Potential Endophytic Fungi from the Leaves of Syzygium

zeylanicum and Their Secondary Metabolite

Syarifah1,2, Elfita3*, Hary Widjajanti4, Arum Setiawan4, Alfia Rahma Kurniawati2

1
Graduate School of Sciences, Faculty of Mathematics and Natural Sciences, University of Sriwijaya,
Jl. Padang Selasa, Palembang, 30139
2
Universitas Islam Negeri Raden Fatah, South Sumatera, Indonesia
3
Department of Chemistry, Faculty of Mathematics and Natural Sciences, University of Sriwijaya,
Indralaya, 30862
4
Department of Biology, Faculty of Mathematics and Natural Sciences, University of Sriwijaya,
Indralaya, 30862
Corresponding Author *: elfita.elfita.69@gmail.com

ABSTRACT
Endophytic fungi are microscopic living organisms that live in plant tissues (leaves, fruits,
seeds, stems and roots) at certain periods by forming colonies without harming their hosts, even
having mutually beneficial relationships. Endophytic fungi generally produce secondary
metabolites that have beneficial biological activities such as antibacterial, antioxidant,
antiviral, antiprotozoal, antidiabetic, and anticancer. Endophytic fungi can be found in various
types of plants, especially medicinal plants, such as Syzygium zeylanicum, one of the many
medicinal plants found in Indonesia. This study aimed to obtain endophytic fungal isolates
from the leaves of Syzygium zeylanicum L., to characterize endophytic fungi, and to test the
antibacterial and antioxidant activity of the obtained endophytic mushroom extracts.
Characterization of endophytic fungi was carried out morphologically and fungi that showed
high bioactivity were characterized molecularly. Antibacterial activity was carried out by disc
diffusion method and antioxidant activity by DPPH method. In this study, 4 isolates of
endophytic fungi were obtained. Based on the morphological characterization of the
endophytic fungi, they are Penicillium citrinum (Code ZL6), Colletotrichum lindemuthianum
(Code ZL8), Aspergillus nidulan (Code ZL9), Scopulariopsis asperula (Code ZL10).
Penicillium mushroom extract (Code ZL6) provided strong antibacterial activity against 4
bacterial pathogens (71% against E. coli; 74% against S. aureus; 76.1% against S. typi and
76.9% against B. subtilis). The antioxidant activity of all fungal endophytic extracts showed a
very strong activity (IC50 ZL6 extract = 7.29 g/mL). The potential endophytic fungus ZL6
based on molecular analysis is Penicillium citrinum.

Keywords: Antibacterial, antioxidant, endophytic fungi, secondary metabolites

1. Introduction
Free radicals are reactive oxygen and nitrogen that go through various physiological
processes in the human body. Uncontrolled radical production can attack lipid membranes,
proteins, enzymes and DNA causing oxidative stress and cell death. Reactive oxygen species
(ROS) play an important role in many human diseases such as diabetes, cancer,
neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, atherosclerosis, aging
and inflammation. The reduction of free radicals can use antioxidant compounds. Antioxidants
are stable molecules that can donate electrons to active free radicals, and end chain reactions
before important molecules are damaged. Elimination of free radicals can delay or inhibit cell
damage (Yang et al., 2018).

Antioxidant activity can be found from various phytochemical studies, one of which is
the plant Syzygium zeylanicum. Research by Nomi et al. (2012) found zelaniin A compounds
in S. zeylanicum leaves which showed strong antioxidant activity of 19.18 ± 0.24 and 4.37 ±
0.29 (mol TE / mol) in the DPPH and ORAC tests. , and nearly 80% lipid peroxidation
inhibition at the level of 100 g/mL (78.76 ± 0.21%). In addition, the plant extract of S.
zeylanicum showed the presence of alkaloids, glycosides, phenolics, flavonoids, steroids,
terpenoids and saponins which showed antioxidant activity from the test results using nitric
oxide of 78.79% (Anoop & Bindu, 2014). The accumulation of bioactive compounds can be
supported by the role of endophytic fungi by helping to increase the production of secondary
metabolites in their host plants (Gupta et al., 2020; Khare et al., 2018; Yuan et al., 2019).
Besides the impact of free radicals, various infectious diseases caused by pathogenic
microbes are also of concern today. Pathogenic microbes include Salmonella thypi, Eschericia
coli, Shigella dysenteriae., Staphylococcus aureus. Microbial infections pose a serious
challenge to human health because of the nature of microbial resistance to antibiotics (Dos
Santos et al., 2015). It takes a strategy to fight pathogenic microbes by finding new anti-
bacteria, where one of the potential sources comes from plants.
Endophytic fungi, especially those that live in medicinal plant tissues, are often used as
a source for the discovery of bioactive compounds. Some plants can degrade the bioactive
compounds they contain to endophytic microbes that grow in their tissues, so that these
endophytic microbes can produce the same compounds as their hosts. In our previous study
(Syarifah et al., 2021) we found an antibacterial compound p-hydroxybenzaldehyde from
endophytic fungi isolated from the root bark of Syzygium zeylanicum. This p-
hydroxybenzaldehyde compound is also produced by its host. To continue our series of
research, in this paper we report the bioactive potential of endophytic fungi from other parts of
the S. zeylanicum plant, namely the leaves and bark. This Syzygium zeylanicum plant has been
used by people in Indonesia as a medicinal plant related to pathogenic bacterial infections and
the effects of free radicals in the body.

2. METHODOLOGY
2.1 Plant Material
Endophytic fungi were isolated from the root bark of S. zeylanicum (L.) from Penukal
Abab Lematang Ilir (Pali) Regency, South Sumatra. Plants have been identified in the
laboratory of the Center for Biological Research – LIPI in the field of botany. Sampling was
carried out in a fresh state in July 2020. Endophytic fungi were isolated from leaf organs and
stem bark. The leaves used are the leaves in the third position of the test. The root bark used is
the root bark that comes from the roots in the soil.

2.2 Isolation of Endophytic Fungi

Isolation of endophytic fungi was started by sterilizing the surface of the leaves and bark
of the guava – rice plant. Fresh plant organs were washed with running water until clean for ±
5 minutes. Then soaked using 70% alcohol for ± 3 minutes. Then rinsed with sterile distilled
water for ± 1 minute, then soaked with Sodium hypochloride (NaOCl) 3% (w/v) for 1 minute.
The surface sterilized leaves were cut aseptically ± 2 cm, while the root bark was cut ± 3 x 0.5
cm. Then the samples were planted in petri dishes containing PDA media, incubated at room
temperature for 3-14 days. Observations were made every day until the fungus was visible.
Fungal colonies growing on PDA medium that showed different morphological properties
(shape, color and size) were further purified. Purification was carried out by transferring the
colonies to a new PDA medium with a single spore isolation method, then incubating at room
temperature for 2 x 24 hours. The separate fungal colonies were purified and then made into
working cultures (in Petri dishes) and stock cultures (in test tubes) by growing on PDA media
(Fitriarni & Kasiamdari, 2018; Hanin & Fitriasari, 2019).

2.3 Identification of Endophytic Fungi

Identification of endophytic fungi was carried out based on their phenotypic characters
macroscopically and microscopically. Observations of colony characteristics included: a)
colony color and reverse side color, b) colony surface: granular, powdery, mountainous,
slippery. c) Presence or absence of exudate drops, d) Presence or absence of radial lines (radial
furrow) from the center of the periphery of the colony, e) presence or absence of concentric
circles. And microscopic observations include the shape of the hyphae or mycelium, the shape
of the spores, the color of the spores, the presence or absence of a septum on the hyphae and
other microscopic characteristics. The phenotypic identification data were then compared with
identification key books such as identification keys Pictorial Atlas of Soil and Seed Fungi
Morphologies of Cultured Fungi and Key to Species (Watanabe, 2010), Larone's medically
important fungi (Walsh et al., 2018) and Fungi and Food Spoilage (Pitt & Hocking, 2009).

2.4 Molecular Analysis of ITS rDNA

Isolates were identified based on the Internal Transcribed Spacer (ITS) area of DNA
(rDNA). Amplification using universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3')
and ITS 4 (5'-TCCTCCGCTTATTGATATGC-3'). DNA sequences of forward and reverse
primers were compiled using the Bioedit program. The sequence results were then identified
to the level of taxa species using the online bioinformatics method of the Basic Local
Alignment Search Tool (BLAST) at the website address http://blast.ncbi.nlm.nih.gov/Blast.cgi.
Furthermore, the sequences were multiple aligned using the MEGA11 program with the
CLUTAL W method and a phylogenetic tree was constructed using the Neighbor-joining tree
method with a bootstrap value of 1000.

2.5 Cultivation and Extraction

All endophytic fungal strains were cultivated by placing 6 blocks of pure culture agar (±6
mm in diameter) into 300 mL Potato Dextrose Broth (PDB) medium. Each isolate was
cultivated as much as 300 mL x 5 Erlenmeyer flasks. The cultures were then incubated for four
weeks at room temperature under static conditions. After the incubation period, the mycelia
were separated from the broth culture using filter paper. Then ethyl acetate solvent was added
to the culture medium (1:1) and extracted by partition (repeated three times). The ethyl acetate
extract was separated from the liquid culture and evaporated using a rotary evaporator to obtain
a clear extract (Budiono et al., 2019; Deepika et al., 2014). The extract was thickened using an
oven at 40°C. The viscous extract and biomass were weighed using an analytical balance.

2.6 Antibacterial Activity Test

Antibacterial activity test using Kirby-Bauer method with NA (Natrium Agar) medium.
There were four test bacteria used, two Gram-negative bacteria (Escherichia coli InaCCB5 and
Salmonella thypi ATCC1048 and two Gram-positive bacteria (Staphylococcus aureus
InaCCB4 and Bacillus subtilis InaCCB1204). The blank disc paper was dripped with 20 L of
endophytic fungal extract with a concentration of 400 g/disk. Dilution of the mushroom extract
using dimethylsulfoxide (DMSO), then left until all the solvent evaporates completely. Positive
control uses Tetracycline 30 g/disk. The disc paper containing the test solution is placed on the
surface of the media that has been inoculated with the test bacteria. Then it is incubated for
1x24 hours at room temperature. 37° C, then observed the inhibition zone. The diameter of the
formed inhibition zone was measured using a caliper. Determination of the antibacterial
activity of the test sample and the criteria for the diameter of the inhibition zone were
determined by the following equation:
A A A
Weak : Bx 100% <50 %; moderate: 50 % <Bx 100% < 70 %; Strong: Bx 100% ˃ 70 %

A: zone of inhibition (mm) test sample

B: zone of inhibition (mm) of standard antibiotics

2.7 Antioxidant Activity Test

Antioxidant activity was determined using the DPPH method. The fractions obtained
from the extraction procedure were dissolved into concentrations of 1000, 500, 250, 125, 62.5,
31.25, 15.625 g/mL. 0.2 mL of each concentration was added 3.8 mL of 0.5 mM DPPH. As a
standard antioxidant ascorbic acid was used and at least three replicates of each concentration
were considered (Goswami & Ray, 2017). The mixture was homogenized and left in a dark
tube for 30 minutes. Absorption was measured using a UVVis spectrophotometer at max 517
nm. In this test, ascorbic acid was used as a standard positive control and methanol as a negative
control. Antioxidant activity can be represented by the value of DPPH absorption inhibition,
which is calculated by the percentage inhibition of DPPH absorption and the IC50 value.
Ak − As
% Inhibition =
As
Ak = Absorbance of control
As = Absorbance of samples

3. RESULTS AND DISCUSSION

3.1 Isolation of Endophytic Fungi
Endophytic fungal isolate colonies showed various physical appearances. Massive
hyphae growth appeared in the root bark samples, with a predominance of white colonies and
yellow pigmentation. The results of the isolation of endophytic fungi from the root bark of
Jambu Nasi Nasi obtained 4 isolates with codes SZL6, SZL8, SZL9, SZL10 (Figure 1). These
fungal colonies showed different morphological properties (shape, color, and size) and were
further purified. Endophytic fungi from root bark showed colony diversity in terms of colony
size, color (white, black, green, and gray) and texture (velvety, cottony and powdery).
 ZL6 colonies with the same front and reverse color characteristics, namely dark green,
velvety textured and wrinkled surface, with a zoning growth pattern. ZL8 with a characteristic
dark green front color and transparent reverse color, has a cotton-like texture with a zoning
pattern. ZL9 with a characteristic dark green front color with white edges and orange white
edges, has a velvet-like texture with a zoning pattern. ZL10 has a characteristic pale brown
front color with white edges and a light orange whitish reverse color, a zoning growth pattern
with a cotton-like texture.

3.2 Morphological Identification of Endophytic Fungi

Figure 1. Colony and microscopic morphology of endophytic fungal species; ZL6(1); ZL8(2); ZL9(3); ZL10
(4); A- pengamatan makroskopis ; B- pengamatan mikroskopis

Table 1. Colony characteristics of endophytic fungi from the leaves and trunk bark of S. zeylanicum
Decription ZL6 ZL8 ZL9 ZL10

Colony Dark green Dark green Drak green Pale brown

Reverse colony Dark green Light green White and orange in Light orange and
Macroscopic

center white
Structure Velvety Velvety Velvety Cottony
Elevation Rugose Flat Flat Flat
Pattern Zonate Zonate Zonate Zonate
Exudate drop No No Yes No
Radial Line Yes No No Yes
Coencentric line No No Yes No
Spore Conidia Conidia Conidia Conidia
Shape Subglobose Phialosporous Phialosporous Phialosporous
Microscopic

Hyphae Septate Septate Septate Septate

Bearing catenulate Conidia simple or Conidial head have Catenulete
Characteristic conidia
in each branch branched, erect short columnarce
basipetally
Genus/Species Cladosporium Colletotrichum Aspergillus nidulan Scoplulariopsis
cladosporia lindemuthianum asperula
Note: (-) = characteristic doesn’t appear; (√) = characteristic appear
 Phenotypic analysis showed that these 4 isolates were grouped into three classes
Dothideomycetes (SZL6), Sordariomycetes (SZL8, dan SZL10), Euriomycetes (SZL9). Table
2. Endophytic fungi isolated from the root bark of S. zeylanicum showed a variety of spore
forms (verticillate, globose and subglobose). Most of the isolates had septate hyphae, except
isolates SZR6 and SZR8 which had coenocytic hyphae.
The identified ZL6 isolate of Penicillium citrinum was also found in the medicinal plant
Stephania kwangsiensis with potential to control phytopathogens. And found citrinin and
emodin compounds (Luo et al., 2019). The ZL8 isolate identified by Colletotrichum was also
found in Buxus sinica plants with antibacterial potential against S aureus, P. aeruginosa, E coli
and B subtilis bacteria. And found compounds chermisione B and colletoreichones (Wang et
al., 2016).
The ZL10 isolate identified by Scopulariopsis was also found in soybean (Glycine max
L. Merr.) and the ZL9 Aspergillus isolate was found in corn (Zea mays L.) with potential as
pest control (Russo et al., 2016). The ZL10 isolate identified by Scopulariopsis was also found
in cotton (Gossypium hirsutum), which had a synergistic effect on the pathogenicity of
Verticillium dahlia (Li et al., 2017).

3.3 Extraction and bioactivity Test

Table 2. Antibacterial and Antioxidant Activities of Endophytic Fungi from S. zeylanicum. leaves
Ethylacetate % Antibacterial activity
Antioxidant
Mushroom Genus/Species extract
activity IC50
code ofIdentification weight E. coli S. aureus S. thypi B. subtilis
(µg/mL)
(gram)
13.9 ± 15.2 ± 16.0 ±
Penicillium 18.3 ± 0.70 7.29
5.8 1.55 1.00 0.30
citrinum *** ****
ZL6 *** *** ***
16.4 ± 12.2 ± 17.4 ±
Colletotrichum 18.2 ± 0.41 3.99
5.2 0.41 0.74 0.62
lindemuthianum *** ****
ZL8 *** ** ***
15.3 ± 12.9 ± 15.0 ±
Aspergillus 14.4 ± 0.43 3.85
5.5 1.64 0.98 0.37
nidulan ** ****
ZL9 *** ** ***
17.1 ± 13.0 ± 14.2 ±
Scoplulariopsis 66.6 ± 0.44 7.69
5.3 0.39 0.65 0.37
asperula ** ****
ZL10 *** ** **
Ascorbic
Tetracycline 30µg/disc Acid 30µg /
Positive control
disc
23.8
19.5 ± 1.4 20.5±1.7 21.0 ± 1.0 2.73
± 2.3
*** *** *** ****
***

Note : * = Low antibacterial or weak antioxidant; ** = moderate antibacterial or antioxidant; *** = strong antibacterial or
antioxidant ; **** = very strong antibacterial or antioxidant.
 ZL6 extract has a broad antibacterial spectrum where its activity is strong on both
Gram+ and Gram-. Phenolic compounds and flavonoids are dominant in plants S. zeylanicum
makes it have strong antibacterial activity (Deepika et al., 2014; Microbiol et al., 2016;
Palanisamy, Ling, Manaharan, & Appleton, 2011; Shilpa & Krishnakumar, 2015).

3.4 Molecular Identification of Selected Endophytic Fungi

Figure 3. Electrophoresis results of ITS rDNA sequences of endophytic fungi

 100 SZL6*
95 MN826202 Penicillium citrinum strain yx-001
75
LC106114 Penicillium citrinum
75
MZ359812 Penicillium citrinum strain JaTpOi(2)
75
MT558921 Penicillium citrinum strain 2010F2
74
LC514694 Penicillium citrinum strain 19MBa-C1-1
61
MN879404 Penicillium citrinum strain AKF2-KU
60
MK791668 Penicillium citrinum strain J
60
MH892829 Penicillium citrinum strain S1
60
MH911355 Penicillium citrinum strain MF22494
60
LC387804 Penicillium citrinum strain IPBCC
60
KT844552 Penicillium citrinum strain ZSF5
60
KC344960 Penicillium citrinum strain a1s2 d37
LC105681 Penicillium citrinum
MN788102 Penicillium citrinum strain DTO 390

47 MK271291 Penicillium citrinum strain 19A

48 MK179258 Penicillium citrinum strain K31
AF506475 Sistotrema coronilla

Figure 4. SZL6 Phylogenetic Tree Construction using the Neighbor-Joining method (Saitou &
Nei, 1987) with bootstrap values (1000 replicates) displayed next to branches (Felsenstein,
1985). This analysis involved 18 nucleotide sequences. There are a total of 540 positions in the
final dataset. Evolutionary analysis carried out in MEGA11 (Tamura et al., 2021).

The results of the phylogenetic tree reconstruction show the relationship between
ZL6 and Penicillium citrinum. Branching on ZL6 with Penicillium citrinum forms a straight
line with a bootstrap value of 100%. The straight line on the branching of the phylogenetic tree
shows that ZL6 with Penicillium citrinum is closely related. According to Fitmawati et al.
(2013) the evolutionary level of a species is indicated by different line lengths in the
phylogenetic tree. The farther evolutionary distance is indicated by the longer line, while the
closer the evolutionary distance of a species is indicated by the shorter line.

3.5 Determination of Chemical Structure

Isolation of bioactive compounds was carried out on selected ethyl acetate extracts with
the highest antibacterial and antioxidant activity, namely isolate ZT2. Concentrated ethyl
acetate extract (2.0 g) was separated by gravity column chromatography (CC) method with a
gradient eluent system, namely 100% n-hexane (100 mL) eluent, a mixture of n-hexane and
ethyl acetate with increasing polarity, (n- hexane:ethyl acetate 9:1 (100 mL), 8:2 (100 mL), 7:3
(100 mL), 5:5 (100 mL); 2;8 (100 mL), 100% ethyl acetate (100 mL) mL), and ethyl acetate
and methanol (9:1 30 mL), 8:2 (30 mL), 7:3 (30 mL)). The stationary phase used was silica gel
60 G (70-230 mesh). The separation results were collected using vials and obtained as many
as 80 vials. The eluate was then analyzed using thin layer chromatography (TLC) with a mixed
eluent of n-hexane and ethyl acetate (5:5). TLC with similar chromatogram patterns were
combined into one fraction. Based on the results of the chromatogram pattern obtained 4
fractions. Namely F1-F4. In the F2 fraction, white crystals were formed and after being purified
with n-hexane, compound 1 was obtained in the form of white crystals weighing 37 mg.
The 1H-NMR spectrum of compound A (Figure 4.38A) shows the presence of eight
proton signals including two aromatic dublet signals with the integration of 2 protons, namely
7.70 (1H, d, J= 8.5 Hz) and 8.17 (1H, d, J= 8.5 Hz). ortho plot setting. This indicates that
compound A is a para-substituted aromatic compound, so it has two pairs of equivalent protons.
In addition, there are six signals on the chemical shift H < 6.5 ppm, namely at H 1.96 (3H, s);
4.21 (1H, m); 4.32 (1H, m); 4.42 (1H, m); 5.23 (1H,s); and 6.32 (1H, d, J= 1.0 Hz), which are
oxygenated protons of methyl, methine and methine. Thus compound A was identified as a
para-substituted aromatic compound having oxygenated hydroxyl, methyl, methine, and
methine groups.

A B

Figure 5. Spectrum of 1H-NMR (A) and 13C-NMR (B) from compound A (1H-500 MHz in aceton)

The 13C-NMR spectrum of compound A (Figure 5) showed the presence of 11 carbon

signals. There are two high-intensity carbon signals indicating the presence of two pairs of
equivalent aromatic carbons. Two other aromatic carbons as quaternary carbons appeared with
low intensity, namely at 147.4 C and 149.8 ppm. Two other carbon signals are in the lowest
field, namely at C 163.8 and 170.1 ppm which are carbonyl ester carbon atoms. Three carbon
signals at C 60.0 – 71.0 ppm (63.1; 66.5; and 70.4 ppm) and one carbon signal at C 54.3 ppm,
each is a methine carbon signal, three of which are oxygenated methine carbon. The analysis
of the 13C-NMR spectrum was corroborated by the data on the HMQC spectrum (Figure 4.39).
The HMQC spectrum showed eight 1H-13C correlations through one bond. Proton signals at
H 4.21 (1H, m) and 4.32 (1H, m) indicate a correlation to the same carbon atom at C 63.1 ppm
indicating a cyclic methylene group. Compound A besides having a substituted benzene ring,
it also has a methyl acetate substituted lactone ring.

Figure 6. Spectrum HMQC of compound A ( 1H-500 MHz; 13C-125 MHz in aceton)

The HMBC spectrum (Figure 6) showed a 1H-13C correlation through two or three
bonds. The aromatic proton signal at H 8.17 ppm correlates with three aromatic carbon atoms
at C 123.1; 147.4; and 149.8 ppm, including the carbon atom equivalent. The aromatic proton
at H 7.70 ppm correlates to two aromatic carbon atoms at C 127.4; 147.4 ppm and oxygenated
carbon in the aromatic substituent at C 70.4 ppm. The methine proton is oxygenated at H 5.23
ppm correlated with the two aromatic carbons at 127.4 and 149.8 ppm. The correlation
indicates that the oxygenated methine group is directly attached to the aromatic ring and is
para-substituted with a hydroxyl group. Furthermore, the correlation of the two methylene
protons namely H 4.21 (1H, m) and 4.32 (1H, m) is seen for the same carbon atom at C 54.3;
70.4; and 170.1 ppm and the correlation of proton methine at H 4.42 ppm to carbon C 63.1
ppm indicating that the methylene proton is bound to the methine carbon and the carbonyl ester
carbon in the open chain. The spectrum also shows the correlation of the methyl proton at H
1.96 ppm to the carbonyl ester carbon atom at C 170.1 ppm which strengthens the indication
of the presence of an ester carbonyl group in the side chain. This HMBC spectrum analysis
indicated that the lactone ring in addition to binding to the aromatic ring and methyl acetate,
also binds to the hydroxyl group. The 1D and 2D NMR spectral data for compound 1 are shown
in Table 3.

Figure 7. Spectrum HMBC of compound A ( 1H-500 MHz; 13C-125 MHz in Aceton)

Based on spectral analysis of 1H-NMR, 13C-NMR, HMQC, and HMBC, it can be

explained that compound A has a para-substituted benzene ring between the hydroxyl group
and the -butyrolactone group. The -butyrolactone ring also binds the hydroxyl and methyl
acetate groups. Thus, the proposed chemical structure of compound A is (4-hydroxy-2-(4-
hydroxyphenyl)- -butyrolactone-3-yl)methyl acetate as shown in Figure 7.

Table 3. Data of NMR from compound A, in 1H-500 MHz; 13C-125 MHz in CDCl3
δH ppm (ƩH. Multiplicity
No C δC ppm Type of C HMBC
(Hz))
2 70.4 CH 5.23 (1H, s) 127.4; 149.8
3 54.3 CH 4.42 (1H, m)
4 66.5 CH 6.32 (1H, d, J= 1.0 Hz)
5 163.8 C
6 63.1 CH2 A. 4.32 (1H, m) 54.3; 70.4; 170.1
B. 4.21 (1H, m)
 7 170.1 C
8 19.9 CH3 1.96 (3H, s) 170.1
1’ 149.8 C
2’ 127.4 CH 7.70 (1H, d, J= 8.5 Hz) 70.4; 127.4; 147.4
3’ 123.1 CH 8.17 (1H, d, J= 8.5 Hz) 123.1; 147.4;
149.8
4’ 147.4 C
5’ 123.1 CH 8.17 (1H, d, J= 8.5 Hz) 123.1; 147.4;
149.8
6’ 127.4 CH 7.70 (1H, d, J= 8.5 Hz) 70.4; 127.4; 147.4

The compound 4-hydroxy-2-(4-hydroxyphenyl)- -butyrolactone-3-yl)methyl acetate has a

hydroxyl group (-OH) and has an aromatic ring structure (phenol). Phenol compounds are
acidic compounds because they easily release H+ ions from the hydroxyl (-OH group) bound
to the aromatic ring (Kusumaningrum et al., 2021).

Figure 8. Structure of Compound A: (4-hydroxy-2-(4-hydroxyphenyl)- ɣ-butyrolactone-3-yl)methyl

acetate

The structure of compound A (4-hydroxy-2-(4-hydroxyphenyl)-ɣ-butyrolactone-3-

yl)methyl acetate) also allows this compound to act as an antioxidant by donating hydrogen
atoms to free radicals in the DPPH compound so that it becomes a more complex form. more
stable. Free radicals are defined as atoms or molecules that have one or more unpaired
electrons. The odd electrons possessed by free radicals cause radicals to become unstable and
highly reactive. The high reactivity of free radicals is able to attract electrons from other
molecules to make them stable (Alugojo, et al, 2014). Compound B has another group at the
para (p) position attached to the benzene ring so that it can stabilize the radicals formed in the
compound by donating free electrons from the group to the aromatic ring, resulting in
resonance and producing a more stable compound because the energy produced is low. This is
due to the resonance effect lowering the electron density at one position (Bendary, et al. 2013).

4. CONCLUSIONS
Endophytic fungi have been successfully cultured and 4 isolates of fungi were obtained
from leaves (coded ZL6, ZL8, ZL9, ZL10). Based on their morphological characterization, the
endophytic fungi were included in the genus Penicillium (1 isolate), Colletotrichum (1 isolate),
Aspergillus (1 isolate), Scopulariopsis (1 isolate). All endophytic fungi have potential
antibacterial and antioxidant activity. The molecular results of the isolate with the highest
antibacterial showed that the isolate ZL6 was identified as Penicillium citrinum with a
compound structure (4-hydroxy-2-(4-hydroxyphenyl)-ɣ-butyrolactone-3-yl)methyl acetate)

ACKNOWLEDGEMENTS
The authors thank to the SP DIPA Kemenristek Republik Indonesia, which provided research
funding through Hibah Disertasi Doktor 2021, with contract no. 057/E5/PG.02.00.PT/2022.

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