Jurnal Kimia dan Kemasan, 43(2),110-116, 2021
©Author(s); http://dx.doi.org/10.24817/jkk.v43i2.6980
RAPID SCREENING OF ANTIBACTERIAL AND ANTIOXIDANT
METABOLITES FROM ENDOPHYTIC FUNGI ISOLATED FROM
Papuacedrus papuana BY TLC-BIOAUTOGRAPHY
Praptiwi1, Muhammad Ilyas2, Kartika Diah Palupi1, Ahmad Fathoni1, Evana1, Marlin
Megalestin Raunsai1 and Andria Agusta1*
1) Research
Center for Chemistry, Indonesian Institute of Sciences
Kawasan PUSPIPTEK, Tangerang Selatan, Banten 15314
2) Research Center for Biology, Indonesian Institute of Sciences
Jl. Raya Bogor Km. 46, Cibinong, Jawa Barat 16911
E-mail: andr002@lipi.go.id
Received : 28 April 2021; revised : 15 Juni 2021; accepted : 23 Juni 2021
ABSTRACT
RAPID SCREENING OF ANTIBACTERIAL AND ANTIOXIDANT METABOLITES FROM ENDOPHYTIC FUNGI
ISOLATED FROM Papuacedrus papuana. Papuacedrus papuana is a plant species that grows in the highland
of Papua. The aims of the study were to determine in vitro antibacterial activity and antioxidant activity of 17
endophytic fungal extracts isolated from P. papuana stem by the TLC-bioautography method. The antibacterial
activity was assessed against Staphylococcus aureus InaCC B4 and Escherichia coli InaCC B5. The antioxidant
activity was assessed by DPPH radical scavenging assay. All of 17 endophytic fungi were grouped into 7 taxa
based on their morphological traits. The results showed that 11 fungal extracts were active against S. aureus
InaCC B4, 15 fungal extracts were active against E. coli InaCC B5, and 6 extracts had antioxidant activity.
Further analysis of active extracts by eluted TLC-bioautography showed several compounds responsible for
antibacterial or antioxidant activity in one extract. The results showed a diversity of endophytic fungi inhabited P.
papuana stem, and these endophytic fungi might be used as a good source of novel antibacterial or antioxidants.
Keywords: Papuacedrus papuana, endophytic fungi, antibacterial, antioxidant.
ABSTRACT
SKRINING METABOLIT ANTIBAKTERI DAN ANTIOKSIDAN JAMUR ENDOFIT YANG DIISOLASI DARI
Papuacedrus papuana. Papuacedrus papuana adalah salah satu tanaman langka yang tumbuh di dataran
tinggi di Papua. Tujuan penelitian adalah untuk mengetahui aktivitas antibakteri secara in-vitro dan aktivitas
antioksidan dari 17 ekstrak jamur endofit yang diisolasi dari batang P. papuana dengan metode Kromatografi
Lapis Tipis (KLT)-bioautografi. Aktivitas antibakteri dievaluasi terhadap bakteri Staphylococcus aureus InaCC B4
dan Eschericia coli InaCC B5. Aktivitas antioksidan dievaluasi dengan uji radikal bebas DPPH. Berdasarkan ciriciri morfologinya, maka 17 jamur endofit tersebut dikelompokkan menjadi 7 genus. Hasil penelitian menunjukkan
bahwa 11 ekstrak aktif menghambat pertumbuhan S. aureus InaCC B4, 15 ekstrak aktif menghambat
pertumbuhan E. coli InaCC B5 dan 6 ekstrak memiliki aktivitas antioksidan. Analisis lebih lanjut dari ekstrak aktif
yang dielusi menunjukkan bahwa ada beberapa senyawa yang menunjukkan aktivitas antibakteri atau
antioksidan. Hasil penelitian menunjukkan bahwa ada keragaman jamur endofit pada batang P. papuana yang
kemungkinan dapat dimanfaatkan sebagai sumber yang potensial sebagai antibakteri atau antioksidan baru.
Kata kunci: Papuacedrus papuana, jamur endofit, antibakteri, antioksidan.
INTRODUCTION
Papuacedrus papuana (syn. Libocedrus
papuana) is one of the plant species that
belongs to Cupressaceae and the only species
in the genus of Papuacedrus. According to
Johns (1995), P. papuana is native to New
Guinea and Maluku. The timber of P. papuana
has been used as a building material, while the
stem bark is used for roof material. Some other
Rapid Screening of Antibacterial and Antioxidant …… Praptiwi et al.
110
species from the conifer family (Cupressaceae)
have been investigated for their bioactivity.
According to Perry and Foster (1994), the extract
of Libocedrus pulmosa, L. bidwillii, and
Cupressus macrocarpa are active against
leukemia cells P-388.
On the other hand, in the last two
decades, many studies focus on the bioactivity
of microbial endophytes. Endophytes usually
colonized the healthy tissue plants (Aly et al.
2010) without causing any harm to the host plant
(Idris, Al-tahir, and Idris 2013). Endophytes may
have the ability to produce bioactive compounds
that are the same or similar to bioactive
compounds derived from the host plant so that
endophytic fungi can serve as an alternative
source of plant metabolites (Tikole, Tarate, and
Shelar 2018). According to Sturz and Nowak
(2000), there are several roles of endophytic
fungi in the host plant such as stimulate plant
growth, increase disease resistance, improve the
ability of the plant to environmental stresses and
recycle nutrients. A wide variety of natural
products have been obtained from endophytes.
Kusari, Lamshöft, and Spiteller (2009) stated
that endophytes usually produce the same
bioactive compounds as the host plant.
Endophytic fungi can be used as a source of
secondary metabolites that are useful for novel
drug discovery (Yan, Sikora, and Zheng 2011;
Guo et al. 2008). The endophytic fungi from
several species of Cupressaceae have been
investigated for their antimicrobial activity.
The search for novel antibacterial and
natural antioxidants has become important. This
is due to increasing resistance by pathogenic
bacteria to commercial drugs (Costelloe et al.
2010). At the same time, the use of natural
antioxidants was also increased lately. This is
due to the use of synthetic antioxidants is
suspected to be carcinogenic (Govindarajan et
al. 2003).
To the best of our knowledge, there is still
no report about the diversity of endophytic fungi
from P. papuana, and also the study of their
activities, especially for the antimicrobial and
antioxidant activity. Therefore, this study aimed
to determine the most potential endophytic fungi
associated with P. papuana as a source of
antibacterial and antioxidant metabolites.
MATERIALS AND METHODS
Materials
Plant Source
Young healthy stems of P. papuana were
collected from Habema, Papua. The plant
voucher was deposited and identified in the
Botany Division, Research Center for BiologyIndonesian Institute of Sciences (LIPI).
Chemicals
Ethanol (70%), NaOCl, ethyl acetate
(Merck), Thin Layer Chromatography (TLC)
plates
(silica
gel
GF254,
Merck),
Dichloromethane (Merck), MeOH (Merck),
Ce(SO4)2 (Merck), Iodonitrotetrazolium p-violet
(INT, Sigma), H2SO4 (Merck), chloramphenicol
(Merck), Mueller-Hinton Broth (MHB, Merck),
2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma),
and (+)-catechin (Sigma).
Methods
Endophytic fungi isolation and cultivation
The fresh young healthy stems of P.
papuana were thoroughly washed under tap
water. Stems were cut about 2 cm in length,
then sterilized by dipping in 70% alcohol for 2
minutes, transferred into NaOCl 5.3% solution
for 5 minutes, then transferred into 70% alcohol
for 1 minute, and washed with distilled water.
Sterilization is performed aseptically in laminar
airflow. The samples that had been surfacesterilized were split with a sterile blade, and the
inner part was placed on Corn Meal Malt Agar
(CMMA) media that has been added with
chloramphenicol 0.05 mg/mL in a petri dish. Petri
dishes were incubated for 7 days at room
temperature. The emerging fungi were isolated
and subcultured on Potato Dextrose Agar (PDA)
media several times to obtain single isolates
(Agusta et al. 2005).
Identification of endophytic fungal isolates
Identification of endophytic fungi isolated
from P. papuana was based on fungal
morphological characters (Domsch, Gams, and
Anderson 2008; Webster and Weber 2007; Ellis
1965). Morphological traits and characteristics
macroscopically and microscopically were
observed from fungal colonies grown on PDA at
room temperature. Color and surface colonies
(granular, such as flour, mounting, slippery),
texture, zonation, growth area, the lines of radial
and concentric, reverse color, and exudate drops
were amongst of macroscopic characters that
observed.
Endophytic fungi cultivation and extraction
A single isolate of endophytic fungi was
inoculated into 200 ml Potato Dextrose Broth
(PDB) and incubated for 3 weeks in a static
condition at room temperature under dark
conditions. After an incubation period, the
biomass and culture broth was extracted with
ethyl acetate. The organic phase was collected
and concentrated with a rotary evaporator.
Analysis of endophytic fungi metabolites
The chemical compounds of ethyl acetate
extract were analyzed by separation with TLC.
The plate was developed using mobile phase
J. Kimia Kemasan, Vol.43 No.2 Oktober 2021 : 110 - 116
111
dichloromethane:
methanol
(10:1).
The
chromatogram was viewed under UV light at
wavelengths of 254 and 366 nm then sprayed
with a staining reagent: 1% Ce(SO4)2/10%
H2SO4, and 1% vanillin/H2SO4.
(1
isolate),
Dematiaceae
(9
isolates),
Coelomycetes (3 isolates), Hyphomycetes (2
isolates), and Sordariomycetes (2 isolates). The
genus Phomopsis is a good source of bioactive
compounds which have remarkable value in
medical application (Xu et al. 2021).
TLC-Bioautography: Antibacterial Assay
Endophytic fungal extracts were screened
for their antibacterial activity against two bacteria
(E. coli InaCC-B5 and S. aureus InaCC-B4). One
hundred µg of endophytic fungal extract (10
mg/mL) were transferred on a TLC plate, then
dipped into a bacteria suspension (10 6 CFU/mL).
The plate was placed on a sterile petri dish and
incubated at 37OC for 18 hours. After an
incubation period, the plate was sprayed with
INT. A clear zone indicated the inhibition zone
against a purple background on the TLC plate.
The active extracts were then observed
for the active compounds responsible for
antibacterial activity. One hundred µg of
endophytic fungi extract (10 mg/mL) was
transferred on a TLC plate, and the compounds
were separated with the mobile phase of
dichloromethane : methanol (10:1) and then
dipped into bacteria suspension. The plate was
placed on a sterile petri dish and incubated at
37oC for 18 hours. After an incubation period, the
plate was sprayed with INT. The spots that are
responsible for antibacterial activity were
indicated by white spot formation.
TLC-Bioautography: Antioxidant potential by
DPPH radical scavenging assay
Ten µl of endophytic fungi extract (10
mg/mL) were transferred to the TLC plate, with
(+)-catechin as positive control and culture
media as negative control were also transferred
to the TLC plate. The plate was then allowed to
air dry, followed by spraying with 1 mM DPPH in
methanol solution. The antioxidant potential was
indicated by the formation of a white-yellow spot
against a purple background. The active extract
(100 µg) was transferred on a TLC plate, then
eluted
with
the
mobile
phase
of
dichloromethane-methanol (10:1). After air
drying, the plate was sprayed with 1 mM DPPH
in methanol solution.
RESULTS AND DISCUSSION
Endophytic fungi isolation and identification
The potential of the endophytic fungi
associated with P. papuana has not been had
much attention yet. Totally 17 filamentous fungi
isolates were obtained from the young stem of P.
papuana (Table 1). Based on their morphological
characters, including macroscopic (Figure 1) and
microscopic characters, these fungi were
classified into 5 taxa, which are Phomopsis sp.
Figure 1. Endophytic fungi associated with P.
papuana stem. The fungal taxa PC-1 – PC-20
are referring to Table 1.
Endophytic fungi, in general, has a wide
range of host. They could be isolated from the
different plants from different taxa and grow
under different ecological and geographical
conditions. However, some endophytic fungi
have only one specific host genera. The result
indicated that P. papuana stem harbored diverse
taxa of fungal endophytes. Qadri et al. (2013)
stated that conifers possessed a broad range of
fungal endophytes. Carroll and Carroll (1978)
observed some specificity concerning the
location of endophyte species within the petiole
of conifers in the Pacific Northwest.
Table 1. List of endophytic fungi isolated from P.
papuana (F. Muell.) H.L.Li. stem.
No.
Fungal
Isolates
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
PC-01
PC-02
PC-03
PC-04
PC-05
PC-06
PC-07
PC-08
PC-11
PC-13
PC-14
PC-15
PC-16
PC-17
PC-18
PC-19
PC-20
Fungal taxa
Dematiaceae
Coelomycetes
Sordariomycetes
Coelomycetes
Phomopsis sp.
Hyphomycetes
Dematiaceae
Dematiaceae
Dematiaceae
Coelomycetes
Dematiaceae
Dematiaceae
Dematiaceae
Sordariomycetes
Hyphomycetes
Dematiaceae
Dematiaceae
Rapid Screening of Antibacterial and Antioxidant …… Praptiwi et al.
Extract
Production
(mg/200 ml
medium)
227.4
687.5
66.6
314.5
127.8
141.5
212.2
2090.0
68.0
814.1
368.2
145.9
122.6
236.4
95.9
114.3
83.8
112
TLC-Bioautography: Antibacterial Activity
The antibacterial and antioxidant assay
was done by TLC-bioautography because this
method was a rapid approach (Rajauria and
Abu-Ghannam 2013). The result from TLCbioautography for antibacterial (Figure 2)
showed that 11 extracts (64.71%) inhibit the
growth of S. aureus, 15 extracts (88.24%) inhibit
the growth of E. coli and 6 extracts (35.29%)
have antioxidant activity (Table 2.). Further
analysis by eluted TLC-bioautography was used
to determine the active spots (metabolites) as
clear zones surrounding the purple background
as shown in Figure 3.
Table 2. The Antibacterial activity and antioxidant
potential of endophytic fungi extract isolated from the
young stem of P. papuana.
No
Isolate
Antibacterial activity
S. aureus E. coli
Antioxidant
potential
1
PC-01
2
PC-02
+
+++
3
PC-03
+
4
PC-04
+
+
+++
5
PC-05
+
+
6
PC-06
+
7
PC-07
+
+
++
8
PC-08
9
PC-11
++
++
+
10
PC-13
+
+
11
PC-14
+
+
12
PC-15
+
+
+
13
PC-16
+
+
+
14
PC-17
+
+
15
PC-18
+
+
16
PC-19
+
17
PC-20
++
++
Note: +++ : strong activity, ++ moderate activity, + weak
activity, - : no activity
Figure 2. Bioautogram of endophytic fungal extracts of
P. papuana against E. coli (a), and S. aureus
(b). The white area showed the inhibition
growth of bacteria. Picture information refers
to Table 1. A: acetone, M: methanol, and C+:
chloramphenicol.
Figure 3. Bioautogram of active extracts developed
with dichloromethane : methanol (10:1), (a)
viewed under 254 nm, (b) viewed under
366 nm, (c) against E. coli, and (d) S.
aureus. White bands indicated the active
chemical compounds in the extracts.
Picture information refers to Table 1.
Table 3. The MIC (antibacterial) and IC50 value
(antioxidant) of endophytic fungi extract from P.
papuana
No
Isolate
1
2
3
4
5
6
PC-02
PC-04
PC-07
PC-11
PC-18
PC-20
MIC (µg/mL)
S. aureus
E. coli
256
256
>256
>256
256
256
>256
>256
>256
>256
256
256
IC50 (ppm)
>1000
345
710
>1000
>1000
>1000
Further analysis of active extracts to
determine their MIC values. The MIC values of
active extracts were in the range of 256 µg/mL
or more (Table 3). It showed that the
antibacterial activity of endophytic fungi extracts
from P. papuana were not strong antibacterial.
Extracts are considered to have significant
antimicrobial activity if the MIC values are lower
than 100 µg/mL (Ríos and Recio 2005; Gibbons
2004).
Results of the present study showed that
endophytic fungal extracts of P. papuana
inhibited the growth of bacteria. The growth
inhibition is indicated by clear zone formation
around the extract or the chemical compounds
responsible for antibacterial activity. Clear zone
J. Kimia Kemasan, Vol.43 No.2 Oktober 2021 : 110 - 116
113
formation indicated the reduction of INT into a
colored formazan that inhibits the growth of
bacteria (Suleiman et al. 2010). In several
previous studies, the crude extract of culture
broth of endophytic fungi showed various
bioactivity such as antibacterial, antifungal, antiinflammatory, antiviral, and anti-tumor activity
(Silva et al. 2006).
The result from the study also showed that
endophytic fungi associated with P. papuana
stem are dominated by Dematiaceae such as
PC-7, PC-11, PC-14, PC-15, and PC-16. The
result showed that these fungi isolates have
antibacterial activity and antioxidant activity. This
result is in harmony with Tsuge et al. (2013), and
Bräse et al. (2009) that Alternaria fungi belong to
Dematiaceae produce metabolites with a variety
of biological activities such as phytotoxic,
cytotoxic,
and
antimicrobial
properties.
Tenuazonic acid produced by Alternaria
alternata was found to be active against
Mycobacterium tuberculosis H37Rv (Logrieco,
Moretti, and Solfrizzo 2009). The result also
showed that PC-05 (Phomopsis sp.) was active
against S. aureus and E. coli. Previous studies
showed that the genus Phomopsis is a rich
source of bioactive compounds as antimalarial
and antitubercular phomoxanthones (Isaka et al.
2001), antifungal phomoxanthone-A (Elsässer et
al. 2005), and antibacterial chromones (Ahmed
et al. 2011).
TLC-Bioautography: Antioxidant potential by
DPPH radical scavenging assay
The antioxidant activity of the endophytic
fungi extracts associated with P. papuana was
assessed by TLC-bioautography with DPPH
radical scavenging assay. The result showed 6
isolates have antioxidant activity (PC-04, PC-07,
PC-11, PC-14, PC-15, and PC-16), which is
indicated by white-yellowish area formation on
the TLC plate (Figure 4).
Figure 4. Bioautogram of endophytic fungal extracts of
P. papuana. TLC was sprayed with 0.2%
DPPH in methanol. The white-yellowish area
showed the antioxidant activity of the extract.
Number 1-17 are extract codes of PC-1 to
PC-20 that refers to Table 1. A: acetone, M:
methanol, and C+: (+)-catechin.
The active extracts were further assay by
separating their chemical compounds on a TLC
plate and developed with mobile phase
dichloromethane-methanol (10:1). This analysis
could be used to determine the active spots
(metabolites) as pale yellow spots surrounding
the purple background as shown in Figure 5.
Figure 5. Chromatogram profile of active extracts for
antioxidant
potential
developed
with
dichloromethane: methanol (10:1), (a)
monitored under UV light 254 nm, (b)
monitored under UV light 366 nm, (c)
sprayed with 0.2% DPPH in methanol.
Extracts no. 4-13 refer to Table 1. White
yellowish bands indicated the active
semipolar compounds in the extract.
The antioxidant activity of the endophytic
fungal extracts was done by DPPH radical
scavenging activity. DPPH in methanol will
produce a purple color, and it reduced to
diphenyl picryl hydrazine gave the yellow color
because chemical compounds in the extract
have antioxidant activity (Mahlo et al. 2016). The
intensity of the yellow color indicates the
potential of free radical scavenging activity by
the extract (Kumar and Pandey 2012) and it
depends on the amount and nature of radical
scavengers in the extract (Qadri et al. 2013).
Further analysis of PC-04 and PC-07 by
serial microdilution was done to determine the
value of IC50. The result showed that the IC50 of
PC-04 and PC-07 were 345 and 710 ppm
respectively (Table 3), indicated that these
extracts have weak antioxidant activity (Blois
1958).
CONCLUSION
A total of 17 isolates of endophytic fungi
were isolated from P. papuana and based on
morphological characters they were classified
into 5 taxa. Among 17 extracts of endophytic
fungi, 11 extracts were active against S. aureus
Rapid Screening of Antibacterial and Antioxidant …… Praptiwi et al.
114
InaCC B4, 15 extracts were active against E. coli
InaCC B5, and 6 extracts have antioxidant
potential. Endophytic fungi from P. papuana
might use as a novel source for antimicrobial
and antioxidant agents which is under further
investigation.
ACKNOWLEDGMENT
The authors thank Dr. Ary Prihardianto
Keim, Research Center for Biology LIPI for
collecting and taxonomical identification of plant
materials.
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