Bioactive Metabolites in
Melaleuca
leucadendra
Leaves Extract:
Phytochemical Profiling and GC-MS Characterization for
Antimicrobial Relevance
Article
Atina
1,5*
, Idha Royani
2,3
, Assaidah
3
, Dedi Setiabudidaya
3
, Miksusanti
4
, Fitri Suryani
Arsyad
2,3
1
Doctoral Program, Universitas Sriwijaya, Palembang-30139, Indonesia
2
Department of Material Science, Universitas Sriwijaya, Palembang-30139, Indonesia
3
Department of Physics, Universitas Sriwijaya, Indralaya-30862, Indonesia
4
Department of Pharmacy, Universitas Sriwijaya, Indralaya-30862, Indonesia
5
Department of Physics, Universitas PGRI Palembang, Palembang-30116, Indonesia
A
bstract
The global escalation of antimicrobial resistance (AMR) necessitates the exploration of natural sources
of
antibacterial
agents.
This
study
examined
the
ethanolic
leaf
extract
of
Melaleuca
leucadendra's
phytochemical composition, chemical profile, and antibacterial activity. Alkaloids, flavonoids, saponins,
tannins,
phenolics,
terpenoids,
and
steroids
were
all
detected
by
phytochemical
screening.
The
quantitative total flavonoid content (TFC) of the extract was 17.78 mg
QE/g, while the total phenolic
content (TPC) was 292.43 mg GAE/g extract. Gas Chromatography–Mass Spectrometry (GC–MS) analysis
identified oxygenated esters (18.86%), oxygenated aromatics (15.22%), phenolic derivatives (13.44%), and
flavonoids (12.86%), as well as methoxylated aromatics and terpenoids (5.34%), phytol, and fatty acid
derivatives. These metabolite classes are widely reported to exert antibacterial effects through multiple
mechanisms such as membrane disruption, enzyme inactivation, and inhibition of nucleic acid synthesis.
Antibacterial testing against Staphylococcus aureus and Escherichia coli using the agar diffusion method
showed a concentration-dependent response, with the highest activity at 80% extract (15.67 ± 0.58 mm
and
15.33
±
1.15
mm
inhibition
zones,
respectively).
Raman
spectroscopy
confirmed
the
interaction
between polymer molecules and secondary metabolite compounds in the extract, thereby potentially
enhancing
antibacterial
properties.
These
findings
highlight
that
the
antibacterial
activity
of
M.
leucadendra is mediated by the synergistic interplay of phenolics, flavonoids, methoxylated aromatics,
terpenoids, and fatty acids. The findings offer compelling proof that
M. leucadendra
is a viable natural
source for the creation of antibacterial compounds that will lessen antibiotic resistance
Keyword
s
:
Antibacterial, b
ioactive compounds, GC
-
MS analysis, Melaleuca leucadendra
*
Corresponding author
Email address:
08013622328001@student.unsri.ac.id
(Atina)
DOI:
https://doi.org/10.22437/chp.v10i1.49078
Received
October 170
th
2025;
Accepted
February 12
nd
2026;
Available online
May 18
th
2026
Copyright © 2026 by Authors, Published by Chempublish Journal. This is an open access article under the CC BY License
(
https://creativecommons.org/licenses/by/4.0
)
19
Atina et al.,
Chempublish Journal, 10(1) 2026, 19-38
Graphical Abstract
Introduction
Microbial resistance (AMR) to antibiotics has
become
one
of
the
greatest
challenges
in
global health [1,2]. Studies show a significant
increase in AMR rates in 76 countries during
2000–2015
[3].
This
issue
is
a
clinical
problem
that
requires
a
multidisciplinary
approach
involving
surgery,
microbiology,
and clinical pharmacy [4]. The World Health
Organization (WHO) has classified AMR as a
serious
threat
that
could
lead
to
a
future
health
crisis
if
not
addressed
immediately
[5], with global health implications including
increased
morbidity,
mortality,
and
healthcare
costs
[6].
Excessive
and
inappropriate
use
of
antibiotics
is
a
major
factor
accelerating
the
emergence
of
resistant bacterial strains [7]. Therefore, the
search
for
and
development
of
new
antibacterial
compounds
[8],
especially
those
derived
from
natural
sources,
has
become an increasingly important research
focus.
Medicinal
plants
are
a
potential
source
of
bioactive
compounds
that
can
be
used
as
alternatives to synthetic antibiotics [9], such
as
curcumin
integrated
into
Phaseolus
vulgaris
polysaccharide
bio-composite
films
that
can
serve
as
a
sustainable
matrix
for
drug
delivery
systems
with
wound
healing
potential [10],
Ricinus communis
(castor bean
leaves)
structurally
optimizes
the
antiproliferative and pro-apoptotic activity of
CuONPs
nanoparticles
for
lung
cancer
therapy
[11].
Through
a
variety
of
mechanisms,
including
compromising
the
integrity
of
cell
membranes,
preventing
protein
synthesis
[12],
and
deactivating
bacterial metabolic enzymes [13], it has been
demonstrated
that
a
variety
of
secondary
metabolites
produced
by
plants,
including
flavonoids, alkaloids, tannins, saponins, and
terpenoids,
have
antibacterial
activity
[14].
The
use
of
medicinal
plants
also
has
advantages in terms of abundant availability,
relatively
low
production
costs,
and
the
possibility of minimal side effects compared
to synthetic antibiotics.
One medicinal plant with great potential as a
source
of
antibacterial
compounds
is
the
gelam leaf (
Melaleuca leucadendra
L.) [15], a
member of the
Myrtaceae
family. This plant
is
widely
distributed
in
tropical
regions,
20
Atina et al.,
Chempublish Journal, 10(1) 2026, 19-38
including Indonesia, and is traditionally used
to
treat
infections,
inflammation,
and
various skin diseases. Various studies have
reported
that
Melaleuca
species
contain
essential
oils
[16]
with
main
components
such as eucalyptol, α-pinene, and terpinen-
4-ol,
which
have
antimicrobial
activity
[17][18].
Previous
studies
have
shown
that
ethanol extracts of plants belonging to the
Melaleuca
genus
contain
secondary
metabolites that are not completely soluble
in
the
essential
oil
fraction,
so
that
polar-
semi-polar solvent extraction methods such
as
ethanol
can
reveal
additional
bioactive
components
that
have
the
potential
to
increase
the
spectrum
of
antibacterial
activity.
Most studies have focused on essential oils,
while
comprehensive
research
on
ethanol
extracts of
M. leucadendra
leaves, including
phytochemical
analysis,
compound
identification using GC–MS, and antibacterial
activity testing, remains limited. This opens
up opportunities to discover new candidate
compounds that have not been identified in
essential
oil
studies
alone.
Based
on
this
background, this study aims to: (1) conduct
phytochemical
screening
of
M.
leucadendra
leaf
ethanol
extracts
to
identify
the
secondary
metabolite
groups
contained
therein, qualitative analysis of total flavonoid
content
and
total
phenolic
content
(2)
analyze the compound profile in the crude
extract
using
Gas
Chromatography
–Mass
Spectrometry
(GC–MS)
instrument,
(3)
assess the antibacterial activity of gelam leaf
ethanol
extract
against
Gram-positive
Staphylococcus
aureus
and
Gram-negative
Escherichia
coli
bacteria
using
the
agar
diffusion
method,
and
(4)
analyze
the
potential interaction of extract components
with
polymers
for
biomedical
use.
It
is
anticipated that this research will aid in the
creation of organic plant-based antibacterial
substances that effectively combat antibiotic
resistance
Materials and Methods
Materials
Fresh
Melaleuca
leucadendra
leaves
were
collected from the Sekojo area, Palembang,
Indonesia.
96%
ethanol
for
analysis
was
purchased
from
PT.
Sumber
Kita
Indah,
Bekasi,
Indonesia.
All
reagents
for
phytochemical screening, including Mayer's
and
Dragendorff's
reagents
(for
alkaloids),
FeCl
₃
solution
(for
phenolics
and
tannins),
Liebermann–Burchard reagent (for steroids
and
terpenoids),
Mg-HCl
reagent
(for
flavonoids), and aquadest, were of analytical
grade.
Mueller–Hinton
Agar
(MHA)
was
obtained
from
HIMEDIA
M173.
Staphylococcus
aureus
(ATCC
25923)
dand
Escherichia
coli
(ATCC
25922)
were
used
as
test
bacteria,
analytical
balance
(accuracy
±0.0001
g),
rotary
evaporator
(Yamato
Scientific
RE301
series),
GC–MS
system
(Thermo
Fisher
Scientific
ISQ
series
with
Trace
1300),
acid
cabinet,
Petri
dishes,
measuring
cups,
Erlenmeyer
flasks,
paper
disks,
autoclave
(UNICLAVE
FD50A),
incubator
(Equitron
Incubator
Stream
Series), micropipette (10–1000 μL), caliper or
ruler (for measuring inhibition zones).
Plants Determination
Gelam (
Melaleuca leucadendra
) was collected
from
swampy
areas
in
Kalidoni
District,
Palembang,
Indonesia
.
Melaleuca
leucadendra
was identified by a botanist at
the
Biology
Laboratory
of
Universitas
PGRI
Palembang.
The
official
letter
of
plant
determination
confirming
the
species
as
Melaleuca
leucadendra
was
issued
under
letter number 133/E.12/FST/UPGRIP/2024.
Extraction
The
extraction
method
using
maceration
followed
the
procedures
[9,19,20]
with
minor changes. Fresh leaves were washed,
dried
at
room
temperature
(25–27°C)
to
a
21
Atina et al.,
Chempublish Journal, 10(1) 2026, 19-38
constant
weight,
and
ground
into
coarse
powder. A total of 500 g of leaf powder was
soaked in 96% ethanol at a ratio of 1:10 (w/v)
in a closed container at room temperature
for
3×24
h,
with
stirring
every
1×24
h.
The
mixture is filtered through Whatman No. 1
filter paper, and the filtrate is collected. The
residue
is
re-macerated
twice
under
the
same
conditions
to
ensure
complete
extraction.
The
combined
filtrate
is
then
evaporated
under
low
pressure
using
a
rotary evaporator at 60°C to obtain a crude
ethanol extract. The crude extract is stored
at
4°C
until
further
analysis.
The
yield
is
calculated using the following question 1.
Y
ield (%) =
Extract
mass
(
g
)
Crude mass (g)
x
100
%
(1)
Qualitative
Analysis
of
Phytochemical
Compounds
Phytochemical
analysis
was
performed
to
identify
the
presence
of
major
secondary
metabolites using color reagents [21,22,23].
Alkaloid: Mayer's reagent, the formation of a
white
precipitate
(Mayer)
indicates
the
presence
of
alkaloids.
Flavonoids:
magnesium
powder
and
HCl,
the
appearance of a red/orange color indicates
the presence of flavonoids. Saponins: foam
test,
foam
that
lasts
more
than
15
min
indicates the presence of saponins. Tannins:
1%
FeCl
₃
solution,
a
blue-black
or
green-
black color indicates the presence of tannins.
Phenolic:
1%
FeCl
₃
solution,
blue
or
green
color
indicates
the
presence
of
these
compounds.
Terpenoid:
Liebermann–
Burchard
reagent,
the
formation
of
a
reddish-brown color indicates the presence
of these compounds. Steroids: Liebermann–
Burchard
reagent,
the
appearance
of
a
green/blue
color
indicates
the
presence
of
these compounds. The results are recorded
qualitatively
as
“presence”
(+)
or
“absence”
(−).
Total
Flavonoid
Content
(TFC)
and
Total
Phenolic Content (TPC)
TFC
were
determined
using
the
methods
described by previous studies [24,25] using
the
aluminum
chloride
colorimetric
assay,
and
the
absorbance
was
measured
at
415
nm. The Folin–Ciocalteu method was used to
evaluate the TPC of the extract as described
in [26-28], with absorbance measured at 765
nm.
Chromatography–Mass
Spectroscopy
(GC-
MS) Analysis
The
chemical
composition
of
ethanol
extracts
was
determined
using
Gas
Chromatography–Mass
Spectroscopy
(GC-
MS) [29-31], with a qualitative method. GC-
MS
analysis
using
Thermo
Fisher
Scientific
ISQ series with trace 1300, TG-5MS column
type, flow rate of 1.200 mL/min, and Helium
as
the
carrier
gas.
Instrument
setup:
The
analysis was performed in GC–MS full scan EI
(electron
ionization)
mode,
positive
ion
polarity, mass range m/z 30–800 with a scan
time
of
0.2
s
per
scan.
The
transfer
line
temperature
was
290
°C
and
ion
source
temperature was 270 °C. Acquisition began
after 2.5 min (segment #1) and was recorded
as
TIC.
Chromatographic
filtering
was
enabled during acquisition.
Oven program
(GC):
The oven was initially set to 60 °C (held
for
1.0
min),
then
heated
gradually
at
10
°C/min to 200 °C (hold for 2.0 min), 220 °C
(hold for 2.0 min), 250 °C (hold for 2.0 min),
and
270
°C
(hold
for
2.0
min).
Maximum
oven temperature 350 °C; cryogenic inactive.
GC
run
time
is
used
as
the
acquisition
duration.
Inlet
&
injection:
Injection
is
performed
on
S/SL
splitless
(inlet
temperature
200
°C),
sample
volume
1.00
µL. Splitless time 0.50 min, then purge at 5.0
mL/min.
Carrier
flow
mode
constant
flow
1.200
mL/min;
gas
saver
10.0
mL/min
starting
at
2.0
min.
Backflush
active
from
1.50 min until the end of the run.
Analysis
22
Atina et al.,
Chempublish Journal, 10(1) 2026, 19-38
data:
The
software
used
in
this
study
was
XCalibur.
The
mass
spectra
of
the
compounds were compared to The National
Institute of Standard and Technology (NIST)
database
in
order
to
determine
their
identities.
Antibacterial Activities
The
antibacterial
activities
of
ethanol
extracts
were
evaluated
using
the
agar
diffusion method [32,33,34] against
S. aureus
(Gram-positive) and
E. coli
(Gram-negative).
Each bacterial strain was grown in nutrient
medium
at
37
°C
for
18–24
h,
adjusted
to
McFarland standard 0.5 (~1,5 × 10⁸ CFU/mL).
Mueller–Hinton
Agar
(MHA)
plates
were
evenly inoculated with bacterial suspension
using sterile cotton swabs. A 6 mm diameter
paper
disk
was
placed
on
top
of
the
MHA
containing
bacteria,
then
10
μL
of
ethanol
extract at concentrations of 10%, 20%, 40%,
and
80%
(w/v
with
ethanol)
was
dropped
onto
the
disk.
Aquadets
was
used
as
the
negative control due to its neutral and non-
antibacterial
nature,
and
30
μg
of
tetracycline
as
the
positive
control
as
a
standard
antibiotic.
The
plates
were
incubated at 37 °C for 24 h, after which the
clear
zones
were
measured.
Each
antibacterial
assay
was
carried
out
in
triplicate
(n
=
3).
Standard
deviation
(SD)
values
were
calculated
using
the
standard
deviation
formula.
Antibacterial
power
(a)
was calculated by comparing the inhibition
of the sample with the inhibition zone of the
positive control [35], according the equation
2.
a
=
inhibition zone of the sample
inhibition zone of the positive control
(2)
a
<
50%
is
weak
antibacterial
activity,
moderate antibacterial activity if 50 ≤ a ≥70,
and strong antibacterial activity if a > 70
[35]
.
Raman Spectroscopy
Raman spectroscopy testing was conducted
to
evaluate
the
potential
of
gelam
leaf
extract as a natural polymer for nanofibers
in
biomedical
applications.
Two
types
of
polymer
solutions
were
prepared:
chitosan/polyvinyl alcohol (CS/PVA) solution
and CS/PVA/ML solution (with 3% gelam leaf
extract
added).
Solution
preparation
followed
the
procedure
of
[36].
Raman
spectroscopy
was
performed
at
a
wavelength of 785 nm on both solutions, and
analysis was carried out by identifying peak
shifts on the graph.
Results and Discussion
Determination
The identification process was carried out by
botanists
in
the
laboratory
to
verify
the
authenticity
of
the
materials
used
in
the
study [37]. The results showed that the plant
belonged
to
the
Myrtaceae
family,
the
Melaleuca genus, with the species identified
as
Melaleuca leucadendra
. The morphological
characteristics of the gelam plant are woody,
with
peeling
grayish-white
to
red
bark,
pointed
green
leaves,
round
flowers
and
inflorescences that are usually white, flower
clusters
with
dangling
stamens,
and
roots
that tend to be shallow and spread out as an
adaptation to wet soil or swamps [38].
Qualitative
Analysis
of
Phytochemical
Compounds
The
extraction
process
from
500
grams
of
dried leaves powder produced 78.03 grams
of
crude
extract
with
a
calculated
yield
of
15.61%. The extraction process is illustrated
in
Figure
1
.
3
liters
of
maserat
were
evaporated at a temperature 60
o
C, and the
crude extract was stored at a temperature of
4
o
C before use.
23
Atina et al.,
Chempublish Journal, 10(1) 2026, 19-38
Figure
1.
Extraction
process
of
Melaleuca
leucadendra
(a)
Melaleuca
leucadendra
leaves;
(b)
crude drugs; (c) maserate; (d) solvent evaporation; (e) crude extract
Phytochemical
screening
indicated
that
ethanol
extracts
of
Melaleuca
leucadendra
leaves
contain
various
secondary
compounds, including alkaloids, flavonoids,
saponins,
tannins,
phenolics,
terpenoids,
and
steroids
(Table
1).
These
findings
are
consistent with previous studies on species
in
the
Melaleuca
genus,
which
have
been
reported
to
produce
bioactive
compounds
with
significant
pharmacological
activity
[16,17,39].
This
diversity
of
phytochemical
classes
may
indicate
that
the
antibacterial
effects of the extract are due to synergistic
interactions
between
different
bioactive
molecules.
Qualitative
phytochemical
analysis of ethanol extracts from
Melaleuca
leucadendra
revealed the presence of several
classes
of
secondary
metabolites.
Table
1
summarizes the results of colorimetric tests
used
to
detect
alkaloids,
flavonoids,
saponins,
tannins,
phenolics,
terpenoids,
and steroids.
Table 1.
Phytochemical analysis of ethanol extract from
M. leucadendra
leaves
No
Phytochemical
Group
Test Method/Reagent
Color in change
Results
1
Phenolics
FeCl
3
Blue
[+]
2
Alkaloids
Mayer’s/ Dragendorff’s
White sediment
[+]
3
Flavonoids
Mg powder + HCl
Orange
[+]
4
Saponins
Foam test
Stable foam
[+]
5
Tannins
FeCl
3
Blue-black
[+]
6
Terpenoids
Liebermann–Burchard
Reddish brown
[+]
7
Steroids
Liebermann–Burchard
Red
[+]
Notes: “+”: presence, “-“: absence
Flavonoids
and
phenolic
compounds,
detected
in
phytochemical
screening,
are
known
to
have
antibacterial
effects
by
disrupting
microbial
cell
membranes
[29],
forming
complexes
with
bacterial
proteins,
and inhibiting nucleic acid synthesis. Tannins
can inactivate microbial adhesins, enzymes,
and cell envelope
transport
proteins, while
saponins
increase
membrane
permeability
by interacting with sterols, causing cell lysis
[17,40].
The
presence
of
terpenoids
and
steroids
can
further
enhance
antibacterial
potential
by
disrupting
the
lipid
bilayer
structure
and
altering
membrane
fluidity,
leading
to
increased
membrane
permeability
and
leakage
of
intracellular
components [23]. The combination of these
mechanisms
may
explain
the
broad-
spectrum
activity
observed
against
Gram-
24
Atina et al.,
Chempublish Journal, 10(1) 2026, 19-38
positive and Gram-negative bacteria in this
study.
Total
Flavonoid
Content
(TFC)
and
Total
Phenolic Content (TPC)
TFC
and
TPC
were
measured
at
a
temperature
of
24.6°C
with
a
humidity
of
61%, a maximum wavelength of 795 nm for
TFC
and
430
nm
for
flavonoids.
Figure
1
shows
the
absorbance
of
Quercetin
and
gallic acid standards for TFC and TPC, with
R
2
=0,997 (TFC) and R
2
= 0,999 (TPC). Based
on the
analysis results, TFC was 17.78 mg
QE/g
extract,
while
TPC
was
292.43
mg
GAE/g extract. These results indicated that
the
extract
is
rich
in
non-flavonoids
phenolic compounds. Phenolic compounds
have significant antioxidant activity and can
support
antibacterial
properties.
Thus,
these
results
show
that
the
extract
has
abundant secondary metabolites, especially
phenolics,
which
are
relevant
to
its
bioactivity potential.
Figure 2.
The standard curve of Quercetin and Gallic Acid
In
contrast,
inhibition
efficiency
decreased
with
increasing
soaking
time,
even
at
constant
inhibitor
concentrations.
This
decline
can
be
attributed
to
the
gradual
degradation or oxidation of active phenolic
compounds
during
prolonged
exposure
to
the
corrosive
environment,
which
reduces
their
adsorption
capability
and
protective
effectiveness.
Additionally,
extended
immersion may lead to partial desorption or
deterioration of the inhibitor film, exposing
portions of the steel surface to the corrosive
medium.
Although
encapsulation
improves
the
stability
of
the
extract,
the
results
indicate
that
its
protective
performance
diminishes over extended soaking periods.
Overall,
the
findings
demonstrate
that
the
corrosion
inhibition
performance
of
encapsulated
cocoa
husk
extract
is
maximized
at
higher
inhibitor
concentrations
and
shorter
soaking
times.
These
results
confirm
the
potential
of
encapsulated
cocoa
husk
extract
as
an
effective
and
environmentally
friendly
corrosion
inhibitor,
while
emphasizing
the
importance
of
optimizing
both
concentration
and
exposure
duration
for
practical applications.
25
Atina et al.,
Chempublish Journal, 10(1) 2026, 19-38
Chromatography–Mass
Spectroscopy
(GC-
MS) Analysis
The
GC–MS
chromatogram
of
the
ethanol
extract
of
M.
leucadendra
leaves
shows
several peaks, indicating a complex mixture
of
compounds.
The
retention
time,
molecular weight, peak area percentage, and
reported
biological
activity
of
the
main
constituents are presented in Figure
2 and
Table 2.
(a)
(b)
Figure
3.
(a)
GC–MS
chromatogram;
(b)
compound
peak
areas
of
GC-MS
analysis
of
M.
leucadendra
leaves
GC–MS analysis of
Melaleuca sp.
leaf extract
shows
a
wide
variety
of
phytochemicals,
which
can
be
classified
into
several
main
groups, including oxygenated phenolics and
aromatics,
aromatic
hydrocarbons,
flavonoid
compounds,
methoxylated
26
F. Toyyibah., et al.
Chempublish Journal, 10(1) 2026, 19-37
aromatics,
terpenoids,
and
fatty
acids
and
their
derivatives
Figure
2(b
)
.
This
chemical
distribution
confirms
the
richness
of
secondary metabolites in the extract and its
potential pharmacological relevance.
Table 2.
Major compounds identified in ethanol extracts of
M. leucadendra
using GC–MS
No
RT (min)
Compound Name
Molecular
Formula
MW
(g/mol)
Peak Area
(%)
1
09.92
Cyclohexane,1-ethenyl-1-methyl-
2,4-bis(1-methyleth
enyl)-,
[1S-
(1à,2á,4á)]-
C
15
H
24
204
1.24
2
10.32
Caryophyllene
C
15
H
24
204
5.34
3
10.74
1,4,7,-Cycloundecatriene,
1,5,9,9-tetramethyl-, Z,Z,Z-
C
15
H
24
204
2.59
4
10.99
2-Isopropenyl-4a,8-dimethyl-
1,2,3,4,4a
,5,6,7-octahydronaphthalene
C
15
H
24
204
1
5
11.15
Naphthalene,
decahydro-4a-methyl-1-
methylene-7-(1
-methylethenyl)-,
[4aR-
(4aà,7à,8aá)]-
C
15
H
24
204
1.89
6
11.26
Naphthalene,
1,2,3,4,4a,5,6,8a-octahydro-4a,8-
dimethyl-2-(1-methylethenyl)-,
[2R-(2à,4aà,8aá)]-
C
15
H
24
204
2.44
7
12.70
2-Propenoic acid,
3-(3-hydroxy-2,6,6-trimethyl-1-
cycloh
exen-1-yl)-,
methyl
ester,
(E)-
C
13
H
20
O
3
302
18.86
8
13.18
1-Naphthalenol,
decahydro-1,4a-dimethyl-7-(1-
methyle
thylidene)-,
[1R-
(1à,4aá,8aà)]-
C
15
H
26
O
222
3.21
9
13.37
Globulol
C
15
H
26
O
222
1.94
10
15.66
4H-1-Benzopyran-4-one,
5-hydroxy-7-methoxy-2-methyl-
C
11
H
10
O
4
206
2.20
11
17.06
10-Methylanthracene-9-
carboxaldehyde
C
16
H
12
O
220
15.22
12
17.31
2,5-Cyclohexadien-1-one,
3,5-dihydroxy-4,4-dimethyl-2-(1-
oxopentyl)-
C
16
H
24
O
5
446
13.44
13
17.44
4H-1-Benzopyran-4-one,
2,3-dihydro-5-hydroxy-7-
methoxy-2-m
ethyl-6-(3-methyl-2-butenyl)-
C
21
H
26
O
4
290
12.86
27
F. Toyyibah., et al.
Chempublish Journal, 10(1) 2026, 19-37
Tabel
2
shows
compounds
derived
from
plants
identified
by
GC-MS
with
a
relative
peak area greater than 1%. GC–MS analysis
of
ethanol
leaf
extracts
of
M.
leucadendra
revealed a variety of secondary metabolites,
including oxygenated phenolic and aromatic
compounds,
flavonoids,
terpenoids,
and
fatty acids and their derivatives. This group
of compounds has been widely documented
for
its
biological
activity,
particularly
in
antibacterial
mechanisms.
Relative
peak
areas
provide
valuable
indications
of
the
potential contribution of each compound to
the overall antibacterial effectiveness of the
extract.
The
main
compound
identified
was
2-
propenoic acid, 3-(3-hydroxy-2,6,6-trimethyl-
1-cyclohexen-1-yl)-,
methyl
ester
(18.86%),
an
oxygen-containing
terpenoid
ester
derivative. Compounds in this class are often
associated
with
membrane-damaging
properties,
increasing
the
permeability
of
bacterial
cell
walls,
and
thus
disrupting
microbial
viability
[41][42].
The
dominance
of
these
compounds
suggests
that
these
metabolites
may
play
a
major
role
in
the
antibacterial activity observed in the extract.
Terpenoid
esters
are
classified
as
phenolic
compounds.
Other
phenolic
compounds
detected
in
relatively high concentrations were phenolic
compounds
derived
from
2,5-
Cyclohexadien-1-one
(13.44%).
The
total
phenolic
compounds
detected
reached
32.3%,
with
the
main
compound
being
2-
propenoic acid, 3-(3-hydroxy-2,6,6-trimethyl-
1-cyclohexen-1-yl)-,
methyl
ester.
The
oxygenated
aromatic
compound
10-
methylanthracene-9-carboxaldehyde
was
detected
at
15.22%.
Phenolic
groups
are
known for their ability to inactivate bacterial
enzymes
through
hydrogen
bonding
and
covalent
interactions,
while
aromatic
aldehydes
can
exert
antimicrobial
effects
through
protein
denaturation
and
disruption
of
cellular
signaling
pathways
[43].
Overall,
these
two
groups
of
compounds
contributed
>40%
of
the
total
peak
area,
highlighting
their
significant
potential in mediating antibacterial effects.
The detection of flavonoid compounds such
as
4H-1-benzopyran-4-one,
2,3-dihydro-5-
hydroxy-7-methoxy-2-methyl-6-(3-methyl-2-
butenyl)-
(12.86%)
further
strengthens
the
biological activity of the extract. Flavonoids
have been widely reported to interfere with
nucleic
acid
synthesis,
inhibit
bacterial
topoisomerase, and damage the cytoplasmic
membrane
[44][45].
The
relatively
high
flavonoid
content
indicates
that
flavonoids
can
act
synergistically
with
phenolic
compounds
in
enhancing
antibacterial
potential.
Terpenoids
are
another
major
group
of
bioactive
compounds,
represented
by
caryophyllene (5.34%), caryophyllene oxide,
globulol.
Spathulenol
and
phytol
were
detected, but with a relative peak area of less
than 1%. Terpenoids are known to integrate
into the lipid layer, disrupting the stability of
bacterial
membranes
and
interfering
with
respiration
[46].
Previous
studies
have
demonstrated
the
strong
antibacterial
effects
of
sesquiterpenes
and
diterpene
alcohols
such
as
phytol
against
Gram-
positive
and
Gram-negative
bacteria.
Although
their
individual
peak
areas
are
moderate
compared
to
phenolics,
their
cumulative
abundance
is
prominent
and
likely
contributes
significantly
to
the
antimicrobial activity of the extract.
Fatty
acids
and
their
derivatives,
including
palmitic
acid
and
methyl
oleate,
were
also
detected
at
<
2%.
These
compounds
generally
have
weaker
antibacterial
effects
than phenolics and terpenoids, but can act
synergistically
by
disrupting
membrane
integrity and facilitating the activity of more
potent
metabolites.
Synergism
between
28
F. Toyyibah., et al.
Chempublish Journal, 10(1) 2026, 19-37
fatty
acids
and
phenolic/terpenoid
constituents
has
been
documented,
whereby
fatty
acids
increase
permeability
and
enhance
the
effectiveness
of
other
antibacterial agents [47,48].
The peak area results of the GC-MS analysis
in
Figure
3(b)
show
that
the
detected
secondary
metabolites
are
dominated
by
phenolic compounds with the highest peak
area
percentage
of
32.3%,
followed
by
hydrocarbons
(15.22%)
and
flavonoids
(12.86%),
while
the
compound
with
the
lowest
contribution
is
fatty
acids
(2%).
The
dominance
of
phenolic
and
flavonoid
compounds
indicates
strong
antibacterial
activity potential, given that both groups of
compounds are known to have antimicrobial
properties
[49,50]
through
mechanisms
of
growth
inhibition
and
damage
to
bacterial
cell membranes. The presence of terpenoids
and
methoxylated
derivatives,
although
in
lower amounts, also enhances the bioactive
activity of the extract, while the hydrocarbon
and
fatty
acid
content
acts
as
compounds
that support stability and solubility [51]. This
profile
overall
confirms
that
the
tested
extract
has
significant
pharmacological
potential, particularly as a candidate source
of natural antibacterial agents.
Overall, the GC–MS profile indicates that the
antibacterial
potential
of
M.
leucadendra
extract is not due to a single compound, but
rather
to
complex
interactions
between
various
secondary
metabolites.
The
high
content of oxygenated aromatic compounds
and flavonoids highlights their central role,
while terpenoids and fatty acids may provide
supporting
or
synergistic
effects.
These
findings are consistent with previous reports
that
plant
extracts
exhibit
antimicrobial
activity
through
the
combined
action
of
diverse
chemical
constituents,
frequently
having
effects
that
are
stronger
than
the
sum
of
the
effects
of
the
individual
substances.
The
data
show
that
ethanol
extracts
of
M.
leucadendra
leaves
contain
a
rich
composition
of
antibacterial
metabolites,
with
phenolics,
flavonoids,
methoxylated
aromatics,
and
terpenoids
as
the
main
contributors.
This
multi-component
profile
provides
a
rational
basis
for
the
broad-
spectrum activity observed against
S. aureus
and
E.
coli
bacteria.
Future
studies should
focus
on
fractionation,
bioassay-guided
isolation,
and
determination
of
minimum
inhibitory
concentrations
(MIC)
to
confirm
the individual and synergistic contributions
of these compounds.
Antibacterial Activities
Antibacterial
analysis
was
conducted
on
S.
aureus
and
E. coli
with extract concentrations
of
10%,
20%,
40% and
80%,
to
identify
the
extract with the highest inhibition zone. The
extract with a concentration of 80% showed
the
greatest
antibacterial
activity
with
an
inhibition
percentage
of
15.67
±
0.58
mm
against
S.
aureus
and
15.33
±
0.58
mm
against
E.
coli
.
The
antibacterial
inhibition
zones
of
the
ethanol
extract
of
M.
leucadendra
leaves are shown in Table 3 and
Figure 3.
Antibacterial
activity
testing
showed
a
concentration-dependent
increase
in
the
inhibition
zone,
with
the
80%
extract
generating the highest antibacterial activity
(Figure 3). This pattern is consistent with the
dose-response
principle,
whereby
higher
concentrations
increase
the
availability
of
active
compounds
to
diffuse
into
the
agar
medium.
Figure
3
shows
that
the
ethanol
extract
of
M.
leucadendra
tested
had
antibacterial activity against
S. aureus
and
E.
coli
with an increase in the diameter of the
inhibition zone as the extract concentration
increased. At low concentrations (10–20%),
E.
coli
has higher sensitivity than
S. aureus
, as
seen from the larger inhibition zone, but at
the
highest
concentration
(80%),
the
29
F. Toyyibah., et al.
Chempublish Journal, 10(1) 2026, 19-37
inhibition
zone
against
S.
aureus
slightly
exceeded
that
of
E.
coli
,
indicating
the
extract's effectiveness against both types of
bacteria. This pattern indicates the presence
of
strong
broad-spectrum
antibacterial
properties,
consistent
with
the
content
of
secondary
metabolites
such
as
phenolics
and
flavonoids
detected
through
GC-MS
analysis.
Figure 4.
Antibacterial activity of ethanolic extract of
M. leucadendra
leaves: (a)
S. aureus
; (b) E.
coli; (c) Inhibition zone
Table 3.
Inhibition zones of ethanolic extract of
M. leucadendra
leaves against test bacteria
|
Concentration (%)
|
Inhibition Zone (mm) and antibacterial power (%)
a
|
|
S. aureus
|
E. coli
|
|
10
|
12.33 ± 0.58
(52.09)
**
|
14.33 ± 0.58
(62.30)
**
|
|
20
|
14.67 ± 0.58
(61.98)
**
|
14.46 ± 0.58
(62.87)
**
|
|
40
|
15.0 ± 0.00
(63.37)
**
|
15.33 ± 0.58
(66.65)
**
|
|
80
|
15.67 ± 0.58
(66.20)
**
|
15.33 ± 1.15
(66.65)
**
|
|
Tetracycline 30 µg
(Positive control)
b
|
23.67 ± 0.58
|
23.00 ± 1.15
|
|
Aquadest
(Negative control)
|
0.00 ± 0.00
|
0.00 ± 0.00
|
Note: a: comparison of the inhibition zone of the test sample with the inhibition zone of the positive control: * weak (<50), ** middle (50-70), ***
strong (>70) [35]; b: inhibition zone of the positive control
30
F. Toyyibah., et al.
Chempublish Journal, 10(1) 2026, 19-37
The
effectiveness
of
the
antibacterial
inhibition zone of the extract is confirmed in
Table 3 and Figure 3. These results support
the
extract's
potential
as
a
naturally
occurring
antibacterial
agent
with
dose-
responsive efficacy.
The
antibacterial
assay
of
the
extract
demonstrated
inhibitory
activity
against
both
S.
aureus
and
E.
coli,
with
inhibition
zones
ranging
from
12.33±0.58
mm
to
15.67±0.58
mm
and
14.33±0.58
mm
to
15.33±0.58 mm, respectively (Table 3). When
compared
to
the
positive
control
(tetracycline,
23-23.67
mm),
the
extract
exhibited
moderate
antibacterial
activity,
while
the
negative
control
produced
no
inhibition
zones,
confirming
that
the
observed
activity
was
attributed
to
the
phytochemicals present in the extract.
The antibacterial power values, expressed as
percentages relative to the positive control,
ranged
from
52.09%
to
66.20%
against
S.
aureus
and 62.30% to 66.65% against
E. coli.
These
values
indicate
that
the
extract
possesses
a
broad-spectrum
antibacterial
effect,
albeit
with
lower
potency
compared
to
standard
antibiotics.
Interestingly,
the
inhibitory
activity
plateaued
beyond
40%
concentration, suggesting a saturation effect
in
which
further
increases
in
extract
concentration did not significantly enhance
antibacterial efficacy.
The slightly higher inhibition zones observed
against
E.
coli
at
lower
concentrations
indicate
that
the
extract
may
contain
bioactive
compounds
with
stronger
activity
toward
Gram-negative
bacteria.
This
is
noteworthy,
as
gram-negative
bacteria
generally exhibit higher resistance to plant-
derived
antimicrobials
due
to
their
outer
membrane barrier. The activity against both
gram-positive
and
gram-negative
bacteria
suggest that the extract metabolites capable
of
disrubting
bacterial
membranes
or
interfering with intracellular processes.
Overall, these finding support the potential
application
of
the
extract
as
a
natural
antibacterial
agent.
However,
its
lower
efficacy compared to tetracycline highlights
the
need
for
further
studies
focusing
on
fractionation,
isolation
of
active
compounds,
and
evaluation
of
their
mechanism
of
action.
Additionally,
testing
against multidrug-resistant bacterial strains
may further establish the clinical relevance
of the extract
Raman Spectroscopy
The
Raman
spectrum
in
Figure
4
shows
a
comparison
between
CS/PVA
(black
line)
and
CS/PVA/ML
(red
line)
solutions.
The
CS/PVA solution is the base solution before
the addition of extract, while CS/PVA/ML is
the
CS/PVA
solution
to
which
Melaleuca
leucadendra
extract
has
been
added.
In
general, both spectra display characteristic
bands
that
indicate
the
presence
of
the
main
functional
groups
that
make
up
chitosan
and
PVA,
but
there
is
a
shift
in
intensity and position of certain peaks after
the addition of
M. leucadendra
(ML) extract,
which indicates the presence of molecular
interactions
between
the
bioactive
compounds
from
the
extract
and
the
polymer
matrix.
The
CS/PVA
spectrum
shows
several
characteristic
bands,
including ~268 cm
⁻
¹ and ~500–600 cm
⁻
¹: C–
C–O
bond
vibrations
from
the
chitosan
polysaccharide
structure;
asymmetric
stretching
vibrations
of
the
C–O–C
ether
group,
originating
from
the
main
chain
of
PVA and the glycosidic units in chitosan at
~892 cm
⁻
¹; weak to moderate bands related
to
–NH
₂
bending
and
C=C
stretching
vibrations,
indicating
the
presence
of
chitosan
amine
groups
and
double
bonds
from
PVA
at
~1460
–1600
cm
⁻
¹,
indicating
carbonyl
C=O
stretching
vibrations,
which
31
F. Toyyibah., et al.
Chempublish Journal, 10(1) 2026, 19-37
may
originate
from
the
remaining
acetyl
group in chitosan or from partial oxidation
of
PVA
~1720
cm
⁻
¹
(peak
shoulder);
C–H
stretching
bands
from
both
polymers
at
~2900–2950 cm
⁻
¹.
Figure 5.
Raman spectroscopy of synthetic polymer solutions for nanofibers
In
the
CS/PVA/ML
spectrum,
there
are
several important changes, namely peaks at
268.05 cm
⁻
¹ and 894.31 cm
⁻
¹ still appear but
with
higher
intensity,
indicating
the
strengthening
of
C–O–C
vibrations
due
to
possible hydrogen interactions between the
PVA
hydroxyl
group
and
phenolic
compounds from the ML extract; there is a
small shift in the peak position from 892.04
→
894.31
cm
⁻
¹,
indicating
a
change
in
the
chemical
environment
of
the
ether
group
due
to
the
formation
of
new
hydrogen
bonds;
the
band
in
the
1500–1700
cm
⁻
¹
region appears to weaken or shift, indicating
that
the
C=O
or
NH
₂
groups
of
chitosan
interact
with
active
compounds
such
as
flavonoids,
terpenoids,
and
fatty
acids
[52]
present in
M. leucadendra
extract; The overall
intensity
of
the
CS/PVA/ML
spectrum
is
weaker
than
that
of
CS/PVA,
indicating
an
increase
in
the
degree
of
amorphousness
due to complex intermolecular interactions
that disrupt the crystallinity of the polymer
matrix
[53].
Changes
in
the
position
and
intensity of the
Raman bands indicate that
the
active
compounds
in
the
gelam
leaf
extract were successfully integrated into the
CS/PVA
matrix
through
non-covalent
interactions,
particularly
hydrogen
bonds
between
the
–OH
or
–NH
₂
groups
of
chitosan/PVA
and
the
phenolic
groups
(aromatic –OH) of the extract.
Raman
spectroscopy
results
show
that
the
solution
was
successfully
formulated
through mixing, resulting in the potential for
increased
release
of
bioactive
compounds
and
strengthening
the
antibacterial
properties of the material. This modification
has
the
potential
to
increase
the
antibacterial
activity
and
biophysical
properties
(such
as
permeability
and
flexibility) of the fibers.
Conclusions
The
ethanol
leaves
extract
of
Melaleuca
leucadendra
contains
various
secondary
bioactive
compounds,
including
phenolics,
flavonoids,
saponins,
tannins,
terpenoids,
steroids, and alkaloids. Quantitative studies
showed
total
flavonoids
of
17.78
mg
QE/g
extract,
while
total
phenolics
of
292.43
mg
32
F. Toyyibah., et al.
Chempublish Journal, 10(1) 2026, 19-37
GAE/g
extract.
GC–MS
analysis
confirmed
the
dominance
of
oxygenated
esters,
oxygenated aromatics, phenolic derivatives,
and
flavonoids,
which
together
accounted
for
more
than
40%
of
the
total
chemical
composition.
This
group
of
compounds
is
known to have antibacterial activity, working
through
mechanisms
such
as
membrane
destabilization,
protein
binding,
and
inhibition
of
enzymatic
and
genetic
processes. Antibacterial testing showed that
the
extract
has
an
inhibitory
effect
on
Staphylococcus aureus and Escherichia coli,
with
the
inhibition
zone
increasing
proportionally
with
concentration.
The
extract
exhibits
broad-spectrum
activity,
demonstrating
its
potential
against
both
Gram-positive
and
Gram-negative
bacteria.
Raman
spectroscopy
confirmed
the
interaction between polymer molecules and
secondary
metabolite
compounds
in
the
extract,
thereby
potentially
enhancing
antibacterial
properties.
These
findings
demonstrate
the
antibacterial
potential
of
M. leucadendra, warranting further studies
to
identify
active
compounds
and
evaluate
their
individual
and
synergistic
effects
through MIC analysis
Acknowledgement
Thank
you
to
the
Directorate
of
Research,
Technology
and
Community
Service
(DRTPM)
of
the
Ministry
of
Education,
Culture,
Research
and
Technology
of
the
Republic of Indonesia for providing funding
for
this
research
through
the
Doctoral
Dissertation Research (PDD) grant for fiscal
year 2024
Author Contributions
The
contribution
of
each
author
to
this
article
is:
Conceptualization,
Atina
and
Fitri
Suryani Arsyad; Methodology, Atina dan Fitri
Suryani
Arsyad;
Formal
Analysis,
Idha
Royani,
Assaidah
dan
Fitri
Suryani
Arsyad;
Writing
–
Original
Draft
Preparation,
Atina;
Writing
–
Review
&
Editing,
Idha
Royani,
Assaidah
dan
Fitri
Suryani
Arsyad;
Supervision,
Dedi
Setiabudidaya
dan
Miksusanti
.
Conflict of Interest
The authors declare that there is no conflict
of interest regarding the publication of this
paper.
Ethical Standards
This
article
does
not
contain
any
studies
involving human or animal subjects.
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