B I O D I V E R S I T A S ISSN: 1412-033X
Volume 22, Number 10, October 2021 E-ISSN: 2085-4722
Pages: 4457-4464 DOI: 10.13057/biodiv/d221039
Antibacterial activity of leaves, flowers, and fruits extract of Etlingera
elatior from Nagan Raya District, Indonesia against Escherichia coli and
Staphylococcus aureus
ERNILASARI
1
, KAMALLIANSYAH WALI L
1
, FITMAWATI
2
, DEWI INDRIYANI ROSLIM
2
, ZUMAIDAR
3
,
SAUDAH
1, ♥
, RAYHANNISA
4


1
Faculty of Teacher Training and Education, Universitas Serambi Mekkah. Jl. Unmuha, Batoh, Banda Aceh 23245, Aceh, Indonesia.
Tel./fax.: +62-651-26160,

email: [email protected], [email protected]
2
Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Riau. Kampus Bina Widya, Jl. H.R. Subrantas Km. 12,5, Simpang
Baru, Tampan, Pekanbaru 28293, Riau, Indonesia
3
Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Syiah Kuala. Jl. Tgk. Syech Abdul Rauf No. 3, Darussalam, Banda
Aceh 23111, Aceh, Indonesia
4
Mathematics and Applied Science Graduate Program, Universitas Syiah Kuala. Jl. Tgk. Syech Abdul Rauf No. 3, Darussalam, Banda Aceh 23111,
Aceh, Indonesia
5
Biology Graduate Program, Faculty of Mathematics and Natural Sciences, Universitas Syiah Kuala. Jl. Tgk Chik Pante Kulu No. 5, Kopelma
Darussalam, Syiah Kuala, Banda Aceh 23111, Aceh, Indonesia
Manuscript received: 6 August 2021. Revision accepted: 26 September 2021.
Abstract. Ernilasari, Walil K, Fitmawati, Roslim DI, Zumaidar, Saudah, Rayhannisa. 2021. Antibacterial activity of leaves, flowers,
and fruits extract of Etlingera elatior from Nagan Raya District, Indonesia against Escherichia coli and Staphylococcus aureus.
Biodiversitas 22: 4457-4464. Etlingera elatior is a medicinal plant that has been used by people in Indonesia, especially Acehnese
people. Based on its secondary metabolites, E. elatior can be used as antibacterial agents against Gram-positive and Gram-negative.
However, to determine the antibacterial activity of E. elatior, the parts of E. elatior that have been used are leaves, flowers, and fruits.
This study was aimed to determine the best concentration of ethanol extract of leaves, flowers, and fruits of E. elatior as an inhibitor
against Escherichia coli and Staphylococcus aureus. The extract was made by the maceration method with 70% ethanol as a solvent.
Antibacterial activity test was carried out by the diffusion agar method using concentrations of 0.5%, 1%, 1.5% and 2%. The results
showed that the fruit extract of E. elatior has antibacterial activity with an effective inhibitory zone at a concentration of 2% is 8.4 mm
(E. coli) and 2.4 mm (S. aureus). Meanwhile, antibacterial activity of the extract of leaves and flowers against E. elatior cannot
determine yet. Identification of leaves, flowers and fruits extract of E. elatior using GC-MS (gas chromatography-mass spectroscopy)
showed 56 compounds were detected.
Keywords: Antibacterial test, Escherichia coli, Etlingera elatior, Staphylococcus aureus, Suak Bugis
INTRODUCTION
Indonesia is one of the countries which is rich in
biodiversity, including medicinal plants that have been
used in healthcare, protect health and disease prevention
since time immemorial (Schroeder 2011; Sofowora et al.
2013). Indonesia is the second-highest number of
indigenous medicinal plants, after the Amazon rain forest
(Elfahmi et al. 2014). Hence, there is a high potential for
pharmaceutical and biotechnological research
opportunities. Recently, using traditional medicine as an
alternative treatment has increased significantly (World
Health Organization 2004). Traditional medicines have no
side effects, are more affordable than most conventional
medicines, as well as availability and compatibility as
plant-derived drugs (Fatima and Nayeem 2016; Juwita et
al. 2018). Etlingera elatior (Jack) R. M. Sm.) is one type of
plant in Indonesia that is widely used as traditional
medicine by local people (Silalahi 2016).
E. elatior Jack. RM is a species of plant belongs to
Zingiberaceae. E. elatior which has various local names,
i.e., Kecombrang in Java, Kincung in North Sumatera,
Honje in West Java, Sikala in Southeast Sulawesi, Bongkot
in Bali, etc (Poulsen 2012; Sabilu et al. 2017; Oktavia et al.
2019). Meanwhile, in Aceh, these species are known as
Bak Kala and Bungong Kala. E. elatior is a medicinal
plant that has been used by people in Indonesia, especially
Acehnese people. Empirically, E. elatior is used as
traditional medicine by the Gayo tribe, Central Aceh
District for the treatment of various diseases such as
coughs, bloating, fatigue and fatigue syndrome, abdominal
pain, as well as postpartum medicine (Saudah et al. 2021).
According to (Jackie et al. 2011; Maimulyanti et al.
2015), E. elatior contains secondary metabolites such as
flavonoids, saponins, phenols, terpenoids, tannins, glycosides,
steroids, which have bioactivities. This bioactive
compound has been reported to exhibit antimicrobial,
antidiabetic, anticancer, antioxidant, antiaging, as well as
anti-inflammatory (Habsah et al. 2005; Lachumy et al.
2010; Zan et al. 2011; Srey et al. 2014; Ghasemzadeh et al.
2015; Nor Asiah et al. 2019). Previous studies reported that
E. elatior leaves from aqueous extract can inhibit the

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growth of Escherichia coli and Staphylococcus aureus
(Ningtyas 2010). Inline to (Sukandar et al. 2011), the
aqueous extract of E. elatior leaves has antibacterial
activity against E. coli (10 mm/100%) and S. aureus (8.663
mm/20%). In addition, Sukandar (2010) also revealed that
the aqueous extract of E. elatior flower has antibacterial
activity against E. coli (4.8 mm/60%) and S. aureus (6.87
mm/20%).
Phenolic and flavonoids had been reported to have
antimicrobial activity through the mechanism of action of
bacterial cell-wall destruction (Manoi 2009; Mahboubi et
al. 2016). The mechanism of action of saponin as an
antibacterial is it can cause leakage of proteins and
enzymes of the cell (Madduluri et al. 2013). Saponins
including glycosides and aglycone portions, while aglycone
portion is steroid and triterpenoid (Manoi 2009). Saponins
are divided into three major groups as a triterpenoid,
steroid or steroidal glycoalkaloid (Mert-Türk 2005).
Another secondary metabolite is tannin, where tannin has
ability to pass through the bacterial cell wall up to the
internal membrane and inhibit the reverse transcriptase
enzyme and DNA topoisomerase so that bacterial cells
cannot be formed (Dabbaghi et al. 2009).
Escherichia coli is a gram-negative bacteria or also
known as E. coli. E. coli is commonly found in the lower
intestine of humans and animals and causes acute diarrheal
diseases in all age groups (Bettelheim 2000). To detect the
presence of bacteria in surface water, E. coli is commonly
used as indicator of fecal contamination. Meanwhile,
Staphylococcus aureus is gram-positive aerobic organism
that causes skin infections, necrosis, abscess formation,
sometimes pneumonia, osteomyelitis and endocarditis
(Jawetz 2005; Bush and Vazquez-Pertejo 2021). S. aureus
is resistant to several antibiotics, including the lactamase,
methicillin, nafcillin, oxacillin and vancomycin groups
(Jawetz et al. 2008). Resistance is a global problem that
often arises in the treatment of infectious diseases. The
increase in bacterial resistance to antibiotics provides a
great opportunity to obtain antibacterial compounds by
utilizing bioactive compounds from the diversity of plants
in Indonesia (Nuria et al. 2009).
Based on the description of E. elatior as medicinal plant
and its potential as antibacterial agent against E. coli and S.
aureus. Therefore, the aim of this study is to determine the
best concentration of ethanol extracts of leaves, flowers
and fruits of E. elatior to inhibit the growth of E. coli and
S. aureus together with identification of its chemical
profiles by GC-MS analysis.
MATERIALS AND METHOD S
Study area
The research was conducted in Suak Bugis, Nagan
Raya District, Aceh, Indonesia (Figure 1). The experiment
was conducted at the Regional Health Laboratory, Jakarta
and Laboratory of Microbiology, Department of Biology,
Faculty of Mathematics and Natural Sciences, Syiah Kuala
University, Banda Aceh, Indonesia, from October to
December 2020.
Procedures
Sample collection
The research was used purposive sampling method.
Samples of E. elatior were collected from seven different areas
of Suak Bugis, Nagan Raya District, Aceh including leaves,
flowers and fruits. The leaves, flowers and fruits were
taken randomly from each stem. The sample was identified by
Saida Rasnovi from Herbarium of the Department of
Biology, Syiah Kuala University as E. elatior. The
coordinate of each location is presented in Table 1.







Figure 1. The collection sites of Etlingera elatior in Suak Bugis, Nagan Raya District, Aceh, Indonesia

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Table 1. The coordinates of the Etlingera elatior sampling area

Species Sample type Sampling area Sampling year The coordinates Information
E. elatior Leaves, flowers, fruit Suak Bugis 2020 96.49311”; 3.850495” Point 1 (T1)
E. elatior Leaves, flowers, fruit Suak Bugis 2020 96.491415”; 3.836671” Point 2 (T2)
E. elatior Leaves, flowers, fruit Suak Bugis 2020 96.473506”; 3.7832” Point 3 (T3)
E. elatior Leaves, flowers, fruit Suak Bugis 2020 96.479313”; 3.777966” Point 4 (T4)
E. elatior Leaves, flowers, fruit Suak Bugis 2020 96.470503”; 3.776872” Point 5 (T5)
E. elatior Leaves, flowers, fruit Suak Bugis 2020 96.47989”; 3.764773” Point 6 (T6)
E. elatior Leaves, flowers, fruit Suak Bugis 2020 96.487234”; 3.761149” Point 7 (T7)



Sample preparation
Leaves, flowers, and fruit were harvested and washed
by distilled water then cut into small pieces. Then air-dried
samples in the open space. After drying process, samples
were stored at 20°C and pulverized into powders.
Extraction
The extract was obtained by the maceration method
with 70% ethanol as a solvent. The extraction process
begins with making simplicial from leaves, flowers, and
fruits that have been pulverized. The simplicial powder of
about 100 grams was inserted into container, then add
solvent until all ingredients were submerged and left for 24
hours. While stirring every 1 x 24 hours, after 24 hours
the mixture was filtered using Whatman filter paper in a
filter funnel. The filtrate was stored and the residue was
soaked in a new solvent for 24 hours and repeated until the
solvent was colorless. The filtrate obtained was then
separated by a vacuum rotary evaporator at 50°C to
separate the solvent from the thick extract filtrate of leaves,
fruits, and flowers. The filtrate was stored in bottles at
20°C for further analysis.
Phytochemical screening
Phytochemical tests were performed on leaves, flowers
and fruit extract of E. elatior to determine the content of
flavonoids, saponins, tannins, alkaloids, triterpenoids, and
steroids qualitatively.
Alkaloid testing
A total of 3 mL of each sample extract was added by
the reagents of Mayer, Dragendorff, and Wagner. The
positive result of Mayer reagent was confirmed by white
precipitate, red precipitate (Dragendorff) and brown
precipitate (Wagner).
Phenolic testing
A total of 3mL of extract was put into a test tube, then
one drop of 1% FeCl3 solution was added to 3 mL of the
extract solution. A positive result of phenolic was indicated
by the formation of a yellow, orange or red color.
Flavonoid testing
A total of 3 mL of sample extract was put into a test
tube, then added 0.1 gram of magnesium powder, 1 mL of
concentrated HCl and 1 mL of amyl alcohol. The mixture
was shaken vigorously and allowed the layers to separate.
The presence of flavonoids is indicated by the formation of
red in the amyl alcohol layer.
Saponin testing
A total of 3 mL of sample extract was put into a test
tube, then shaken vigorously and left for 10 minutes. The
presence of saponin is indicated by the formation of stable
foam.
Tanin testing
A total of 3 mL of sample extract was put into a test
tube, then add 3 drops of 1% FeCl3. The formation of blue
or greenish-black color indicates the presence of tannins
compounds.
Steroids and triterpenoids testing
A total of 3 mL of sample extract was dissolved in hot
ethanol, heated for 5 minutes, then the solution was
filtered. The filtrate result was dried by heating, then 1 mL
of diethyl ether was added. A total of 3 drops of the ether
fraction were transferred to a drip plate, then add 3 drops of
acetic anhydride and 1 drop of concentrated sulfuric acid.
Formation of reddish or violet color indicates the presence
of triterpenoids, whereas the formation of green or blue
color indicates the presence of steroids.
Gas Chromatography-Mass Spectroscopy (GC-MS) test
The analytical method using Gas Chromatography-
Mass Spectroscopy (GC-MS) can measure the type and
content of compounds in a sample both qualitatively and
quantitatively. Leaves, flowers and fruits extract of E.
elatior were analyzed using GC-MS Agilent 19091S-436
HP-5MS. 1 µL of the extract was injected into GC-MS
which was operated using a glass column length of 25 m,
diameter: 0.25 mm and thickness of 0.25 µm with the CP-
Sil stationary phase. 5CB with an oven temperature
between 70°C up to 325°C with an increased rate of 10°C/
min, a carrier gas of helium with a pressure of 16.30 kPa, a
total rate of 40 mL/min and a split ratio of 1:50.
Antibacterial activity test by disc diffusion method
Antibacterial activity test was carried out by the disc
diffusion method using paper discs (about 6 mm in
diameter) for E. coli and S. aureus bacteria. The antibacterial
testing was carried out in three repetitions. The paper discs
were immersed in samples with concentrations of 0.5%,
1%, 1.5% and 2%, then placed on culture medium (Mueller
Hinton Agar) that has been inoculated with a suspension of
the pathogen. Incubated at 37
o
C for 2x24 hours.

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Observations were made by measuring the inhibition zone
diameter of inhibition zone based on the paper discs
(Banjara et al. 2012; Chusri et al. 2011). The negative
control used in this study is DMSO 10% (v/v).
Data analysis
The presence of bioactive compound was analyzed
by Gas Chromatography-Mass Spectroscopy (GC-MS) and
antibacterial activity test was analyzed by quantitative
method. The measurement of inhibition zone was
determined by a caliper in each concentration.
RESULTS AND DISCUSSION
Identification of active compounds
Phytochemical test is a qualitative test of the content of
the groups of secondary metabolites in a sample. Analysis
of the chemical content of the leaves, flowers, and fruits of
E. elatior was conducted at the Regional Health
Laboratory, Jakarta by observing the presence or absence
of precipitation reactions and color changes in the test tube.
The analysis of phytochemical screening is presented in
Table 2.
Antibacterial activity test against E. coli
The results of the antibacterial activity test of the
ethanol extracts of E. elatior fruit on the growth of E. coli
are presented in Table 3. Inhibition zones around disc paper
were indicated antibacterial activity. The observation of
the inhibition zone was carried out by diffusion agar
method. The results of the antibacterial activity test on the
three test extracts with concentrations of 0.5%, 1%, 1.5%,
and 2% on E. coli were shown in Figure 2.
Antibacterial activity test against S. aureus
The results of the antibacterial activity test of the
ethanol extracts of E. elatior fruit on the growth of S.
aureus were presented in Table 4. The results of the
antibacterial activity test on the three test extracts with
concentrations of 0.5%, 1%, 1.5% and 2% on S. aureus can
be seen in Figure 3.
GC-MS analysis
The results of the GC-MS analysis of E. elatior leaf,
flower and fruit extracts are presented in Table 5, Table 6
and Table 7.
Discussion
The results of phytochemical analysis showed that
the leaves extract of E. elatior contained alkaloids, phenolic,
flavonoids, saponins, tannins and steroids contents.
The flowers extract of E. elatior contains alkaloids,
phenolic, flavonoids, saponins, tannins and terpenoids.
While the fruit extract of E. elatior contains alkaloids,
phenolic, flavonoids, tannins and terpenoids (Table 2).
These results following with (Effendi et al. 2019), where
the results of phytochemical testing showed that the leaves
powder of E. elatior contains alkaloid, flavonoid, saponin,
steroid, tannin and phenolic. Kecombrang flower (E.
elatior) contains flavonoid, saponin, tannin and terpenoid
(Farida and Maruzy 2016). Meanwhile, the phytochemical
compounds contained in the Kecombrang fruit (E. elatior),
i.e., alkaloids, polyphenols, flavonoids, saponins and
essential oils (Handayani 2010). Ahmad et al. (2015) also
revealed that the total phenolic content for E. elatior fruit
was 2.29 mg GAE/g extract, while the total flavonoid
content for E. elatior fruit was 1.78 mg QE/g extract.
The presence of alkaloids, phenolic, flavonoids,
saponins, tannins and terpenoids in this study indicated that
extract of leaves, flowers and fruit of E. elatior has the
potential as an antibacterial and antimicrobial. In line with
Abdallah (2011) and Sadeek and Abdallah (2019), the
bioactive compounds in E. elatior are known as potential
sources for antibacterial agents. According to Juwita et al.
(2018), the high potential antimicrobial activity possessed
by E. elatior is due to the presence of phenolic, terpenoid
and flavonoid contents.


Table 3. The results of antibacterial activity tests of the ethanol
extracts of Etlingera elatior fruit against Escherichia coli

Concentrati
ons (%)
Average
(mm)
Diameter zone of inhibition to
nearest mm
0.5 0 Weak
1 2.5 Weak
1.5 7.8 Intermediate
2 8.4 Intermediate


Table 4. The results of antibacterial activity tests of the ethanol
extracts of Etlingera elatior fruit against Staphylococcus aureus

Concentrations
(%)
Average
(mm)
Diameter zone of inhibition
to nearest mm
0.5 0 Weak
1 0.7 Weak
1.5 1.9 Weak
2 2.4 Weak


Table 2. Qualitative phytochemical analysis Etlingera elatior

Phytochemical
screening (extract)
Alkaloids
Phenolic Flavonoids Saponins Tannins Steroids Terpenoids
Mayer Dragendorff Wagner
E. elatior fruits + + + + + - + - +
E. elatior leaves + + + + + + + + -
E. elatior flowers + + + + + + + - +

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Table 5. The results of GC-MS analysis of leaves extract of Etlingera elatior

Peak Retention time Quality Chemical components Content (%)
1 26.045 94 Neophytadiene 2,35
2 27.258 47 Hexadecanoic Acid, Methyl Ester 1,48
3 27.803 42 Ethyl 4-methyl octanoate 5,81
4 28.307 99 n-Hexadecanoic acid 3,73
5 28.541 89 n-Hexadecanoic acid 1,68
6 28.706 70 phytol 4,05
7 29.334 89 2-Aminoethanethiol Hydrogen Sulfate (Ester) 3,71
8 29.513 49 Myrrhine 2,11
9 29.706 702 Phenol, 2-(1-methyl-2-butenyl)-4-methoxy- 1,86
10 29.989 46 Phenol, 2-(1-methyl-2-butenyl)-4-methoxy- 3,04
11 30.934 53 Undeca-3,4-diene-2,10-dione,5,6,6-trimethyl- 7,02
12 31.134 58 (+)-(9.Beta.H)-Labda-8 (17), 13 Є-Diene-5-OL 2,90
13 31.458 90 Z,E-3,13-Octadecadien-1-olacetate 2,77
14 31.733 99 Squalene 3,92
15 32.002 58 1-Benzyl-4,6-Dimethoxy-2,3-Diphenylindole 1,61
16 32.120 48 Cholesta-4,6-Dien-3-OL, 6-Fluoro-(3.Beta)- 1,93
17 32.451 46 1,3-Dithiolen, 2-(28-Norurs-12-EN-17-YL)- 1,16


Table 6. The results of GC-MS analysis of flower extract of Etlingera elatior

Peak Retention time Quality Chemical components Content (%)
1 6.773 38 Erythritol 7,66
2 10.875 93 2-Methoxy-4-Vinylphenol 1,14
3 15.302 95 1-Decene 4,68
4 19.232 81 Tridecyl acetate 3,09
5 27.183 96 Hexadecanoic Acid, Methyl Ester 4,76
6 27.989 38 8-Thiabicyclo [3,2,1]octan-3-ol-6-methoxy-, (3-endo, 6-exo)- 9,39
7 28.238 99 n-Hexadecanoic acid 5,15
8 28.479 74 Cis-13-Octadecenoic acid, emthyl ester 9,30
9 29.217 99 9,12-Octadecadienoic acid 5,79
10 29.520 70 1,3;2,5-Dimethlene-4-methyl-d-rhamnitol 3,31
11 29.872 92 2-Dodecen-1-yl(-)succinic anhydride 3,47
12 30.292 60 2,15-Hexadecanedione 2,28
13 30.672 97 1-Docosane 3,87
14 30.982 44 10H-Phenothiazine 3,54
15 31.444 78 14-.Beta.-H-Pregna 6,99
16 31.865 78 Methanone, (2-Aminophenyl) (2-Methoxyphenyl)- 6,49
17 32.265 56 Hexadecanoic acid, 2-hydroxy-, methyl ester 1,23
18 32.499 96 1-Docosane 4,57
19 33.016 64 Anthranilic Acid, N-Methyl-N-Phenyl- 1,65
20 33.568 47 1,3-Dioxolone, 2,2,4-trimethyl-5-tridecyl- 2,40
21 33.795 97 (9Z)-9,17-Octadecadienal 2,53
22 3 (5)-(2hydrophenyl)-5(3)-Phenylpyrazole 1,55
23 49.013 56 Tricosane 1,70


Table 7. The results of GC-MS analysis of fruit extract of Etlingera elatior

Peak Retention time Quality Chemical components Content (%)
1 5.538 27 1-Methyl-4-Methylenecyclohexane 1,42
2 6.428 72 6-Azabicyclo [3.2.1] octane 3,98
3 7.407 38 3-Ethoxy.Gamma.Butyrolactone 6,14
4 11.392 37 2-N-Propoxyamphetamine 1,11
5 12.785 27 1-Cyclopentene-1-1methanol, .Alpha.-Ethyl- 3,77
6 25.762 30 (Tetrahydroxy Cyclopentadienone) Trcarbonyliron (0) 2,51
7 27.045 50 (3Z)-3-Ethyl-2-Methyl-1,3-Hexadiene 7,45
8 27.189 97 Hexadecanoic Acid, Methyl Ester 5,09
9 27.762 64 Ethyl 3-Cyclohexylpropanoate 5,01
10 28.279 95 n-Hexadecanoic acid 10,01
11 28.493 99 9-Octadecanoic acid (Z)-, methylester 8,21
12 28.865 55 2-Aminoethanethiol Hydrogen Sulfate (Ester) 5,24
13 29.244 98 Oleic Acid 16,31
14 30.706 59 Cis-11-Hexadecal 4,86
15 30.934 91 Octadecenoate 2,67
16 31.451 95 14-.Beta. –H-Pregna 5,09

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Figure 2. The diameter of the inhibition zone of Escherichia coli from the fruit, leaf and flower extract of Etlingera elatior with
concentrations of 0.5%, 1%, 1.5% and 2%.




Figure 3. The diameter of the inhibition zone of Staphylococcus aureus from the fruit, leaf and flower extract of Etlingera elatior with
concentrations of 0.5%, 1%, 1.5% and 2%.



In this study, E. elatior was obtained at various of
latitudes (Table 1). Where the latitude can affect the
production of secondary metabolites in plants. These
results following with Demasi et al. (2018), which stated
that the distance of sea (latitude) had an influence on both
morphological and phytochemical traits. The contents
of active substances in a plant were influenced by their
environment (Liu et al. 2016). The presence or absence of
certain secondary metabolites in medicinal plants are
influenced by a variety of factors such as light, climate,
temperature, groundwater, soil fertility and salinity (Giweli
et al. 2013). The concentrations of phenolic compounds in
plant were influenced by temperature, soil composition,
ultraviolet radiation and rainfall (Kouki and Manetas 2002;
Monteiro et al. 2006). The tannin's content can be
influenced by environmental changes and development of
the plant (Hatano et al. 1986; Salminen et al. 2001).
The data in Table 3 showed that the fruit extract of E.
elatior has antibacterial properties to inhibit the growth of
E. coli with an average zone of inhibition size of 8.4
mm/2%, 7.8 mm/1.5% and 2.5 mm/1%. The fruit extracts
of E. elatior are seen by the diameter of inhibition formed
around the paper discs (Figure 3). However, the best
extracts of E. elatior fruit to inhibit the growth of E. coli
was at a concentration of 2% (8.4 mm). Those
values translated to categories of intermediate. A higher
concentration of ethanol extract, which may will produce a
larger zone of inhibition. According to Morales et al.
(2003), the activity of the antimicrobial inhibition zone was
grouped into four categories, i.e., weak activity (<5 mm),
intermediate (5-10 mm), strong (>10-20 mm) and very
strong (>20-30 mm). Previous study revealed that the
Kecombrang fruit peel formula has antimicrobial activity
to inhibit the growth of E. coli bacteria with ranges from
24,103-26,877 mm (Naufalin 2013).
The data in Table 4 showed that the fruit extract of E.
elatior also has antibacterial properties to inhibit the
growth of S. aureus with an average zone of inhibition size
of 2.4 mm/2%, 1.9 mm/1.5% and 0.7 mm/1%. However,
those values translated to categories of weak. In line with
Pan et al. (2009), the inhibition zone of 0-3 mm includes
the weak category, 3-6 mm good, and ≥6 mm strong.
Simangunsong (2019) reported that the fruit extract of E.
elatior has antibacterial activity to inhibit the growth of S.
aureus at a concentration of 500 mg/mL with the diameter
of clear zone of 22.06 mm. Meanwhile, the negative
control used in this study is DMSO 10% (v/v). DMSO is a
surfactant that can dissolve polar and nonpolar materials. It
also showed no antibacterial activity in Figure 3 and Figure
4 (Brito et al. 2017).
The difference in diameter of inhibition zones at each
concentration possibly was due to differences in the
magnitude of active substances contained in the extract. In
addition, the diameter of the inhibition zone was also
influenced by the level of sensitivity of the bacteria. E. coli
had higher antibacterial activity than S. aureus. This is
related to the differences in the constituent components of
the cell structure. Gram-positive bacteria contain 90%
peptidoglycan and a thin layer of negatively charged
teichoic acid and teichuronic acid. In Gram-negative
bacteria, there is an outer layer of the cell wall that contains
5-20% peptidoglycan. This layer is the second lipid layer

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called the lipopolysaccharide layer. This layer is composed
of phospholipids, polysaccharides, and proteins (Madigan
et al. 2000).
The result of GC-MS analysis of leaves extract of E.
elatior in Table 5, it showed that the main components of
the extract are undeca-3,4-diene-2,10-dione,5,6,6-
trimethyl-with content of 7,02% and ethyl 4-methyl
octanoate with content of 5,81%. The main components of
the extract of E. elatior flower are 8-Thiabicyclo
[3,2,1]octan-3-ol-6-methoxy-, (3-endo, 6-exo)- with
content of 9,39% and cis-13-Octadecenoic acid, methyl
ester with content of 9,30% (Table 6). In addition, the main
components of the extract of E. elatior fruit are oleic acid
with content of 16,31% and n-Hexadecanoic acid with
content of 10,01%. GCMS is a method of separating
organic compounds using gas chromatography (GC) and
mass spectrometry (MS). The principle of separation is
based on differences in the volatile ability of the compound
and based on interactions with the stationary phase
(capillaries).
ACKNOWLEDGEMENTS
The authors would like to thank WRI (World Resources
Institute). Our special thanks to all staff members in the
Laboratory of Microbiology, Department of Biology,
Faculty of Mathematics and Natural Sciences, Syiah Kuala
University, Banda Aceh, Indonesia. This research was
funded by the Indonesian Ministry of Research,
Technology and Higher Education (Directorate of Research
and Community Service-DRPM 2021, grant number
025/LL13/LT/AKA/2021–090/LPPM-USM/VII/2021).
This research was supported by the Ministry of Research
and Technology/National Research and Innovation Agency
of the Republic of Indonesia (Kemristek-Brin) 2021
University Cooperation Research (PKPT) scheme.
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