Vol. 12 | No.

3 |1463 - 1469| July - September | 2019

ISSN: 0974-1496 | e-ISSN: 0976-0083 | CODEN: RJCABP
http://www.rasayanjournal.com
http://www.rasayanjournal.co.in

CYTOTOXIC AND ANTIBACTERIAL ACTIVITIES OF

MARINE SPONGE-DERIVED FUNGUS Aspergillus nomius NC06
Muh. Ade Artasasta1, Yanwirasti1, Muhammad Taher2, Akmal Djamaan3
and Dian Handayani3,*
1
Departement of Biomedical, Faculty of Medicine, Andalas University, 25163 Padang, Indonesia
2
Faculty of Pharmacy, International Islamic University Malaysia,
25200 Kuantan, Pahang, Malaysia
3
Laboratory of Sumatran Biota, Faculty of Pharmacy, Andalas University,
25163 Padang, Indonesia
*E-mail: [email protected]
ABSTRACT
Sponge-derived fungi have attracted recent attention due to its important source of interesting biologically active
compounds. In our previous study, we have obtained 13 fungi from marine sponge Neopetrsiachaliniformis. Among
them, only Aspergillus nomius NC06 showed cytotoxic activity with the percentage of viability113.9 % and 70.31 %
of Vero cell and WiDr colon cancer cell, respectively. This study aimed to isolate the cytotoxic compound from the
ethyl acetate extract of N. nomius NC06 using chromatography method. A total of 5 fractions of the extract obtained
using vacuum liquid chromatography. These fractions were tested against HCT 116 colon cancer cell and ten human
pathogenic bacteria. Fraction II, III, IV, and V showed cytotoxic activity with IC50 of 5.28, 15.82, 10.27, and
45.57 µg/mL, respectively. In antibacterial testing, fraction II and III were potential because of their ability to inhibit
the growth of ten pathogenic bacteria with the diameter of inhibition zone more than 12 mm.
Keywords: Sponge-derived fungi, Aspergillus nomius, Cytotoxicity, Antibacterial
© RASĀYAN. All rights reserved

INTRODUCTION
The sponge is known as the host for various microorganisms such as bacteria and fungi.1,2 But the
association relationship and ecological function of microorganism in sponge body are remaining unclear3.
However, microorganism associated with a sponge is known as natural resources to produce the potential
bioactive compound. Especially marine sponge-derived fungi repeatedly show the potential bioactive
compounds against various diseases such as cancer and pathogenic microbial infection.3-5
Thiel et al., (2007) figured out that the sponge-derived microorganisms mostly exist in cortex and
endosome layers of sponge nevertheless the location of associated-fungi in the sponge is still unknown6.
In addition, Kjeret al., (2010) published a protocol to isolate marine fungi from sponges and other marine
macroorganisms. This protocol focused to isolate fungal to the marine sponge7. However, there are a lot
of unknown interactions between sponges to its associated fungi. Furthermore, several studies have
suggested that sponge-derived fungi have shown to exhibit interesting new bioactive sources that were
previously unknown to originate from terrestrial starins8.
Aspergillus nomius was successfully isolated from marine sponge N. chaliniformis in our previous study.
This fungus showed selectivity between cell normal Vero and WiDr colon cancer cell with the percentage
of viability of 113,9 % and 70,3 %, respectively.11 It is potential fungus to be continued to explore its
bioactive compound.
EXPERIMENTAL
Sponge Material, Isolation, Cultivation, and Extraction of Secondary Metabolites from Marine
Sponges-Derived Fungi
These stages have been conducted due to our previous study12. Marine sponge N. chaliniformis was taken
at Mandeh Island, West Sumatra, Indonesia using scuba diving method. Furthermore, isolation of fungi
Rasayan J. Chem., 12(3), 1463-1469(2019)
http://dx.doi.org/10.31788/RJC.2019.1235284
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from N. chaliniformiswas conducted using Sabouraud Dextrose Agar as a medium and incubated at a
temperature of 27-29 °C for 5-7 days then purified by using the scratch method. We obtained A. nomius
from this sponge and cultured in big scale by using rice as a medium for 4 to 8 weeks7. After this fungus
overgrown on rice, then it was extracted using ethyl acetate (1:1).

Fractionation of Secondary Metabolites

Isolation compounds of 27.53g of crude extract from A. nomius were done by vacuum liquid
chromatography with n-hexane, ethyl acetate, dichloromethane, and methanol as mobile phase and silica
gel 60(0.063-0.2 mesh) as the stationary phase. Thin-layer chromatography (TLC) was used for
monitoring every vials-collected and combining as one fraction that has the same spot pattern on TLC. In
this study, we successfully collected 5 fractions. Furthermore, these fractions were submitted to the HPLC
to be characterized by every compound in one fraction.

MTT Assay
Sample Screening
Cytotoxic activity of HCT 116 as a colon adenocarcinoma cell line was conducted using MTT assay.
These cell lines were obtained from the Laboratory of Biotechnology and Cell Culture Pharmacy Faculty,
International Islamic University Malaysia. HCT 116 was cultured in DMEM GibcoTM. This cell was
seeded in 96-well plates (density: 6x103 cells/well) and incubated at 37ºC, 98% relative humidity with
5% CO2. After overnight incubation (confluence), the fraction of Ethyl acetate A. nomius extract was
added with concentrations of 100 µg/mL, 10 µg/mL, 1 µg/mL and 0.1 µg/mL.Then 100 µLMTT (5
mg/mL) was added and incubated for 4 hours. The absorbance was measured using Tecan Microplate at
560 nm using DMSO as blank and Doxorubicin as a positive control. The absorbance of each fraction
against HCT 116 colon cancer cells was expressed as viability percentage.13

Antibacterial Activity
Salmonellatyphosa, Pseudomonas aeruginosa, Vibrio cholera, Escherichiacoli as Gram-negative bacteria
and Enterococcus faecalis, Staphylococcus aureus, Staphylococcusmutans, Bacillus subtilis,
Micrococcusluteus, Staphylococcusepidermidis as Gram-positive bacteria had been prepared for this
study. Every fraction was diluted being 5 % with DMSO as a test solution. One piece of sterile disk paper
(6mm) was soaked in the test solution. DMSO was used for negative control and chloramphenicol disk as
a positive control. Zone of inhibition (mm) was measured after incubation at a temperature of 37 oC for
24 hours.14

High-Performance Liquid Chromatography

Every fraction was diluted with methanol HPLC (1:1), and then pipetted out 50 µL to the HPLC vial and
added with methanol HPLC until the total volume of 500 µL. Furthermore, diluted sample on HPLC vial
was submitted to UltiMateTM 3000 UHPLC with column C8, 4.6 x 150 mm, 5 µm, mobile phase A: H2O
with 0.1 % TFA; B: methanol, flow rate: 1 mL/min. Gradient elution from0 to 35 min was 10 - 100 % B
while 35 to 60 min was 100 – 10 % B.

RESULTS AND DISCUSSION

Aspergillus nomius is a species of fungi as a potential source for cytotoxic and other bioactivity15. In our
study, we successfully isolated. nomius from marine sponge N. chaliniformis. As much as 27.53 g, crude
ethyl acetate extract of A. nomius was obtained. The result of VLC column was obtained 5 fractions that
we collect based on the same spot pattern on TLC. These fractions were characterized based on its
retention time of compounds contained in each fraction by using HPLC. In Figure 1, HPLC
chromatogram for every fraction is shown. Every peak that appeared in 235 nm were representative why
these fractions were different from each other.
Based on HPLC chromatograms, the same main peak was observed in fraction II to V, with the retention
time around 2.67 min. On the other hand, every fraction exhibited different main peak such as a peak in
the retention time of 18.34 and 31.74 which were only observed in fraction I. For fraction III and IV there
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were also 2 main peaks in retention time around 22.15 and 22.95 min. The peak in retention time around
26.37 min appeared as the main peak in fraction IV and V.
Cytotoxic screening from a natural product is important to be conducted due to the prospecting of
potential anticancer agents16. In this study, MTT assay was used to evaluate the cytotoxic activity of the
fifth fractions. Here, we reported cytotoxic activity of the fifth fractions against HCT 116 colon cancer
cell line. Figure 2 showed fraction II was the most potent cytotoxic activity with IC50 of 5.28 µg/mL
followed by fraction IV, fraction III, fraction V then fraction I.Doxorubicin was used as a positive control
against HCT 116 colon cancer cell with IC50of0.47 µg/mL.

250 AR181106 #5 FI UV_VIS_1

mAU WVL:235 nm

FI

8 - 31,743
2 - 10,820
1 - 10,110

100 3 - 18,340

7 - 30,710
4 - 27,570
5 - 28,343
6 - 29,860

9 - 35,770
0

-150 min
0,0 10,0 20,0 30,0 40,0 50,0 60,0

350 HW181221 #9 FII UV_VIS_1

mAU WVL:235 nm
FII
1 - 2,740

200

100
3 - 12,123
2 - 7,703

6 - 24,067
4 - 22,493
5 - 23,197

7 - 25,097

8 - 27,233

9 - 30,170

-150 min
0,0 10,0 20,0 30,0 40,0 50,0 60,0

1.000 AR181106 #7 FIII UV_VIS_1

mAU FIII WVL:235 nm
1 - 2,687

800
7 - 22,157
8 - 22,953

600
11 - 26,577

400
19,550

12 - 26,943
43- -20,003

10 - 24,890

13 - 27,910
9 - 23,730
5 - 20,543
6 - 21,370
2 - 17,863

14 - 30,083

200

-200 min
0,0 10,0 20,0 30,0 40,0 50,0 60,0

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1.400 AR181107 #6 FIV UV_VIS_1

mAU WVL:235 nm

1 - 2,673
FIV

11 - 26,367
1.000

9 - 22,943
750

8 - 22,193
500

10 - 25,533
19,550
7 - 21,483
65--19,997

12 - 27,903
2 - 4,790

4 - 18,320
3 - 13,227
250

min
-200
0,0 10,0 20,0 30,0 40,0 50,0 60,0

600 AR181107 #7 FV UV_VIS_1

mAU WVL:235 nm

FV
375
1 - 2,670

5 - 26,457

250
4 - 23,003
3 - 22,297
2 - 10,050

125

min
-200
0,0 10,0 20,0 30,0 40,0 50,0 60,0

Fig.-1: HPLC Chromatogram of Fraction I to Fraction V; Column C8, 4.6 x 150 mm, 5 µm, Mobile Phase A: H2O
with 0.1 % TFA; B: Methanol, Flow Rate: 1 mL/min.

Fig.-2: Cytotoxic Activity of Fraction I-V of A. nomius extract against HCT 116 colon Cancer.

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Antibacterial activity test is listed in Table-1. In this study, fraction II and III showed potential
antibacterial activity that can inhibit ten pathogenic bacteria with inhibition zone more than 10 mm. In
Table-1 also showed Fraction III can inhibit Gram-positive and Gram-negative bacteria with inhibition
diameter zone (mm) of 15.43±0.27, 15.38±0.33, 16.7±0.55, 15.41±0.28, 15.98±0.29, 13.95±0.58,
14.76±0.94, 15.86±1.5, 17.53±0.62, 15.71±0.45againstS. typhosa, P. aeroginosa, V. cholera, E. faecalis,
S. epidermidis, S. aureus, E. coli, S. mutans, B. subtilis, M. luteus, respectively.
Table-1: Antibacterial Activity of Fraction I-V of A. nomius extract
Pathogenic Bacteria Inhibition Zone (mm) ± Standard Deviation (SD)
Fraction I Fraction II Fraction III Fraction IV Fraction V
S. typhosa 10.83±1.41 13±0.7 15.43±0.27 - 7.28±0.12
P. aeroginosa 8.2±0.08 12.66±0.79 15.38±0.33 - 7.85±0.56
V. cholerea 12.75±0.55 14.31±0.20 16.7±0.55 - -
E. faecalis 9.78±0.54 14.86±0.57 15.41±0.28 - -
S. epidermidis 9.55±0.35 12.4±1.01 15.98±0.29 7.28±0.18 8.18±0.11
S. aerus 8.33±0.15 12.16±0.05 13.95±0.58 - -
E. coli 10.73±1.31 13.38±1.32 14.76±0.94 7.38±0.12 8.4±0.26
S. mutans - 10.6±0.43 15.86±1.5 - -
B. subtilis 9.55±1.3 12.85±1.58 17.53±0.6 - 7.4±0.57
M. luteus 10.21±0.45 14.05±1.21 15.71±0.45 6.83±0.32 7.53±0.15

The result of cytotoxic and antibacterial activity showed that fraction II and III were potential fractions to
be furtherly studied. Due to of HPLC chromatogram in these fractions, there was some peak that showed
as main peak such us retention time of 2.74 min, 22.16 min, 22.95 and 26.58 min. Based on library hits of
UV spectra of this main peak were probably eudesmic acid (2.74 min), spongiciadin E (22.16 min), and
Notoamide E (26.58 min). Another main peak of these fractions in the retention time of 22.95 min was
undetected or no spectra library hits found (Figure 3). Further study is needed to prove what compounds
are contained in these fractions using the spectroscopy method. However, these main peaks might be
responsible for the potential cytotoxic and antibacterial activity of fraction II and III.
Peak #1 100% at 2.74 min Peak #7 100% at 22.16 min
70,0 70,0
% Library Hit: Eudesmic acid 972.23 % Library Hit: Spongiacidin E 954.14
366
214.5 365.1
362.9
368.4
a b
268.5

nm -10,0 nm
-10,0
200 250 300 350 400 450 500 550 595 200 250 300 350 400 450 500 550 595

Peak #8 100% at 22.95 min Peak #11 100% at 26.58 min

70,0 70,0
% No spectra library hits found! % Library Hit: Notoamide E 958.92

240.3 237.4
c d
360.9
314.7

-10,0 nm nm
-10,0
200 250 300 350 400 450 500 550 595 200 250 300 350 400 450 500 550 595

Fig.-3: UV Spectra of Main Peaks from Fraction II and III. (a) Library Hit of UV Spectra of the Compound in
Retention Time 2.74 min (eudesmic acid), (b) 22.16 min (spongiacidin E), (c) 22.95 min (no spectra library hit), (d)
26.58 min (notoamide E).
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Lack of information on secondary metabolites of A. nomiusas cytotoxic and antibacterial activity.

However, the study of Gloeret al., (1989) and Staub et al., (1992) reported bioactive compounds from A.
nomius, isolated from pine sawfly Diprionsimilis, such us nomine, and aspernomine which are indole
diterpenoids. This study reported that aspernomine exhibits cytotoxicity against solid tumor cell such us
A-549 lung carcinoma, MCF-7 breast adenocarcinoma, and HT-29 colon adenocarcinoma with IC50
values of 3.09, 4.93, and 3.08 µg/mL, respectively17,18. Compared with A. nomius that we isolated from
marine sponge N. chalinifromis may produce different secondary metabolite due to the different
environment19.
Antibacterial from Aspergillus genus repeatedly showed strong activity. Study of Li et al., (2012)
successfully reported aspergiterpenoid A, sydonol, and sydonic acid from Aspergillus sp. had potential
antibacterial activity towards S. albus, B. subtilis, B. cereus, S. lutea, E. coli, and M. tetragenus with MIC
range values of 1.25 to 5 µM20. Moreover, a new cyclic hexapeptide and a new isocoumarin isolated from
sponge-associated fungus A. similanensis showed potential antibacterial activity against S. aureus, B.
subtilis, E. coli, and P. aeruginosa with MIC value of below2.56 µg/mL21.

CONCLUSION
Aspergillus nomiuswhich was isolated from marine sponge N. chaliniformis showed potential cytotoxic
and antibacterial activities. Fraction II and III were found most cytotoxic against HCT 116 colon cancer
(IC50< 20 µg/mL) and antibacterial activity (inhibition zone > 12 mm) against S. typhosa, P. aeroginosa,
V. cholera, E. faecalis, S. epidermidis, S. aureus, E. coli, S. mutans, B. subtilis, M. luteus. Further study is
recommended to identify the active compound that is responsible for cytotoxic against HCT 116 colon
cancer and ten pathogenic bacteria.
ACKNOWLEDGMENT
Authors thank Prof. Dr. Peter Proksch and Mrs. Ni P. Ariantari (Institute of Pharmaceutical Biology and
Biotechnology, Heinrich Heine University Düsseldorf, Germany) for HPLC analysis of fractions. This
research was funded by The Ministry of Research and Technology, Indonesia through PMDSU Research,
059/SP2H/LT/DRPM/IV/2018.
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