then tested for MBC, namely
at the concentrations in the table and concentrations
one level above it. The MBC test was carried out by
taking 10 μL of the selected extract concentration
and then dropping it on a petri dish containing NA
media. Then incubated for a day (24 hours) at room
temperature. After 24 hours of incubation, it was
observed whether there was bacterial growth. If there
is no bacterial growth, the concentration is expressed
as the MBC value (Priyanto et al., 2022).
RESULTS AND DISCUSSION
G. japonica is a plant that is often found in the
yard and is easy to cultivate. Indonesian people
know this plant as "Daun Dewa" because it has
many health benefits such as anti-inflammatory,
antioxidant, treating high blood pressure, antidiabetic, and anti-cancer (Wu et al., 2011) (Xu &
Zhang, 2017). G. japonica leaves are also used
as a source of natural antioxidant compounds
that can inhibit the adverse effects of oxidative
stress. Traditional medicinal plants such as G.
japonica leaves have free antiradical compounds
such as polyphenols, flavonoids, and phenolic
compounds ( Tan et al., 2020). In this study, the

Umi Fatmawati, et al.
antioxidant, anti-aging and antimicrobial activities
of the ethanol extract of G. japonica leaves were
tested. The results of this research imply that G.
japonica leaves extract can be developed as a
health supplement candidate to scavenge free
radicals,
anti-aging, as well as an alternative
therapy for bacterial infections, especially against
antibiotic-resistant bacteria.
Total Phenolic Content (TPC) and Total Flavonoid
Content (TFC)
Detection of total phenolic content in 70%
ethanol extract and 96% ethanol extract of
G.japonica leaves were 13.14±1.46 mg GAE/g
and 22.11±2.63 mg GAE/g extract, respectively.
While the total flavonoid content of 70% ethanol
extract and 96% ethanol extract of G. japonica
leaves
were 8.04±1.22 mg
QE/g
and
14.70±1.05 mg QE/g extract, respectively (Table 1).
The more concentrated of the solvent used the
greater the phenolic content detected. Phenolics are
one of the most abundant compounds and main
active constituents obtained from plants of the
genus Gynura (Meng et al., 2021). Phenolic
compound
were suggested to be responsible
mainly for the antioxidant (Chen et al., 2015),
antibacterial activities of Gynura plants (Saha et
al., 2023). The use of ethanol solvents also
greatly influences the extraction of phenolic
compounds where ethanol has high polarity and
good solubility (Abo et al., 2012). Ethanol is a safe
solvent for natural products extraction and is used in
the food and drug industry. Absolute ethanol and
dilute ethanol are widely used for the extraction of
antioxidant compounds derived from phenol from
natural products with good results. The presence of
phenol and flavonoid compounds in G. japonica
leaves extract indicates that this plant contains
natural antioxidants that can reduce or inhibit cell
oxidative activity.
Antioxidant Activity of G. japonica Leaves Extract
Based on the results of the in vitro antioxidant
test using the DPPH method on samples of G.
japonica leaves extracted with 70% and 96% ethanol
solvents, they showed antioxidant activity with an IC 50
values
of
1411.36±56.35
µg/mL
and
1470.00±41.82 µg/mL respectively (Table 2). In this
method, ascorbic acid and trolox were used as
positive controls. The IC50 values of the two solution
concentrations are not much different. This indicates
that differences in solvent concentration have no
effect on the antioxidant activity of plant samples on
DPPH free radicals. The test results for the
antioxidant activity of dewa leaf extract are still
relatively low. The type of plant organ extracted also
influences antioxidant activity. (Krishnan et al., 2015)
stated that the antioxidant activity of leaf extracts is
much lower than that of root extracts. The IC50 value
is used to interpret the results of the DPPH method
and is a substrate concentration that can cause a
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Molekul, Vol. 20. No. 2, July 2025: 330 – 338
Table 1. The total content of phenolic and flavonoid compounds of G. japonica leaves extract
Sample

G. japonica leaf with 70% ethanol
G. japonica leaf with 96% ethanol

Phytochemical contents
TPC (mg GAE/g extract)
13.14±1.46
22.11±2.63

50% reduction in DPPH activity as a free radical
(Proestos et al., 2013). The smaller the IC50 value,
the less amount of substrate needed to inhibit the
DPPH free radical, thus showing higher antioxidative
abilities such as positive control Trolox (2.17±0.06
µg/mL) and ascorbic acid (1.06±0.28 µg/mL). DPPH
is a free radical that can accept electrons or
hydrogen radicals to become stable molecules.
DPPH is known as a stable lipophilic free radical
model. The chain reaction of lipophilic radicals is
initiated by lipid autooxidation. The presence of
natural exogenous antioxidants can react with DPPH
to reduce the number of available hydroxyl groups
which is indicated by a color change from purple to
yellow when read with a spectrophotometer at 517
nm (Proestos et al., 2013). The higher the ability of a
plant extract to turn DPPH purple color to yellow, due
to the formation of 1,1-diphenyl-2-picrylhydrazine,
the higher its antioxidant power (Figure 1).
The results of this study show that the 70% and
96% ethanol extracts of G. japonica leaves are
detected to have antioxidant activity by the DPPH
method, which means that there are compound
components in the G. japonica leaf extract that can
capture free radicals through electron or hydrogen
donation mechanisms. Even
though the IC50 value is still high compared to the
control, the results of this study make it possible that
the G. japonica leaf can be used as a therapeutic
agent to capture free radicals that cause cell
damage.
The antioxidant activity of G. japonica leaves
tested using the ABTS method showed an IC50 value
of 2516.10 + 18.77 µg/mL in samples extracted
with 70% ethanol and 3598 + 27.98 µg/mL
in samples extracted with 96% ethanol. The use of
extract solutions of different concentrations also
affects the antioxidant activity of the samples.
Ethanol, as a polar solvent, is effective in extracting
polar and semi-polar compounds. Compounds such
as flavonoids, alkaloids, tannins and glycosides
dissolve well in ethanol. This is because ethanol is

TFC (mg QE/g extract)
8.04±1.22
14.70±1.05

able to penetrate cell walls and dissolve these
compounds efficiently (Yulianti et al., 2021). Plants
extracted with ethanol solvent at a lower
concentration showed lower antioxidant activity when
compared to plant samples extracted with ethanol
solvent with a higher concentration. On the other
hand, the use of different concentration of ethanol
solvent, revealed the same antioxidant activity
(Spigno et al., 2007). The IC50 values detected by the
ABTS method tend to be higher than those detected
by the DPPH method (Abo et.al, 2012). The use of
DPPH method in detecting antioxidant activity is
more often used than ABTS. Proton radical
scavenging is very important in antioxidant activity
which can reduce the binding of ABTS free radicals
by plant extracts indicating that there are compound
components in the extract that are able to bind free
radicals via the electron/hydrogen donation
mechanism and can protect cells from degradation
caused by free radicals (Batubara et al., 2020).
Anti-aging of G. japonica Leaves Extract with the
Streak Method
Based on the anti-aging test using the streak
method, the viability of S. cerevisiae cells can be
seen based on their ability to grow on the surface of
the agar medium. Adding H2O2 to the medium
creates environmental stress for yeast cells and tests
their ability to tolerate oxidative stress. For the
negative control, yeast cells were supplemented with
1% DMSO without adding extracts.
Figure 2. showed that S. cerevisiae cells added
with G. japonica leaves extract with 70% and 96%
ethanol can grow on YPD plate medium with the
addition of 3 mM H2O2 and 6 mM H2O2 after 48
hours of incubation. The difference is that in Figure
2. the cells without the addition of extract were then
streaked on YPD medium with 6 mM H2O2 which did
not grow (Figure 2a), whereas at 3 mM H2O2 the
cells could still grow (Figure 2b). This indicates that
the addition of G. japonica leaf extract which was
previously detected to have antioxidant activity was
able to maintain the number of S. cerevisiae cells

Figure 1. DPPH reaction with natural antioxidant (Arce-Amezquita et al., 2019)
334

Antioxidant, anti-aging and antibacterial activity

Umi Fatmawati, et al.

Table 2. Data on antioxidant activity using the DPPH and ABTS methods
Sample

G. japonica leaf with 70% ethanol
G. japonica leaf with 96% ethanol
Ascorbic acid
Trolox

Antioxidant Activities
DPPH IC50±SD (µg/mL)
ABTS IC50±SD (µg/mL)
1411.36±56.35
2516.10±18.77
1470.00+41.82
3598.41±27.98
1.06±0.28
4.30±0.40
2.17±0.06
14.30±6.28

under oxidative stress of H2O2 up to a concentration
of 6mM. Meanwhile, S. cerevisiae cells that were not
treated with G. japonica leaves extract could still
grow on YPD medium with the addition of 3 mM
H2O2 (Figure 2c). This shows that giving G. japonica
leaf extract extracted with 70% or 96% ethanol
solvent can extend cell life and prevent cell death
due to H2O2 oxidative stress response. H2O2 is
considered as a harmful molecule that impairs the
integrity and functionality of cells. In oxidative stress,

it is a crucial redox signaling molecule that is
hazardous to a variety of organisms (Saputra et al.,
2024). Several potential mechanisms for the
antioxidant activity of Gynura are suggested as
follows: suppression of ROS, suppression of lipid
peroxidation, modification of enzymatic antioxidant
synthesis or activities, and manipulation of GSHrelated parameters are some of the hypothesized
mechanisms underlying Gynura's antioxidant action
(Tan et al., 2020).

(a)

(b
)

(c)

(d)

Figure 2. G. japonica leaves extract from 70% and 96% ethanol extract showed anti-aging activity in the
model organism of S. cerevisiae after culture was incubated for 4 days in aging assay; (a). S. cerevisiae
without addition extract streaked on YPD plate medium containing 6 mM H 2O2, (b) S. cerevisiae with addition
extract streaked on YPD plate medium containing 6 mM H 2O2, (c) S. cerevisiae without addition extract
streaked on YPD plate medium containing 3 mM H 2O2, and (d) S. cerevisiae with addition extract streaked on
YPD plate medium containing 3 mM H2O2. A. G. japonica with 70% ethanol solvent extraction; B. G. japonica
with 96% ethanol extraction
Table 3. Screening for antibacterial activity in several ethanol extracts of herbal leaves using the disc method
Extract
Concen
tration
(%)
*

Bacterial Inhibition Zone (mm)

S. aureus

E.coli

P. aeruginosa

B. subtilis

Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol Control
70%
96%
70%
96%
70%
96%
22.5 + 25.0+6. 10.0 + 12.5+3. 10.0+2. 20.0+3.
100
+
4.1
5
2.5
4
9
7
22.5+
10.0+3. 10.0+1. 10.0+4.
75
5.0+2.1 +
1.9
7
8
6
3.5 +1.
10.0+
50
15.0+ 3.2 5.0+1.5 +
3
3.5
* Extract concentration in percent; **Concentration of ethanol solution; tetracycline used for control (+)
Ethanol
70%

Ethanol
96%
10.0+
22.5+ 2.5
4.8
5.0+
10.0 + 3.1
1.8
*

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Molekul, Vol. 20. No. 2, July 2025: 330 – 338
Table 4. MIC and MBC values of G. japonica leaves extracts
MIC/MBC values (µg/mL)
Extract
70% ethanol
96% ethanol

S. aureus

E. coli

P. aeruginosa

Bacillus subtilis

390.62/>781.25
390.62/>781.25

˃ 25,000/˃ 25,000
˃ 25,000/˃ 25,000

˃25,000/˃ 25,000
˃25,000/˃ 25,000

˃25,000/˃ 25,000
˃25,000/˃ 25,000

Screening of Antibacterial Activity
The antibacterial activity of the ethanol extract of
G. japonica leaves was screened using the well
diffusion method. Based on the screening results, it
was found that the G. japonica leaf extract with 70%
and 96% ethanol had bacterial inhibitory activity
against all test bacteria at extract concentrations of
100%, 75%, and 50% (w/v) (Table 3). The inhibition
zone formed by the 70% ethanol extract tends to be
higher than that of the 96% ethanol extract. The
inhibition test was then confirmed by the Minimum
Inhibition Concentration (MIC) test using microplate
well 96 in MHB medium.
MIC test results showed that G. japonica leaves
extract with 70% or 96% ethanol solvent could inhibit
the growth of S. aureus at a minimum concentration
of 390.62 µg/mL. Meanwhile, the concentration of
other inhibiting bacteria in E. coli, P. aeruginosa, B.
subtilis was still high (> 25,000 µg/mL) (Table 4).
This indicates that G. japonica leaves extract has
antibacterial activity which is sensitive to S. aureus.
On the other hand, the Minimum Bacterial
Concentration (MBC) value of G. japonica leaves
extract against S. aureus is 781.25 µg/mL. The
same thing was also reported by (Ashraf et al.,
2020) who states that the ethanol extract of Gynura
leaves is more effective at inhibiting S. aureus
bacteria
than other types such as Methicillinresistant Staphylococcus aureus. G. japonica leaves
extract was also reported to have antibacterial
activity against S. aureus and P. aeruginosa
(Jiangseubchatveera et al., 2015). The antibacterial
activity of Gynura leaves is caused by the
presence of several flavonoid compounds and
phenolic compounds contained in its constituents
(Wan et al., 2011). The results of this research
indicate that the ethanol extract of G. japonica
leaves can be developed herbal medicines or
phytopharmaceuticals to prevent diseases caused by
oxidative stress. The results of the antiaging activity
test of G. japonica leaf extract on Saccharomyces
cerevisiae cells showed the potential to protection
from cells damage due to exposure to free radicals
H2O2. The antibacterial test results of the ethanol
extract of Gynura leaves have the potential to be
developed as an alternative natural antibiotic.
CONCLUSIONS

Gynura japonica ethanol extract was detected to
contain phenolic and flavonoid compounds which
are responsible for antioxidant activity. The results of
the antioxidant activity test on DPPH and ABTS free

radicals showed that Dewa Leaf extract at both types
of solvent concentrations showed antioxidant activity
that was not much different in vitro. The results of the
anti-aging test using S. cerevisiae as a model
organism showed that the addition of leaf extract
was able to maintain the number of S. cerevisiae
cells under H2O2 oxidative stress up to a
concentration of 6 mM. The results of the
antimicrobial test showed that the ethanol extract of
G. japonica leaves showed antibacterial activity
against S. aureus. The results of this study imply that
the ethanol extract of G. japonica leaves has the
potential to be developed as a therapeutic agent,
especially as an antioxidant and antimicrobial agent.
ACKNOWLEDGMENTS
This work was supported by Institute of Research
and Community Service (LPPM) Universitas Sebelas
Maret Surakarta through “Hibah Grup Riset (HGR)”
2022 to Umi Fatmawati with number of contract
254/UN27.22/PT.01.03/2022.
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