Molekul, Vol. 20. No. 2, July 2025: 404 – 414

Articles

MOLEKUL

eISSN: 2503-0310

https://doi.org/10.20884/1.jm.2025.20.2.16366

Antioxidant and Antibacterial Activities of Plant and Endophytic Fungi Extracts from
Syzygium myrtifolium Walp, with LC-HRMS Profiling of Active Extracts
Sintia Frisky Efendi1, Suryati1*, Dwinna Rahmi2, Praptiwi2, Muhammad Ilyas3, Andria Agusta2
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Andalas University.
Padang, West Sumatra, Indonesia
2
Research Center for Pharmaceutical Ingredients and Traditional Medicine, National Research, and Innovation
Agency (BRIN), KST Soekarno, Cibinong, West Java, Indonesia.
3
Research Center for Biosystematics and Evolution, National Research and Innovation Agency (BRIN),
KST Soekarno, Cibinong, West Java, Indonesia.
1

*Corresponding author email: suryati_chemua@yahoo.com
Received May 14, 2025; Accepted July 11, 2025; Available online July 20, 2025
ABSTRACT. The emergence of antibiotic resistance and oxidative stress-related diseases highlights the urgent need for
novel bioactive compounds. This study investigates the potential of Syzygium myrtifolium Walp. and its endophytic fungi as
sources of antibacterial and antioxidant agents. Sixteen endophytic fungi isolates were obtained from six plant parts and
identified morphologically. Thin layer chromatography (TLC)-based chemical profiling demonstrated comparable
secondary metabolite patterns between the plant and its endophytic fungi, indicating possible shared biosynthetic
pathways. Antibacterial screening using TLC-bioautography and minimum inhibitory concentration (MIC) assays
demonstrated that both the plant and endophytic fungi extracts inhibited the growth of Staphylococcus aureus and
Escherichia coli, with Xylaria sp. showing notable activity. (SmDh4) exhibiting the strongest activity (MIC = 64 µg/mL).
Antioxidant activity determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay indicated high to very high radical
inhibition capacity, especially in SmRTd (AAI = 25.91) and SmAk1 (AAI = 24.97). Liquid chromatography-high resolution
mass spectrometry (LC-HRMS) analysis unique secondary metabolites on endophytic fungi, including L-α-palmitin, αeleostearic acid, and 8-methylnaphthalene-1,2-diol, which were detected exclusively in the endophytic fungi extracts,
highlighting their potential as alternative antibacterial and antioxidant agents.
Keywords: Antibacterial, antioxidant, endophytic fungi, secondary metabolites, Syzygium myrtifolium Walp.

INTRODUCTION
The increasing threat of antibiotic-resistant
pathogens and the harmful effects of oxidative stress
have created an urgent need for natural compounds
with antibacterial and antioxidant activities (Ho et al.,
2025; Milatovic et al., 2021). This urgency
encourages the exploration of new bioactive
metabolites, one of which is derived from the plant
Syzygium myrtifolium Walp., a species belonging to
the Myrtaceae family, which is widely distributed in
tropical and subtropical regions (Pillai & Sreekala,
2023).
Syzygium myrtifolium Walp. is rich in various
bioactive compounds with potential pharmacological
properties. Its leaves are known to contain
flavonoids, phenolics, alkaloids, saponins,
and
triterpenoids (Ahmad et al., 2021; Amanah et al.,
2023). These compounds exhibit antioxidant,
antimicrobial, antiviral, anticancer, and antidiabetic
activities (Amanah et al., 2023). Overexploitation of
plant and cultivation constraints have driven

advances
in
pharmaceutical
sciences
and
biotechnology, leading to the discovery of
environmentally friendly endophytic fungi that can
rapidly produce the same or more potent secondary
metabolites through biotransformation from their
host plant (Zakariyah et al., 2024). This shift began
in 1993 with the discovery of paclitaxel from
Taxomyces andreanae, an endophyte of Taxus
brevifolia, highlighting the potential of endophytes as
alternative sources of plant-derived bioactive
compounds, such as taxol (Tiwari & Bae, 2022).
Since then, endophytic fungi have been
recognized for forming symbiotic relationships with
medicinal plant, significantly influencing their
secondary metabolism and metabolite production
(Alam et al., 2021). These fungi produce a variety of
bioactive
compounds,
including
phenolics,
flavonoids, steroids, polyketides, saponins, and
alkaloids, which help plant cope with biotic and
abiotic stresses and enhance their immune systems
(Jha et al., 2023). Several endophytic fungi exhibit

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Sintia Frisky Efendi, et al.

diverse biological activities such as antibacterial,
antioxidant, anticancer, antiviral, antidiabetic, antiinflammatory, and antiparasitic effects (Hashem et
al., 2023).
The close mutualistic symbiosis between medicinal
plant and their endophytic fungi has drawn
significant research interest in exploring their
antioxidant and antibacterial activities. This study
focuses on S myrtifolium Walp. due to its rich profile
of bioactive compounds, aiming to assess these
biological activities in both the plant and its
associated endophytes, as well as to identify the
active
secondary
metabolites
using
liquid
chromatography-high resolution mass spectrometry
(LC-HRMS). The findings are expected to contribute to
advancements in pharmaceutical sciences and
biotechnology.
EXPERIMENTAL SECTION
Material
The plant parts of S. myrtifolium Walp. The roots,
stems, young petioles, mature petiole, mature leaves
(green), and young leaves (red) were used and
obtained from Padang, West Sumatra, Indonesia.
Chemicals used in this study include dimethyl
sulfoxide/DMSO (Merck), Folin-Ciocalteu (Merck),
ethanol P.A (Merck), quercetin (Sigma-Aldrich),
Dragendroff (Merck), vanillin (Sigma-Aldrich),
hexanes,
ethyl
acetate, methanol,
acetone,
dichloromethanes, aquadest, chloramphenicol, and
Iodonitrotetrazolium/INT (Sigma) and media such as
Mueller Hinton Agar/MHA (Criterion), Mueller Hinton
broth/MHB (Criterion), Potato Dextrose Broth/PDB
(Difco™), Potato Dextrose Agar/PDA (Difco™), Corn
Meal Malt Agar/CMMA (Difco™).
Plant Identification and Preparation
Syzygium myrtifolium Walp. was taxonomically
identified at the ANDA Herbarium, Department of
Biology, Faculty of Mathematics and Natural
Sciences, Andalas University, Padang. The plant parts
were then air-dried, ground into a fine powder using
a grinder, weighed, and extracted.
Isolation of Endophytic Fungi
Each plant part was stored at low temperature,
surface-sterilized using ethanol and sodium
hypochlorite, and then dried aseptically. Sterilized
samples were cut into 1×1 cm² pieces and placed on
CMMA
supplemented
with
0.05
g/L
chloramphenicol,
then
incubated
at
room
temperature for 5 days. Emerging fungi colonies
were subcultured on PDA to obtain pure isolates.
Each pure isolate was then morphologically identified
(Mahmud et al., 2020).
Identification of Endophytic Fungi
Fungi identification was carried out based on
morphological characteristics according to a previous
study (Ilyas et al., 2019). Morphological identification
was performed by observing both macroscopic and
microscopic phenotypic characteristics. Macroscopic

characterization included observations of colony
color, shape, surface, texture, exudate droplets, and
reverse color. For microscopic observation, fungi
mycelia were mounted in a drop of 1% lactophenol
blue solution. Microscopic characterization was
conducted using a light microscope to observe
hyphae, hyphal pigmentation, septation, clamp
connections, spores, and other reproductive
structures.
Cultivation of Endophytic Fungi
Each isolate was cultivated in 200 mL of potato
dextrose broth (PDB; HiMedia™) in 500 mL culture
flasks and incubated in the dark for 21 days.
Isolation and cultivation of endophytic fungi were
carried out aseptically. The isolation and cultivation
of endophytic fungi were conducted aseptically
(Mahmud et al., 2020).
Extraction of Endophytic Fungi and Plant
Plant extraction was performed by maceration
using ethyl acetate with three repetitions. In addition,
endophytic fungi isolated from the plant were
harvested after incubation, along with their growth in
medium and biomass, then blended and macerated
using the same solvent. The filtrate was concentrated
by rotary evaporation and stored at 20°C. (Mahmud
et al., 2020).
Chemical Compounds Analysis by Thin Layer
Chromatography (TLC)
Chemical compounds present in plant and
endophytic fungi extracts were analyzed using silica
gel thin layer chromatography (TLC). Extracts were
prepared at 10 mg/mL in ethyl acetate, and then 10
µL of the extract was applied to TLC plates and eluted
with dichloromethane:methanol (30:1). Separated
compounds were visualized under UV light at  254
and  366 nm, followed by spraying with vanillinsulfuric acid (heated at 110°C), Dragendorff’s, and
Folin-Ciocalteu reagents to detect different classes of
metabolites (Kumari et al., 2021).
TLC-Bioautography for Screening Antibacterial and
Antioxidant Activities
Screening of antibacterial and antioxidant
activities of plant and endophytic fungi extracts (10
mg/mL) was performed using TLC-bioautography. A
total of 10 µL of each extract and chloramphenicol as
a positive control were applied to TLC plates and
dried. Antibacterial screening was performed by
dipping the plates in bacterial suspension, incubating
at 37°C for 18 hours, then with iodonitrotetrazolium
(INT). The white zone indicated bacterial inhibition.
Active extracts were developed with CH₂Cl₂:MeOH
(30:1), dried, and sprayed again with INT.
Antioxidant screening was carried out by spraying
plates containing extracts and catechin (positive
control) with 0.02% DPPH in methanol. Yellow spots
on a purple background indicated antioxidant
activity. Active extracts were developed, dried, and
sprayed again with DPPH (Wang et al., 2021).

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Determination of Minimum Inhibitory Concentration
(MIC)
The minimum inhibitory concentration (MIC) of all
extracts was determined by serial microdilution in a
96-well plate (Charria-Giron et al., 2021). Extracts
(10240 µg/mL) were serially diluted with MHB to a
final concentration of 256 µg/mL. Each well was then
inoculated with 100 µL of bacterial suspension (106
CFU/mL). Chloramphenicol and broth served as
positive and negative controls, respectively. Plates
were incubated at 37°C for 24 hours, after which 10
µL of INT (4 mg/mL) was added. MIC was defined as
the lowest concentration showing no visible bacterial
growth.
Determination of IC50 of Active Extract
Antioxidant activity was measured using the DPPH
method through serial microdilution in a 96-well
plate (Kumari et al., 2021). Extracts (10240 µg/mL)
were diluted to a final concentration of 128 µg/mL,
and then DPPH solution (61.50 µg/mL) was
subsequently added. Catechin and methanol served
as positive and negative controls. After 90 minutes of
incubation in the dark, absorbance was recorded at l
517 nm. The IC₅₀ value was calculated using the
following formula:

AAI (Antioxidant Activity Index) was calculated as the
ratio of the DPPH concentration to the IC₅₀ value.
RESULTS AND DISCUSSION
Plant and Endophytic Fungi Identification
Identification revealed that this plant belongs to the
family Myrtaceae and genus Syzygium, identified as
S. myrtifolium Walp. Six extracts were obtained from
the plant, which facilitated the isolation of 16
endophytic fungi strains, as shown in Table 1 and
Figure 1. These results align with previous studies
showing that a single plant species can be colonized
by various endophytic fungi, reflecting the diversity of
species capable of forming symbiotic relationships
with host plant (Habisukan et al., 2021). The diversity
of endophytic fungi in S. myrtifolium demonstrates
their significant potential as sources of bioactive
compounds
for
therapeutic
development,
emphasizing the important
role these fungi play in producing such compounds.
for example, Acremonium chrysogenum, which has
been used in the industrial production of
cephalosporin antibiotics (Ibrahim et al., 2024).

Figure 1. Macroscopic view of representative endophytic fungi inhabiting Syzygium myrtifolium Walp., in vitro
culturing on PDA, 7-10 days incubation at 27°C: (A) Diaporthe sp. SmAk1 (B) Xylaria sp. SmBt1(C)
Acremonium sp. SmBt2 (D) Colletotrichum sp. SmRTd1 (E) Neopestalotiopsis sp. SmRTd2 (F) Xylaria sp.
SmTd2 (G) Xylaria sp. SmDh1 (H) Arthrinium sp. SmDh3, and (I) Neofusicoccum sp. SmDm2.
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Table 1. List of endophytic fungi isolated from plant parts of S. myrtifolium Walp.
No
1.

Plant part
Root

Plant Code
SmAk

No
Isolate Code
Fungi taxa based on morphology*
1.
SmAk1
Diaporthe sp.
2.
SmBt1
Xylaria sp.
2.
Stem
SmBt
3.
SmBt2
Acremonium sp.
4.
SmRTd1
Colletotrichum sp.
Young
5.
SmRTd2
Neopestalotiopsis sp.
3.
SmRTd
petiole
6.
SmRTd3
Colletotrichum sp.
7.
SmRTd4
Colletotrichum sp.
8.
SmTd1
Colletotrichum sp.
Mature
4.
SmTd
9.
SmTd2
Xylaria sp.
petiole
10.
SmTd3
Neopestalotiopsis sp.
11.
SmDh1
Xylaria sp.
Mature
12.
SmDh2
Arthrinium sp.
5.
Leaf
SmDh
13.
SmDh3
Arthrinium sp.
(green)
14.
SmDh4
Xylaria sp.
Young
15.
SmDm1
Arthinium sp.
6.
SmDm
leaf (red)
16.
SmDm2
Neofusicoccum sp.
(*): Cultures were growth on potato dextrose agar (PDA), 5-7 days incubation at 27°C

Figure 2. Chromatograms of plant and endophytic fungi extracts with eluted CH₂Cl₂:MeOH
(30:1, v/v), visualized under (A) UV  254 nm, (B) UV  366 nm, (C) vanillin–H₂SO₄, (D)
Dragendorff, and (E) Folin–Ciocalteu. Black boxes indicate plant codes; white boxes indicate
endophytic fungi codes.

Figure 3. TLC-bioautograms of endophytic plant and fungi with eluted CH2Cl2: MeOH (30:1) (A).
Plant (E. coli), (B). endophytic fungi (E. coli), (C). Plant (S. aureus), (D). endophytic fungi (S.
aureus). White zones indicate bacterial inhibition and represent antibacterial activity. Black boxes
indicate plant codes, and white boxes indicate endophytic fungi codes.
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Phytochemical Profile of Extracts by Thin Layer
Chromatography (TLC)
The chemical compound profiles of plant extracts
and endophytic fungi from S. myrtifolium Walp. were
analyzed using TLC (Figure 2). TLC offers a
preliminary overview of the diversity and similarity of
compounds present in both plant extracts and
endophytic fungi, making it a valuable initial method
for mapping secondary metabolites (Wilson & Poole,
2023). Separated chemical compounds were
visualized under UV light at  254 nm and  366
nm. Spots observed at  366 nm indicate the
presence of compounds containing chromophore
groups or conjugated double bond systems (Raunsai
et al., 2023).
Figure 2 shows that Folin–Ciocalteu reagent
detects phenolic compounds as blue-black spots,
Dragendorff’s reagent indicates alkaloids with
orange spots, and vanillin-H₂SO₄ reveals diverse
secondary metabolites through various colored
spots (Fathoni et al., 2021). TLC analysis revealed
similar retention patterns between the extract of S.
myrtifolium Walp. and its associated endophytic
fungi, suggesting the possibility of shared biosynthetic
pathways. This is evident in the SmDh and SmDh2
extracts, where phenolic compounds were detected at
Rf = 0.87, indicated by blue-black spots. A similar
pattern was observed in the SmRTd extract and its
associated endophyte SmRTd2, with phenolic
compounds appearing at Rf = 0.12, also marked by
blue-black coloration. The metabolites produced by
the fungi appeared structurally simpler, likely due to
the absence of chlorophyll-derived compounds
(Vaishnav & Demain, 2011). Endophytic fungi exhibit
significant pharmacological potential, as indicated by
the presence of alkaloids in the extracts SmDh1 (Rf =
0.20) and SmDh4 (Rf = 0.27). Additionally, a greater
production of phenolic compounds was observed,
particularly in the SmAk1 extract (detected at Rf ≤
0.61), and in extracts SmDh1, SmDm1, and SmDm2
(detected at Rf = 0.87), as illustrated in Figure 2.
Through horizontal gene transfer, endophytic fungi
can produce bioactive metabolites similar to the host
plant or new compounds that have potential as
alternative sources (Bielecka et al., 2022).
Antibacterial Activity
Screening of antibacterial activity using TLC-dot
blot and TLC-bioautography showed that four host
plant extracts (SmRTd [3], SmTd [4], SmDh [5], and
SmDm [6]) inhibited the growth of both E. coli
and S. aureus. Among the endophytic fungi extracts,
three isolates (SmDh4 [14], SmDm1 [15], and
SmDm2 [16]) inhibited E. coli, while seven extracts
(SmBt1 [2], SmDh1 [11], SmDh2 [12], SmDh3 [13],
SmDh4 [14], SmDm1 [15], and SmDm2 [16])
showed activity against S. aureus.
TLC-bioautography was conducted to separate
and identify these bioactive compounds based on

their antibacterial activity. The white zone indicates
the presence of active antibacterial compounds,
resulting from the absence of dehydrogenase enzyme
activity in dead bacterial cells (Raunsai et al., 2023),
whereas the purple background is produced by the
reduction of INT to formazan by bacterial
dehydrogenase (Wang et al., 2021). Based on
previous findings, S. zeylanicum and its endophytic
fungus Penicillium brefeldianum, isolated from the
plant, produce the same compounds, such as phydroxybenzaldehyde,
and
exhibit
similar
antibacterial activity. This suggests that the similarity
in secondary metabolites contributes to their
comparable antibacterial effects (Syarifah et al.,
2021).
The MIC values presented in Table 2, show that
the endophytic fungus Xylaria sp. isolate SmDh4
exhibited strong antibacterial activity against
Staphylococcus aureus (MIC = 64 μg/mL) and
moderate activity against Escherichia coli (MIC =
128 μg/mL), according to the classification by
(Dzotam et al., 2018), who defined MIC values
<100 μg/mL as strong and 100–500 μg/mL as
moderate. This is consistent with the TLCbioautography result, where SmDh4 displayed a
white
zone,
qualitatively
indicating
strong
antibacterial activity (Figure 3), which is further
supported by its MIC values. Based on the MIC
values, endophytic fungi demonstrate the ability to
produce secondary metabolites with notable
antibacterial activity. For instance, the SmDh extract
showed an MIC of 256 μg/mL, whereas its
endophyte SmDh4 exhibited a stronger activity with
an MIC of 64 μg/mL, suggesting that the endophyte
may produce distinct, more potent antibacterial
compounds than its host plant. A similar pattern was
observed in SmRTd and its endophyte
SmRTd1
against S. aureus, with MIC of 256 μg/mL and 128
μg/mL, respectively. These findings are consistent
with the TLC-bioautography results, where SmRTd
displayed only two active spots, while SmRTd1
showed a broader inhibition zone. However, in some
cases, the host plant may exhibit stronger bioactivity,
as observed in SmTd (MIC 128 μg/mL) compared to
its endophyte SmTd1 (MIC 256 μg/mL). These results
sugges t a mutualistic relationship between S.
myrtifolium Walp. and its endophytic fungi, in which
both partners contribute bioactive compounds that
inhibit the growth of S. aureus and E. coli.
Previous studies have shown that, endophytic
fungi such as Xylaria sp. can produce potent
bioactive compounds with significant antibacterial
activity. For instance, Xylaria sp. isolated from
Ginkgo biloba leaves was found to produce 7amino-4-methylcoumarin,
exhibiting
strong
antibacterial effects against S. aureus and E. coli with
MIC values of 16 μg/mL and 10 μg/mL, respectively
(Liu et al., 2008).

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Table 2. Minimum inhibitory concentration (MIC) of extracts from Syzygium myrtifolium Walp. and its
endophytic fungi.
No

Extract code

1
Chloramphenicol**
2
SmAk*
3
SmAk1
4
SmBt*
5
SmBt1
6
SmBt2
7
SmRtd*
8
SmRTd1
9
SmRTd2
10
SmRTd3
11
SmRTd4
12
SmTd*
13
SmTd1
14
SmTd2
15
SmTd3
16
SmDh*
17
SmDh1
18
SmDh2
19
SmDh3
20
SmDh4
21
SmDm*
22
SmDm1
23
SmDm2
(*): plant extract, (**): positive control

MIC (μg/mL)
Activities
E.coli
Strong
2
NT
NT
NT
Moderat
256
NT
Moderat
Moderat
256
NT
NT
NT
Moderat
256
Moderat
NT
NT
Moderat
Moderat
Moderat
Moderat
Strong
128
Moderat
128
NT
Moderat
-

S.aureus
2
256
256
128
128
256
256
256
256
256
64
128
256

Antioxidant Activity
The antioxidant activity was evaluated to assess
the ability of plant extracts and endophytic fungi
extracts from S. myrtifolium Walp. to inhibit free
radicals, using the DPPH method. This method is
based on the donation of hydrogen atoms from
antioxidant compounds to stabilize free radicals,
converting them into non-radical compounds (1,1diphenyl-2-picrylhydrazine), as indicated by a color
change from purple to yellow (Kusmiati et al., 2018).
According to the qualitative analysis presented in
Figure 4, using TLC-bioautography assay, plant
extracts demonstrated strong antioxidant activity, as
evidenced by the numerous yellow spots formed
on the TLC. In contrast, six endophytic fungi
extracts (SmBt1, SmBt2, SmRTd1, SmTd2, SmDh1,
and SmDh4) exhibited weak antioxidant activity.
This difference is likely related to variations in

Activities
Strong
NT
NT
NT
Moderat
NT
NT
Moderat
NT
NT
NT
Moderat
NT
NT
NT
NT
NT
NT
NT
Moderat
Moderat
NT
NT

secondary metabolite profiles produced by the plants
and
their
associated
endophytic fungi. The
symbiotic metabolic relationship was further
supported by the similar antioxidant activity observed
in root extract, where both the plant extract SmAk
and the endophytic fungi extract SmAk1 showed
activity in polar compounds, indicated by active spots
at Rf ≤ 0.39. A comparable pattern was observed
in young leaf extracts, with plant extract SmDm and
endophytic extracts SmDm1 and SmDm2 exhibiting
antioxidant activity across nearly all detected
compounds. These results suggest the presence of
phenolic compounds, consistent with the data
shown in Figure 2, which revealed high phenolic
content in SmAk, SmDm1, and SmDm2 extracts.
Following qualitative TLC analysis, IC50 and AAI
values of the extracts were determined using the
DPPH method.

Figure 4. TLC-bioautograms of S. myrtifolium Walp. extracts using CH₂Cl₂: MeOH (30:1). Black
boxes indicate plant codes, and white boxes indicate endophytic fungi codes.
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Table 3. Antioxidant activity of S. myrtifolium Walp. plant extracts and endophytic fungi extracts,
expressed as IC50 (μg/mL) and AAI.
No

Extract code

R2

1
Catechin**
0.9784
2
SmAk*
0.9937
3
SmAk1
0.9958
4
SmBt*
0.9936
5
SmBt1
0.9964
6
SmBt2
0.9995
7
SmRtd*
0.9926
8
SmRTd1
0.9859
9
SmRTd2
0.9945
10
SmRTd3
0.9979
11
SmRTd4
0.997
12
SmTd*
0.9949
13
SmTd1
0.9997
14
SmTd2
0.9977
15
SmTd3
0.994
16
SmDh*
0.9955
17
SmDh1
0.9969
18
SmDh2
0.9927
19
SmDh3
0.9913
20
SmDh4
0.9802
21
SmDm*
0.9999
22
SmDm1
0.9916
23
SmDm2
0.9926
(*): plant extract, (**): positive control
Antioxidant activity was evaluated based on IC₅₀
and AAI values. IC₅₀ indicates the concentration of a
compound required to inhibit 50% of DPPH radical
activity, reflecting its effectiveness in preventing
oxidation (Yusuff et al., 2019). AAI is used to
standardize antioxidant activity in DPPH-based
assays, with classifications as follows: weak (AAI <
0.5), moderate (0.5 ≤ AAI ≤ 1.0), strong (1.0 < AAI
≤ 2.0), and very strong (AAI > 2.0) (Abarca-vargas
et al., 2019).
The results showed that four plant extracts,
namely SmRTd, SmTd, SmDh, and SmDm, exhibited
very strong antioxidant activity with AAI values of
25.91; 11.34; 17.15; and 6.55, respectively. This
strong activity was also reflected in the TLCbioautography, where antioxidant-active spots
appeared at Rf ≤ 0.24, indicating the presence of
polar antioxidant compounds. Among the endophytic
fungi extracts, three isolates SmAk1 (Diaporthe sp.),
SmDm1
(Arthrinium
sp.),
and
SmDm2
(Neofusicoccum sp.) also demonstrated very strong
antioxidant activity with AAI values of 24.97; 9.15,
and 15.38, respectively. TLC-bioautography revealed
that SmAk1 contained active antioxidant compounds
at Rf ≤ 0.39, while SmDm1 and SmDm2 showed
activity across a broad range of compounds, from
polar
to
non-polar.
In
addition,
SmTd3
(Neopestalotiopsis sp) showed strong antioxidant
activity with an AAI value of 2.31. The root extract of
the plant, SmAk, exhibited strong antioxidant activity

IC50

AAI

0.833
10.34
1.23
47.49
282.73
144.26
1.19
330.63
121.88
167.4
177.9
2.71
287.93
325.82
13.34
1.79
563.03
31.06
95.55
303.59
4.69
3.36
2.00

36.9
2.97
24.97
0.65
0.11
0.21
25.91
0.09
0.25
0.18
0.17
11.34
0.11
0.09
2.31
17.15
0.05
1.01
0.32
0.1
6.55
9.15
15.38

Category base on
AAI Value
Very strong
Strong
Very strong
Weak
Weak
Weak
Very strong
Weak
Weak
Weak
Weak
Very strong
Weak
Weak
Strong
Very strong
Weak
strong
Weak
Weak
Very strong
Very strong
Very strong

with an AAI of 2.97. Supported by TLC results
showing dominant polar antioxidant compounds at
Rf ≤ 0.39.
Strong antioxidant activity has previously been
reported for Neopestalotiopsis sp. (IC₅₀: 22.92
µg/mL) and Diaporthe sp. (IC₅₀: 37.61 µg/mL)
isolated from Cinnamomum loureiroi, with eugenol,
lauric acid, myristaldehyde, and caprylic acid
identified as key antioxidant compounds. Similarly,
this study found strong antioxidant activity in
Neopestalotiopsis sp. (SmDm2) and Diaporthe sp.
(SmAk1) isolated from S. myrtifolium, supporting
their potential as natural antioxidant sources
(Tanapichatsakul et al., 2019).
Analysis of Plant and Endophytic Fungi Extracts Using
LC-HRMS
Extracts of plant and their associated endophytic
fungi that exhibited strong antioxidant and
antibacterial activities were selected for LC-HRMS
analysis to compare their secondary metabolite
profiles. The selected extracts included SmAk, SmAk1,
SmDh, SmDh1, SmDh4, SmDm, and SmDm1 (Figure
5). SmAk and SmAk1 were selected for their strong
antioxidant activity; SmDh for its strong antioxidant
and moderate antibacterial activities; SmDh1 and
SmDh4 for their moderate to very strong antibacterial
activities; SmDm for its moderate antibacterial and
very strong antioxidant activity; and SmDm1 for its
very strong antioxidant activity. These seven extracts

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Antioxidant and Antibacterial Activities

Sintia Frisky Efendi, et al.

were selected for LC-HRMS analysis to compare their
secondary metabolite profiles. The comparison was
based on the five most intense compounds in each
extract, selected for their efficiency, biological
relevance, and representativeness of the extract’s
chemical profile. These major constituents are likely
to significantly contribute to the extracts biological
activity, making them suitable for further
pharmacological or biotechnological analysis. The
analysis of SmAk and SmAk1 extracts showed four
similar compounds and one compound that was
only
present in SmAk1. Two compounds were
known, and three were new compounds with
retention times of 14.363, 9.838, 9.916, 9.828,
and 7.086 minutes. These compounds are L-αpalmitin (C19H38O4), C14H31NO, 2-amino-1,3,4octadecanetriol (C18H39NO3), C14H31NO found in
SmAk and SmAk1 extracts, while C37H62N10O2 is only
found in SmAk1. The chromatograms of these
compounds are presented in Figure 5, and their
chemical structures are shown in Figure 6.
L-α-palmitin is the major compound found in the
SmAk1 extract (Diaporthe sp.), which likely
contributes to its high antioxidant activity (AAI =
24.97), as also reported by previous researchers
who observed strong antioxidant properties of L-αpalmitin isolated from S. litoralle (Hidajati et al.,
2018). In addition to this major compound, the
presence of minor compounds such as 4methoxycinnamic acid (8.79 min), (7E,7′E)-5,5′diferulic acid (7.80 min), and ellagic acid (5.37

min) in the SmAk1 extract may also contribute
synergistically to its antioxidant activity (Zheng et al.,
2024).
Extracts SmDh, SmDh1, and SmDh4 showed
five
peaks
with
the highest intensity. One
compound is known to be present in SmDh, SmDh1,
and SmDh4. The compound is
(-)-caryophyllene
oxide (C15H24O), the retention time is 8.108 min.
Previous
research
demonstrated
that
βcaryophyllene, an isomer of caryophyllene oxide,
exhibits strong antibacterial activity, particularly
against S. aureus, with MIC values ranging from 3 to
14 µM (Dahham et al., 2015). Three compounds
were found in extract SmDh and SmDh4, these
compounds
are
1,2,3,4-tetramethyl-1,3cyclopentadiene
(C9H14),
IPMP
(2-isopropyl-5-methylphenol) (C10H14O), and NP011220 (C11H18N2O2) respectively at retention times
of 9, 131; 9,132; and 5,023; min. IPMP (2isopropyl-5-methylphenol) likely contributes to the
antibacterial
activity
of
SmDh1 and SmDh4
extracts. Previous studies showed that IPMP
synergistically
inhibits
biofilm formation by S.
mutans through antimicrobial action and suppression
of biofilm-related genes, supporting its potential
as an effective antibiofilm agent (Korenaga et al.,
2024). In addition, α-eleostearic acid (C₁₈H₃₀O₂)
was detected exclusively in the SmDh1 and
SmDh4 extracts, with a retention time of 14.673
minutes (Figure 5), and its chemical structure is
shown in Figure 6.

Figure 5. Chromatogram of plant extracts and endophytic fungi from Syzygium myrtifolium Walp obtained by
LC-HRMS analysis. (A) SmAk, (B) SmAk1, (C) SmDH, (D) SmDH1, (E) SmDH4, (F) SmDm, (G) SmDm1. The
boxes indicate shared secondary metabolites between the plant and its endophytic fungi.
411

Molekul, Vol. 20. No. 2, July 2025: 400 – 414

L-α-palmitin

2-amino-1,3,4-octadecanetriol

1,2,3,4-tetramethyl-1,3cyclopentadiene

IPMP

NP-011220

α-eleostearic acid

α-linolenic acid

Indole-3-acetic acid

(-)-caryophyllene oxide

8-methylnaphthalene-1,2-diol

Quinoline

Figure 6. Structure of major secondary metabolite compounds from SmAk, SmAk1, SmDh, SmDh1, SmDh4,
SmDm, and SmDm1 extracts.
LC-HRMS analysis of SmDm and SmDm1 extracts
showed five peaks with the highest intensity. two
compounds were detected in both extracts: αLinolenic acid (C₁₈H₃₀O₂) at 14.66 minutes, and 2amino-1,3,4-octadecanetriol (C₁₈H₃₉NO₃) at 9.912
minutes. In addition, Three other compounds were
found exclusively in the SmDm1 extract: 8methylnaphthalene-1,2-diol (C₁₁H₁₀O₂), indole-3acetic acid (C₁₀H₉NO₂), and quinoline (C9H7N) with
retention times of 10.411, 6.528, and 6,529
minutes, respectively (Figures 5 and 6). α-linolenic
acid present in the young leaf extract exhibits strong
antioxidant activity, which is consistent with the
findings of studies conducted by (Yammine et al.,
2024).
CONCLUSIONS
In this study, plant extracts and endophytic fungi
isolates were successfully obtained from S.
myrtifolium Walp. The SmTd and SmDm extracts
exhibited moderate antibacterial activity, while the
endophytic
fungi
isolate SmDh4 (Xylaria sp.)
showed strong antibacterial effects. The highest
antioxidant activity was observed in the SmRTd
extract, followed by SmAk1 (Diaporthe sp.), which
also demonstrated very strong antioxidant potential.
Based on LC-HRMS analysis, the compounds (−)caryophyllene oxide and IPMP were identified as
contributors to the antibacterial activity, whereas L-αpalmitin and α-linolenic acid were associated with
antioxidant effects. These findings suggest that S.
myrtifolium and its endophytic fungi are promising
sources of bioactive compounds for further
pharmacological development.

ACKNOWLEDGMENTS
The authors would like to thank Mr. Andi Saptaji
Kamal for his excellent assistance with the laboratory
work. Thank you to the priority grant from the Drug
and Vaccine Research Organization for Health of
National Research and Innovation Agency (BRIN), the
Science and Technology Research Partnership for
Sustainable Development (SATREPS) from the Japan
Agency for Medical Research and Development
(AMED), and the Japan International Cooperation
Agency (JICA) for their partial support of this
research.
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