Cytotoxic and Antimicrobial Activities of Ethyl Acetate Extract from Fungus Trichoderma reesei strain JCM 2267, Aspergillus flavus strain MC- 10-L, Penicillium sp, and Aspergillus fumigatus Associated with Marine Sponge Stylissa flabelliformis
1Faculty of Pharmacy, Gadjah Mada University, Yogyakarta, Indonesia.
2Department of Pharmaceutical Biology, Faculty of Pharmacy, Gadjah Mada University, Yogyakarta, Indonesia.
3Department of Pharmacology, Faculty of Pharmacy, Gadjah Mada University, Yogyakarta, Indonesia.
*Corresponding Author E-mail: erna_prawita @ ugm.ac.id
This study was to investigate the cytotoxic and antimicrobial activities of ethyl acetate extract yielded from fermented fungi Trichoderma reesei strain: JCM 2267, Aspergillus flavus strain MC-10-L, Penicillium sp, and Aspergillus fumigatus associated with Stylissa flabelliformis sponge, and also to investigate the significant component in ethyl acetate extract of it. Antimicrobial experimentation was performed to various microbes with liquid microdilution method. The assessment of antimicrobials was undertaken by observing the value of MIC50. Cytotoxic testing was performed using MTT assay method. The cytotoxic activity evaluation was conducted by finding the value of IC50. The compound analysis in the ethyl acetate extract was used GC-MS. The result showed that in the antimicrobial experimentation, there was a variation in the percentage of inhibition where the smallest MIC50 values were found in Aspergillus fumigatus fungi ethyl acetate extract against Candida albicans ATCC 10231 at 1.3mg/mL. The result of the cytotoxic test against tumor cell line T47D, the lowest IC50 was found in Penicillium sp fungi with 111mg/mL value. Aspergillus flavus strain MC-10-L fungi, Penicillium sp, and Aspergillus fumigatus obtained from sponge isolation Stylissa flabelliformis have antimicrobial and cytotoxic activity with various range. All fungi extracts are not toxic against normal cells (Vero cells). The class of compounds in ethyl acetate fungi extract Trichoderma reesei strain JCM 2267 are mostly cyclohexane.
KEYWORDS: Trichoderma reesei strain JCM 2267, Aspergillus flavus strain MC-10-L, Penicillium sp,
Aspergillus fumigatus, Stylissa flabelliformis, bioactivities.
Marine sponge by weight consists of 40-60%
microbes (Brickmann et al., 2017). Marine sponge and other invertebrates
contain various types of microorganism, in which sometimes
benefit from various
chemical compounds produced
by the microorganism that are protecting the sponge or other inhabitants against predators or pathogen.
Marine microbes are also known to produce many secondary metabolites which can be used for pharmaceutical purposes (Devi et al, 2014; Jayaprakashyel et, 2017; Larichev et al, 2017; Ashwini et al, 206; Pitamber et al, 2014; Muralihdaran et al, 2017, 2019; Sarkar et al, 2020)
The increase of the evidence has implications on microbial symbionts as the actual source from compounds coming from marine organism, making the marine microbial symbionts an important object of marine microbiology and marine natural products for their potency to solve problems such as stagnant supply of marine products naturally. There are many evidences showing that most of bioactive metabolites, originally regarded as an original product of an animal, are often synthesized by its symbiotic microorganisms (Mehbub et al., 2014; Sharmakumar et al, 2017). Some anticancer metabolites from marine sponge has been developed up to preclinical phase testing or clinical trials, and discodermolide, halichondrin B, and bryostatin 1 are considered as products derived from their symbiotic microbes (Tores et al., 2017). Recently, from 20 compounds originated from (or separated by) natural marine products in clinical trials to cure cancer, 15 are isolated from sponge, tunicates, and molluscs, and only 5 are derived directly from microorganisms (Martins et al., 2014). In the search of new antibiotics, sponge-related microbes have been assessed as a promising source, for sponges have a number of unknown phylogenetic microbial species. Hirsutanol A, a new cyclic sesquiterpene isolated from fungus separated from Indo- Pacific Haliclona sp. sponge, has significantly gone against Bacillus subtilis, a furan carboxylic sumiki's acid isolated from fungus derived from an active Cladosporium herbarum acid sponge which had went against Bacillus subtilis and Staphylococcus aureus (Jadulco et al., 2001). Selvin et al (2009) had found an organic solvent and water-soluble antimicrobial compound of actinomycetes derived from Nocardiopsis dassonvillei. Despite the unclear way of work, many of the crude extracts obtained from sponge-derived microbial species fermented broths evaluated against the most common pathogenic bacteria shows that this extract greatly inhibited the pathogenic bacteria Escherichia coli while significantly go against some others. This result shows that detailed research regarding such extract will open a way to an invention of a new antimicrobial drug (Gopi et al., 2012).
Sponges contain microbes about 50% of their body mass. This led to the notion that the secondary metabolites they contained were microbial products associated with sponges. Previous research has shown that the Stylissa flabelliformis sponge contains various fungi that have been identified by the PCR method and have antimicrobial effects with agar diffusion method. Earlier research had stated that 10 Stylissa flabelliformis fungi sponge association taken from West Bali National Park, Indonesia, had been isolated, identified, and conducted antimicrobial precursor by diffusion method (Setyowati et al., 2017). This research aimed to acknowledge the antimicrobial activity with microdilution and cytotoxic selectivity of cancer cells from ethyl acetate extract fermented from fungi Trichoderma reesei strain JCM 2267, Aspergillus flavus strain MC-10-L, Penicillium sp, and Aspergillus fumigatus associated with S. flabelliformis sponge.
All chemicals used in this research were of
analytical grade. Fungi Trichoderma reesei strain JCM 2267, Aspergillus flavus strain MC-10-L, Penicillium
sp, and Aspergillus fumigatus associated
with S. flabelliformis sponge. T47D cancer cells, WiDr cells, 4T1
cells, and vero cell of central
university laboratory (LPPT) UGM collection. Microbes
testing Escherichia coli (EC) ATCC 25922, Staphylococcus aureus (SA) ATCC 25923,
Bacillus subtilis (BS) ATCC 6633, Enterococcus faecalis (EF) ATCC 29212, and Candida albicans (CA) ATCC 10231 (Dexa),
dimethyl sulfoxide (DMSO,
Merck), Roswell Park Memorial Institute (RPMI) 1640, Medium 199 (Sigma), Sabouroud Dextrose Broth
(SDB) (Oxoid), Nutrient Agar (NA) (Oxoid), Brain Heart Infusion
(BHI, Oxoid), Sodium hydrogen carbonate
(NaHCO3), [3-(4,5-dimetiltetrazoliumbromide) (MTT) (Sigma
Aldrich), Phosphate Buffer Saline (PBS), Fetal Bovine Serum (FBS, Sigma) 10%, 0.25% Trypsin-
EDTA (Gibco, Invitrogen Canada), Sodium Dodecyl
Sulfate (SDS, Merck-Schuchardt, Germany) 10% in 0.01 N HCl, Penicillin-Streptomycin (Gibco,
Invitrogen Canada), Fungizone
0.5 % (Gibco), Dulbecco’s modified Eagle’s medium (DMEM)
(Sigma Aldrich).
A microplate reader (Bio-Rad, Japan), Haemocytometer (Neubauer, Germany), CO2 incubator (Heraeus®, Germany), an inverted microscope (Olympus, Japan)
Fermentation method and fungi extraction associated sponge was performed based on a method previously conducted by Setyowati et al. (2017). In the mentioned research, T. reesei strain TV221 fermented for 11 days to produce optimal secondary metabolites was used. The fermentation in this study to produce secondary metabolite on T. reesei strain JCM 2267 was performed for 6 days, while Aspergillus flavus strain MC-10-L for
11 days, Penicillium sp for 5 days, and Aspergillus fumigatus for 5 days.
Antimicrobial test was performed with liquid micro dilution method to determine the inhibitory percentage values (MIC50 and MIC90) against various microbial tests. The medium used were BHI (bacteria) and SDB (yeast). Microbes were inoculated into the medium at 37oC for 24 hours. The microbial suspension experimentation would be inoculated into the medium in which the Optical Density (OD) value was read with spectrophotometer at λ600nm for bacteria, and λ520nm for yeast (Kolarevic et al., 2016; Chhibber et al., 2015). The samples (fungi fermented ethyl acetate extract) were dissolved with DMSO solvent, then made into series of
concentrations of 1000, 500, 250, 125, 62.5, and 30 μg/mL. Each series of concentration were added with microbial suspension test before put in to 96-microplate. Each mixed suspension were added into 96-microplate as much as 200μg/mL. Streptomycin-positive controls for Gram negative bacteria, kanamycin for Gram positive bacteria, and nystatin for yeast were used. Plate was incubated at 37oC for 24 hours. Microbial absorbance was measured using microplate reader by observing absorbance at λ600nm for EC and SA, and λ520nm for CA. The values of MIC50 was calculated with live cell percentage using Microsoft Excel (Linear regression of concentration logs) (CLSI, 2006; Balouiri et al., 2015).
Cells with density of 2x104 cells/wells in complete cell culture medium were put into 96-well plate wells, each 100μL. Cells were incubated in the incubator with 5% CO2 at 37oC for 24 hours. Series of concentration sample (2000, 1000, 500, 250, 100, 50, 25, and 12.5μg/mL)
were made for treatment with DMSO solvent. Plates that contain cells from CO2 incubator was taken up. Cell media were discarded. Cells were washed with 100μL PBS at each well, then PBS was discarded. Series of concentration samples were put into the well. The controls used were cell control and media control. Plate was incubated in the CO2 incubator 5% at 37oC for 24 hours. The cell medium was removed and then washed with PBS, and 100μL reagent MTT (0.5mg/mL) was added into each well. Cells were incubated for 2 - 4 hours in the CO2 incubator. The cell condition was examined with inverted microscope and observed whether formazan had been formed. Stopper 100μL SDS 10 % (0.01 N HCI) was then added to each well. Plate was incubated in the dark at room temperature overnight. Plate absorbance was read using ELISA reader with λ550 nm. Absorbance graph (after subtracted with media control) vs concentration was made. Living cell
percentage and IC50 value analysis were calculated with Microsoft Excel (CCRC, 2013; Maboni et al., 2015). The formula which was used to calculate percent inhibition is as follows (Quave et al., (2008):
(1-OD sample-OD sample blank)
% Inhibition =--------------------------------------------------- x 100%
OD DMSO-OD DMSO blank
Selectivity Index (SI) cytotoxicity (Badisa et al., 2009): Selectivity index was determined using following formula:
IC50 on cell vero
SI =
IC50 on tumor cell lines
Selectivity Index (SI) antimicrobe (Nunes et al., 2016) The relation between cytotoxicity and antifungal activity is determined by the Selectivity Index (SI), which is calculated with the logarithm ratio of cytotoxic concentration (IC50) and MIC50 value for strain (SI = log [IC50] / [MIC50])
A method of analysis was performed based on a method previously conducted by Setyowati et al (2017).
The antimicrobial activity test was performed by determining the value of MIC50 from ethyl acetate extract fermented from fungi associated with S. flabelliformis (Fig. 1). The MIC50 value and selectivity index (SI) from fungi's ethyl acetate are shown in Table 1.
(a) (b) (c) (d)
Figure 1. Morphology fungi (a) Trichoderma reesei strain JCM 2267, (b) Aspergillus flavus strain MC-10-L, (c) Penicillium sp, (d) Aspergillus fumigatus associated with S. flabelliformis (Setyowati et al., 2018).
Table 1. MIC50 value and selectivity index (SI) ethyl acetate extract of fungi associated with S. flabelliformis sponge (Erawan, 2018; Rahmanti, 2018; Hanum, 2018; Devi, 2018)
|
No |
Name of fungus |
SA ATCC 25923 |
Vero cell |
SI |
EC ATCC 25922 |
Vero cell |
SI |
CA ATCC 10231 |
Vero cell |
SI |
|
MIC50 |
IC50 |
log (IC50/MIC) |
MIC50 |
IC50 |
log (IC50/MIC) |
MIC50 |
IC50 |
log (IC50/MIC) |
||
|
1 |
a |
344 |
2908 |
0.93 |
144 |
2908 |
1.31 |
2.9 |
2908 |
3 |
|
2 |
b |
942 |
5227 |
0.74 |
557 |
5227 |
0.97 |
21 |
5227 |
2.4 |
|
3 |
c |
1346 |
5585 |
0.62 |
719 |
5585 |
0.89 |
6.9 |
5585 |
2.91 |
|
4 |
d |
856 |
4143 |
0.68 |
1203 |
4143 |
0.54 |
1.3 |
4143 |
3.5 |
|
No |
Name of fungus |
EF ATCC 25923 |
Vero cell |
SI |
BS ATCC 6633 |
Vero cell |
SI |
|
MIC50 |
IC50 |
log (IC50/MIC) |
MIC50 |
IC50 |
log (IC50/MIC) |
||
|
1 |
a |
63 |
2908 |
1.67 |
516 |
2908 |
0.75 |
|
2 |
b |
363 |
5227 |
1.16 |
1169 |
5227 |
0.65 |
|
3 |
c |
800 |
5585 |
0.84 |
1333 |
5585 |
0.62 |
|
4 |
d |
339 |
4143 |
1.09 |
1137 |
4143 |
0.56 |
Note: a=Trichoderma reesei strain JCM 2267, b=Aspergillus flavus strain MC-10-L, c= Penicillium sp, d=Aspergillus fumigatus. SA=Staphylococcus aureus ATCC 25923, EC=Escherichia coli ATCC 25922, CA=Candida albicans ATCC 10231, EF=Enterococcus faecalis ATCC 29212, BS= Bacillus subtilis ATCC 6633.
According to Rio and Recio (2005), an extract sample is said to have antimicrobial activity when it has MIC50 under 1000mg/mL. Therefore, when the data is observed from Table 1, it means T. reesei strain JCM 2267 is fungi which have the most potent antimicrobial activity, for its MIC50 value for all microbes is the least and under 1000mg/mL, except for C. albicans ATCC 10231 microbes in which MIC50 value (2.9mg/mL) is bigger than the MIC50 value of A. fumigatus fungi (1.3mg/mL). Meanwhile, its toxicity towards vero cells can be seen from the result of its selectivity index. Positive values indicate that selectivity sample against microbes is somewhat Vero cell, while negative values indicate toxicity to vero cells. Table 1 shows all fungi extracts are not toxic against normal cell (vero cell).
On the table 2, cytotoxic activity was present in range 111-550g/mL. It shows that fungi associated with S. flabelliformis sponge have cytotoxic activity. According to Prayong (2008), extracts of natural products are declared potentially cytotoxic if having IC50 value less than 100mg/mL and moderate cytotoxic if IC50 value is
between 100 to 1000mg/mL. Table 2 also shows that examined samples are not toxic against normal cell (Vero cell). Therefore, it can serve as a source to look for cytotoxic compounds from fungi associated sponges. According to Badisa et al. (2009) and Sutejo et al. (2016) extracts are said to have high selectivity if the SI value ≥ 3.
T47D cells are positive ER/PR breast cancer cells because such cells express estrogen receptor and progesterone receptor. Moreover, T47D cells include cancer cell lines that are responsive to endocrine and chemotherapy (Holliday and Speirs, 2011; Dogan et al., 2015). The T47D breast cancer cells express a p53 protein that undergoes missense mutation. This mutation occurs at a residue of 194 (in the zinc-binding domain, L2). It causes p53 protein not being able to bind to the response element in the DNA so that the ability of p53 to regulate cell cycle is reduced and even disappeared (Schafer et al., 2000). P53 protein is a transcription factor with pro apoptosis function (Ozaki and Nakagawara, 2011)
Table 2. IC50 value against tumor cell line (T47D) and Selectivity index (SI) ethyl acetate extract of fungi associated S. flabelliformis
(Erawan, 2018; Rahmanti, 2018; Hanum, 2018; Devi, 2018)
|
S No |
Name of fungus |
T47D cell |
Vero cell |
SI |
|
IC50 (mg/mL) |
IC50 (mg/mL) |
(IC50 vero cell /(IC50 T47D cell) |
||
|
1 |
a |
158 |
2908 |
18.4 |
|
2 |
b |
550 |
5227 |
9.5 |
|
3 |
c |
111 |
5585 |
50.3 |
|
4 |
d |
523 |
4143 |
7.9 |
Note: a=Trichoderma reesei strain JCM 2267, b=Aspergillus flavus strain MC-10-L, c=Penicillium sp, d=Aspergillus fumigatus
Table 3. Metabolite compound of a. Trichoderma reesei strain JCM 2267, b. Aspergillus flavus strain MC-10-L, c. Penicillium sp, and d. Aspergillus fumigatus associated with sponge S. flabelliformis: Class of compounds identification of ethyl acetate extract by Gas Chromatography Mass Spectrometry (GC-MS Shimadzu type QP2010SE):
a. Trichoderma reesei strain JCM 2267
|
R Time |
Name of compound |
% Area |
|
10.917 |
1,2-Ethanediol, 1-phenyl- (CAS) Styrene glycol |
13.44 |
|
11.24 |
Acetamide, N-butyl-(CAS) N-butylacetamide |
3.09 |
|
13.62 |
1,4-diaza-2,5-dioxo-3-isobutyl bicyclo[4.3.0]nonane |
10.58 |
|
13.861 |
Hexadecanoicd acid, methyl ester (CAS) Methyl palmitate. |
2.37 |
|
14.14 |
1,4-diaza-2,5-dioxo-3-isobutyl bicyclo[4.3.0]nonane |
3.55 |
|
14.217 |
1,4-diaza-2,5-dioxo-3-isobutyl bicyclo[4.3.0]nonane |
5.26 |
|
15.413 |
2,5-Piperizedione-3,6 bis(2methylpropyll) |
5.13 |
|
16.473 |
3-benzyl-1,4-diaza-2,5-dioxobiacycl o(4.3.o) nonane |
4.05 |
|
16.942 |
1,2-Benzenedicarbocyclyc acid diisooctyl ester (CAS) Isooctylphthalatee |
52.52 |
b. Aspergillus flavus strain MC-10-L
|
R Time |
Name of compound |
% Area |
|
11.417 |
Phenol, 2,4-bis (1,1dimethylyethyll)-(CAS) 2,4-di-trtier rbutylphenoll |
2.67 |
|
13.596 |
1,4-diaza-2,5-dioxo-3-isobutyl bicyclo[4.3.0]nonane |
3.45 |
|
13.858 |
Hexadecanoid acid, methyl ester (CAS) Methyl palmitate |
4.43 |
|
14.121 |
1,4-diaza-2,5-dioxo-3-isobutyl bicyclo[4.3.0]nonane |
2.23 |
|
14.2 |
1,4-diaza-2,5-dioxo-3-isobutyl bicyclo[4.3.0]nonane |
1.89 |
|
14.789 |
9-octadecenoic acid, methyl ester (CAS) Methyl octadec-9-eno |
7.38 |
|
16.936 |
1,2-Benzenedicarbocyclyc acid, bis (2ethylyhexyll) ester (CAS) bis (2-ethyl) |
77.5 |
c. Penicillium sp
|
R Time |
Name of compound |
% Area |
|
13.605 |
1,4-diaza-2,5-dioxo-3-isobutyl bicyclo[4.3.0]nonane |
3.72 |
|
13.860 |
Hexadecanoid acid, methyl ester (CAS) Methyl palmitate |
7.82 |
|
14.204 |
1,4-diaza-2,5-dioxo-3-isobutyl bicyclo[4.3.0]nonane |
3.49 |
|
14.792 |
9-octadecenoic acid, methyl ester (CAS) Methyl octadec-9-eno |
6.87 |
|
16.435 |
Demecolcinee |
0.97 |
|
16.937 |
1,2-Benzenedicarbocyclyc acid diisooctyl ester (CAS) Isooctylphthalatee |
77.14 |
d. Aspergillus fumigatus
|
R Time |
Name of compound |
% Area |
|
8.336 |
2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one |
13.06 |
|
9.129 |
1,2-ethanadiol, 1-(-2-furanyl)-(CAS) dihidroxyethyl-1-furan |
8.26 |
|
9.215 |
2-Furancarboxaldehyde, 5-(hydroxymethyl)-(CAS) HMF |
18 |
|
13.063 |
N-acetyl-4-hydroxyphenylacetamide |
3.47 |
|
13.625 |
1,4-diaza-2,5-dioxo-3-isobutyl bicyclo (4.3.0) nonane |
7.51 |
|
13.864 |
Hexadecanoid acid, methyl ester (CAS) Methyl palmitate |
3.8 |
|
14.146 |
1,4-diaza-2,5-dioxo-3-isobutyl bicyclo (4.3.0) nonane |
2.7 |
|
14.225 |
1,4-diaza-2,5-dioxo-3-isobutyl bicyclo (4.3.0) nonane |
2.96 |
|
14.795 |
1,4-diaza-2,5-dioxo-3-isobutyl bicyclo (4.3.0) nonane |
3.57 |
|
16.942 |
1,2-Benzenedecarbocyclyc acid bis (2-ethylhexyl) ester (CAS) bis (2-ethyl) |
35.97 |
c. Metabolite compound of the fungus associated with sponge S. flabelliformis:
Estimation of compound content classification in ethyl acetate extract of the fungi related Stylissa flabelliformis is identified with GC-MS Spectrometry method. The analysis result can be seen in Table 3 (a, b, c, d).
From the data result in Table 3 (a, b, c, d), it is shown that all ethyl acetate extracts contain cyclohexanone compound. Cyclohexanone compound is also present in ethyl acetate extract of T. reesei strain TV221 fungi associated S. flabelliformis sponge, as investigated in previous researches (Setyowati et al., 2017; 2018). It is interesting because every fermented fungus extract contains mentioned compounds, and it needs further investigation.
Research on investigating active compound derived from fungi associate sponge had been widely practiced. It is because the microbial content in the sponge is quite high, and there is a possibility that the active compound produced is a product of microbes associated with sponges (Indraningrat et al., 2016). Microorganism living in relationship with sponges and other marine invertebrates have been claimed as a real source for some metabolites found in their hosts (Proksch et al., 2002). As in the case of Bryostatins, it has been proven that metabolites considered initially to be taken from host organisms are derived from microbes (Dobretsov et al., 2006; König et al., 2006; Egan et al., 2008; Rungprom et al., 2008). Thus, more compounds that were initially considered biosynthesis by sponges or other marine microorganisms are now found to be produced by their associated microorganisms (Hentschel et al., 2006). The following examples demonstrate various activities shown by specific sponge microbial association derived from fungi.
Fungi A. versicolor resulted from isolated marine sponge Petrosia sp taken from the shores of Jeju Island, South Korea, shows the presence of aromatic polymeric derivative compounds and five anthraquinoneindirubinin. Sterigmatocystin compound, averantin, methyl-averantin, and nidurufin showed antiproliferative activity against five tumor cells in humans. Averantin and nidurufin compounds also showed antibacterial activity against Gram-positive bacteria (Lee et al., 2010).
From Aspergillus insuetus (OY-2017) fungi of the Mediterranean psammocinia sponge, three new meroterpenoids are found, namely insuetolides A-C and four sesquiterpenes, including a new derivative. Isuetolida has a new carbon skeleton formed from the cyclization of the farnesyl and 3,5-dimethylorselonate. Insuetolida A showed weak antifungal activity against Neurospora crassa, whereas insuetolida C showed mild cytotoxic activity against human leukemia cells MOLT-4 (Cohen et al., 2011).
Five new sesterpenoid ophiobolin were isolated with A. ustus from S. domuncula sponge (Liu et al., 2011). The cytotoxic activity of sesterpenoid ophiobolin compound derived was then examined against murine lymphoid cells L5178Y with results that did not show cellular growth inhibition activity.
Aspergillus flavus strain MC-10-L fungi, Penicillium sp, and Aspergillus fumigatus obtained from sponge isolation Stylissa flabelliformis have antimicrobial and cytotoxic activity with various concentration range. All fungus extracts are not toxic against normal cells (Vero cells). The class of compounds in ethyl acetate fungi extract Trichoderma reesei strain JCM 2267 are mostly cyclohexane.
The author would like to acknowledge the funding support from PUPT-DIKTI Indonesia No: 2732/UN1/DITLIT/DIT-LIT/LT/ 2020
The authors declare no conflict of interest.
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Received on 19.09.2020 Modified on 21.10.2020
Accepted on 09.11.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(10):5126-5132.
DOI: 10.52711/0974-360X.2021.00893