Antibacterial Compound from Aspergillus elegans SweF9 an Endophytic Fungus from Macroalgae Euchema sp

Email: riznatd@gmail.com The antibiotic resistance of phatogenic bacteria has become a serious health concern and encouragement to search for novel and efficient antimicrobial metabolites. On the other hand, endophytic fungi have great potential as a natural source for antimicrobial agents. The objective of this study was to isolate antibacterial compound from endophytic fungi A.elegans SweF9. The fungus was stationarily cultured at 30°C for 12 days in potato malt peptone (PMP) medium, then the filtrate was extracted with ethyl acetate. The antibacterial activities of the extract were evaluated by agar diffusion method against Gram-poitive (Bacillus subtilis and Staphylococcus aureus) and Gramnegative (Escherichia coli) bacterial strains. The broth extract at a concentration of 1% was able to inhibit the growth of E. coli, S. aureus and B. subtilis with an index of antibacterial activity 84.6%, 91.6%, and 90% compared to streptomycin sulfate at the same concentration. The active compound (1) was purified to yield amorphous white and identified using FTIR, NMR, and EI-MS analyses, revealed identified as (+) epi-Epoformin. The compound showed an antibacterial activity index of against E. coli, S. aureus and B. subtilis bacterial were 48%, 45%, and 47%, respectively, at concentration 1%.


INTRODUCTION
Resistance to antimicrobial (AMR) agents is a relevant problem faced by health services and has become a serious concern around the world (Elisha et al., 2017;Santos et al., 2015). Natural products such as fungi from terrestrial, marine, and endophytic are considered an essential source for novel antibacterial compounds because of their abundant species diversity, rich in secondary metabolites and the improvements in their genetic breeding and is it relevant with natural products as an essential source for novel antibacterial compounds (Calvo et al., 2002). Endophytic fungi have proved to be a rich source of bioactive secondary metabolites, and recently several novel bioactive substances have been isolated from these microorganisms such as Epipolysulfanyldioxopiperazines (derivated of leptosins), were extracted from the endophytic fungus Leptosphaeria sp. isolated from the brown alga Sargasssum tortile showed the cytotoxicity against P-388 leukemia, and A. wentii pt-1 of marine red alga Gymnogongrus flabelliformis gifted another set of new xanthone derivatives, yicathin A C, exhibiting inhibitory activity against E. coli, S. aureus, and C. lagenarium (Sarasan et al., 2017).
The antimicrobial activities of an increasing number of fungi living in unique environments have been investigated for the discovery of new antibacterial compounds, such as endophytic fungi from wild plants and marine fungi. In the last decade, many novel bioactive natural products from marine fungi have been discovering that possess cytotoxic, anticancer, antiviral, antibacterial or antifungal activities (Thomas et al., 2010;Rateb et al., 2011). Although the active constituents may occur in lower concentrations, endophytic fungi may be the better sources of new and active antibacterial compounds than synthetic drugs to combat infectious diseases (Kalyanasundaram et al., 2015). It is due to their characteristic properties regarding temperature, 218 Volume 30 Issue 3 (2019) nutrients, competition, and salinity, they have developed specific or unique secondary metabolites which have different chemical structure compared with terrestrial fungi and synthetic compounds (Hasan et al., 2015).
Although the association of macroalgae and fungi has been described regarding pharmacological issue, there is a lack of studies about Indonesian marine seaweed endophytic fungi. Srikandace and Andayani (2015), have reported sicx endophytic fungi from marine Euchema sp, namely SF1 (coral reef), SF3 (sponge), SwF5 (sea worm), SweF9 (seaweed), SweF10 (seaweed), SweF11 showed antibacterial activity. Furthermore, extract of SweF9 (A. elegans) showed potential antibacterial activity with inhibition zones of 20-25mm at concentrations of 0.313mg/mL and 2.50mg/mL, compared to other ethyl acetate extract of isolated endophytic fungi. Therefore, the objective of this research was to isolate the antibacterial compound from endophytic fungi A. elegans SweF9. Bioautography-TLC and agar diffusion method were conduct to run antibacterial activity against gram-negative and gram-positive pathogen bacterias.

Materials
The fungal A. elegans SweF9 culture collection of the Research Center for chemistry -LIPI and test microorganisms used were S. aureus (InaCC-B4), B. subtilis (InaCC-B334), and E. coli (InaCC-B5) bacteria collected of Indonesian Culture Collection of the Research enter for Biology -LIPI. was obtained from the Indonesian. Culture media PDA, PDB, malt extract, peptone, Czapek-Dox broth were purchased from Difco. Streptomycin sulphate from Sigma. Silica gel 70-230 mesh and TLC plate were obtained from eMerck. All solvents used were analytical grade and distilled before use.
UV Spectra were recorded in MeOH on an Agilent Techn. Carry 60 spectrophotometer. The 1 H NMR spectra were recorded in CDCl3 on Agilent 500MHz with DD2 console system, while 13 C NMR at 125MHz. The chemical shift values (δ) are given in parts per million (ppm) and coupling constant (+) (J) in Hz. ESI-MS analysis was conducted by LCMS/MS Xevo G2-XS QTOF (Waters). Column chromatography was performed on silica gel (E. Merck, 70-230 mesh). TLC separation were performed on precoated plates (Silica gel 60, PF254, 0.2mm, E. Merck) and spots were detected under UV light.

Optimization of Culture Medium
The strains were prepared on PDA plates and incubated at 30ºC for seven days in the dark. A. elegant SweF9 was cultivation in three different media: potato dextrose broth (PDB), potato malt peptone (PMP) and Czapek-Dox broth (CDB) incubated for ten days in room temperature at static condition. The culture media and mycelium were separated by filtration. The mycelium (B) and filtrate (F) was extracted with EtOAc. Each of organic extract was dried over anhydrous Na2SO4, evaporated under reduced pressure to afford a brown oily gummy residue. All extracts were tested for their antibacterial activity.

Determination of the incubation period
The strain A. elegan SweF9 was inoculated into PMP medium, incubated up to 20 days in stationary condition at room temperature. Their growth and secondary metabolite production were determined every two days by comparing mycelial weight and antibacterial activity.

Extraction and Isolation
Secondary metabolites extraction was carried out by culturing A. elegans SweF9 in PMP (10 L) incubated for 12 days at room temperature at static condition. The culture were filtered to separate (filtrate) and mycelia (residual cake). The filtrate (F) was ecxtracted with EtOAc (4x5L) to give a brown gum (6.35g). The F extract (4.5g) was subjected to column chromatography (CC) on silica gel (70-230 mesh) using a stepwise gradient nhexane: EtOAc, to EtOAc: MeOH to obtain twelve fractions (F1-F12) based on TLC profile analysis. The fraction F2 showed highest inhibition zone on bacterial tests and Furthermore, TLC analysis of F2, showed major spot with 2 minor other spots. For isolation of antibacterial compounds, semi preparative TLC was used. To obtain bigger amounts of fractions, 150µL of F2 in a 15cm band was applied on TLC plate and developed to an 8cm distance with phase mobil of n-hexane: EtOAc (2:1). Separated fractions was scraped off together with silica gel, then was extracted with MeOH and concentrated. Recrystallization of the extract from methanol and n-hexane to yield F2.1 (20mg) as an active compound (1).

Antimicrobial activities assay Agar disk-diffusion method
Extracts, fractions and isolated compound were tested by disc diffusion method (Kalyanasundaram et al., 2015). The sterile nutrient agar (NA) media plates were prepared and inoculated with the test organism. Ten microliters of samples (1%) was added on to a sterile 6 mm paper disc using a micropipette and allowed to dry. Discs containing compounds were placed on the surface of the medium. The experiment was carried out in triplicates. Streptomycin sulphate (1%) was used as positive control and 10µL of DMSO was used as negative controls. The plates were incubated at ±35 for 12-24h. The diameter of the inhibition zone (mm) around the disc was measured by using the scale. The value of activity was adjusted to Activity Index= (Zone of inhibition of extract/Zone of inhibition of antibiotic) x 100%.

Thin layer chromatography (TLC)bioautography
The procedure autobiographic method is similar with agar difusion method. TLC plate (Merck Silica Gel 60 F254) was loaded with 10µL of fraction F2. The solvent system used was n-hexane: EtOAc (2:1). The chromatogram was kept for evaporation of the solvent then placed on the bacterial inoculated agar surface for a few minutes to allow diffusion. Next, the plate is removed and the agar plate was incubated. The zone of inhibition growth appears in the places, where the antimicrobial compounds were in contact with the agar layer . The antimicrobial activity was observed by inhibition zone and the Rf value was defined.

Optimization of culture condition
To examine the effect of culture medium on antibacterial activity of secondary metabolite, the strain A. elegans SweF9 was cultured in three different mediums, PDB, PMP, and CzB under static condition for ten days at room temperature. all extracts obtained were tested for antibacterial activity (Table I). Based on the result, the antibacterial activity of extracts of PDB and PMP does not differ significantly. However, extract (F) PMP shows stronger antibacterial activity, where the inhibition zone at the lowest concentration (1%) was wider than inhibition zones of the other extracts.  (Figure 1). Based on that result, A. elegan SweF9 could produced antibacterial agent after the fungus reached its stationary phase and decreased up to 12 days of incubation and remaining almost constant from 14 to 20 days' incubation. Based on these results, for scale up production to isolation antibacterial agent, A. elegan SweF9 was cultured in PMP medium (10L) at the stationary condition for 12 days.

Isolation and characterization of antibacterial compound
Approximately ~ 6.5g filtrate EtOAc extract (F) was obtained from the culture broth (10L). The extract (F) was examined against pathogen bacterial by TLC-bioautographic test. The bioautographic revealed clear zones of bacterial growth inhibition for E. coli, S. aureus, and B. subtilis were 30, 27, 29mm (Figure 2). Bioautographic belongs to microbiological screening methods commonly used for the detection of antimicrobial activity from several organic extracts, mainly plant extract (Jesionek et al., 2015). In this study, we used agar diffusion-TLC bioautographic also known as an agar contact method. It involved the transfer of the antimicrobial agent by diffusion from TLC to an agar plate previously inoculated with microorganism test . It is one of the simplest and cheapest methods for detecting antimicrobial compounds in extracts or fraction because the method is easy to run, reproducible, highly sensitive and requires less equipment (Jesionek et al., 2015).
Separation of the extract F (±5.5g) was conducted by vacuum column chromatography with gradual polarity increase to obtain 12 fractions (F1-F12) based on TLC profile. Each fraction obtained was tested for its antibacterial activity. Fraction F-2, F-3, and F-4 have wider inhibition zona than other fractions against three bacteria tests (Table II). Based on that result, F-2 and F-3 fraction were further purified, whereas F4 was not purified due to the limited of sample (<100mg). The F3 fraction was purified by chromatography column, but no pure active compound was isolated. Preparative-TLC and recrystallization of F2 with hexane and methanol to yield powder white (20mg) referred to as compound 1. In agar diffusion assays (Table III), compound 1 was active against E. coli, S. aureus, and B. subtilis with an activity index of 48, 45 and 47%, respectively.
Spectroscopic analysis was conducted to elucidate the structure of the active compound. Compound 1 was obtained as powder white and showed FTIR absorption bands at 3336 and 1678cm-1 which indicated the presence of hydroxyl (-OH) and carbonyl (C=O) groups, respectively. (Cala et al., 2018). The molecular formula was deduced to be C7H8O3 with two unsaturation's from EI-MS (m/z 141).
According to the spectroscopic data and literature as comparisons (Cala et al., 2018;Nagasawa et al., 1978), compound 1 was determined as 5-hydroxy-3-methyl-7-oxabicyclo [4.1.0] hept-3-en-2-one, trivial name is epiepoformin compounds. (+) -Epi-Epoformin was isolated firstly from unidentified fungus, which was separated from the diseased leaf of crapemyrtle (Lagerstroemia indica L.) and showed the marked inhibition against the germination of lettuce seeds, Lactuca sativa L. (Nagasawa et al, 1978). Recently, this compound was also used as a fungicide to inhibit the growth of Puccinia pathogenic fungi and  223 Uromyces which are pathogenic in legumes (Barilii et al., 2016). The biological activity of epiformin is better known as an antifungal or fungicide, while antibacterial activity has not been reported before. Furthermore, this compound has not been reported to be isolated from A. elegans isolated from marine biota.

CONCLUSION
In summary, endophytic fungi A. elegans SweF9 isolated from Euchema sp, possess antibacterial activity against tested bacteria, and revealed (+)-Epi-Epoformin as the active compound. Our results suggest that the fungal endophytes of A. elegans SweF9 could be an appropriate source of bioactive secondary metabolites.

ACKNOWLEDGEMENT
This work was supported by from Indonesian Institute of Sciences (LIPI). The author also thanks to Mrs. Andini Sundowo, M.Si for the mass spectra measurement, Dr. Nina Artanti and Dr. Sofa Fajriah for valuable discussion.