Antioxidant and antimicrobial screening of an unidentified yellow pigmented marine-derived fungus

The exploration of fungal secondary metabolites has increased significantly since the first discovery of penicillin. In addition, pigment derived from marine fungi attracts plenty of attention due to its complex and unusual chemical structures, which have tremendous antioxidants and antimicrobial activities. Hence, our current work aimed to characterize the crude pigment extract of an unidentified fungus SUCCF0001 and its potential as an antioxidant and antimicrobial agent. The fungus was cultivated on two semi-solid media namely MEA and PDA for 14 days at 25 °C, and two different liquids media namely MEB and PDB for 14 days at 25 °C with agitation at 150 r.p.m. The semi-solid medium and the fungal mycelium from the broth culture were extracted with methanol, while the liquid medium using ethyl acetate. The crude extracts were characterized using TLC plates and then visualized using DPPH, vanillin-H2SO4, and FeCL3. The presence of carotenoid pigment was detected using TLC with β-carotene as a marker. Antioxidant bioassay was screened using the DPPH method; while antimicrobial activity bioassay was conducted using the paper disc method against Bacillus subtilis, Escherichia coli, Micrococcus luteus, Pseudomonas aeruginosa and Candida tropicalis. The results showed that only the PDB extract had radical scavenging activity (RSA) > 50% for antioxidant activity. On the other hand, antimicrobial bioassay showed that only MEB extract had antimicrobial activity against B. subtilis. TLC analysis indicated the presence of antioxidants, terpenoids and phenols in both PDB and MEB extract. However, the result of fungal pigment characterization using TLC concluded that the pigment was not a β-carotene.


Introduction
The study of fungal secondary metabolites has been increasing significantly since the discovery of penicillin from Penicillium notatum [1].Fungal secondary metabolites display an incredibly broad range of biological activities, many important pharmaceutical products have been discovered and have contributed on improvements in human health [2].Several prior studies discovered bioactive pigment 2 from Indonesia's marine microorganisms including bacteria and fungi.For in instance, Sibero et al. reported antibacterial activity of a yellow pigmented coral-associated bacterium against Klebsiella pneumoniae, ESBL Escherichia coli, and MRSA strain MDR [3].In addition, a red-pigmented fungi exhibit antibacterial activity against several multidrug-resistant (MDR) bacteria [4].Sibero et al. [5] also stated that sponge-associated fungi produce antimicrobial substances, which is a pigmented fungus from sponge-derived Fusarium [6].
Many microbial secondary metabolites have been found to have biological activities such as anticancer, anti-inflammatory, antioxidant, and antibiotic properties.Interestingly, some of biological activities can also be associated with pigments produced by fungi [7].Fungal pigments are grouped into carotenoids, polyketides, melanin, azaphilones (polyketide derivatives), etc [8].Fungal pigments have demonstrated potential significance to human health, boosting interest in understanding and developing fungal pigments for therapeutic applications.Pigment derived from marine fungi attracts plenty of attention due to its complex and unusual chemical structures, which have tremendous antioxidants and antimicrobial activities [34].
Furthermore, a study conducted by Lagashetti et al. [9] reported that crude pigment extract of Gonatophragmium triuniae had antioxidant activity.Fungus Monascus purpureus and Penicillium mallochii were also known to produce several pigments with antibacterial and antioxidant properties [35,36].Thus, it is concluded that the fungal pigment is a promising source of bioactive compounds for future application in pharmaceutical industry.Moreover, as a mega-biodiversity country, Indonesia harbours enormous marine fungi that potentially produce bioactive pigments.Hence, our current work aimed to characterize the crude pigment extract of an unidentified fungal isolate SUCCF0001 and its potential as an antioxidant and antimicrobial agent.

Microorganisms
This research utilized an unidentified fungus (SUCCF0001) that was previously isolated from seaweed collected from Sumba Island, East Nusa Tenggara.For antimicrobial assay, several clinical human pathogens such as Bacillus subtilis, Eschericia coli, Micrococcus luteus, Pseudomonas aeruginosa, and Candida tropicalis were provided from a local hospital in Semarang.

Fungal metabolite production
Fungus was cultivated on Malt Extract Agar (MEA), Potato Dextrose Agar (PDA), Malt Extract Broth (MEB), and Potato Dextrose Broth (PDB) HiMedia for 7 days and incubated at 25 o C. The fungal isolate was recultivated for 7 days in 5 ml MEB and PDB as a seed culture.After 7 days, MEA & PDA cultures were inoculated into a new MEA & PDA medium, while 5 ml MEB and PDB seed cultures were transferred into 150 ml MEB and PDB for 14 days and incubated at 25 °C with agitation at 150 r.p.m [4,5].

Secondary metabolites extraction
Secondary metabolite extraction was done using the maceration method.MEB and PDB were filtered to separate filtrate and mycelium using Whatman's No. 1 filter paper.The agar media (MEA and PDA) and the mycelia from MEB and PDB were extracted by methanol (MeOH).Whereas MEB and PDB filtrate was extracted by ethyl acetate (EtOAc).Filtrate and solvent were separated using a separatory funnel and shaken vigorously then left to stand until two layers appeared.Afterwards, the methanol solvent was evaporated using a rotary evaporator at 40 °C, while the ethyl acetate solvent at 37 °C.Then, the crude extract was then weighed, wrapped with aluminium foil, and stored in a freezer [11].

Thin layer chromatography (TLC)
The crude extract was subjected to Silica Gel 60 F254 Aluminium TLC Plate for metabolites profiling as the stationary phase, while chloroform and ethyl acetate as the mobile phase (9:1, 8:2, 7:3).The extracts were deposited onto the TLC plates using a capillary tube.Then the metabolites spot was marked under UV lamps 245nm and 365 nm.After the visualization was carried out, followed by detection using specific reagents with the addition of 2,2-diphenyl-1-picrylhydrazyl (DPPH), vanillin-H2SO4, and FeCL3 reagents.Afterwards, the spot's Rf value was calculated [7,8,9].In addition, a -carotene was prepared by dissolving it in chloroform.This pigment was utilized as a marker to detect the presence of carotenoid pigment [3].The following formula was used to calculate the Rf value:

Antioxidant activity assay
The antioxidant assay was performed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method with several modifications as described by Budiono [13], Fadhillah [14] and de Torre [15].The crude extract was dissolved to 1000 ppm in methanol.After that, 0.75 mL of crude extract was added to 0.75 mL of DPPH solution with three replications.The solution mixture was homogenized and left in the dark for 30 minutes before measuring absorbance using a UV-Vis spectrophotometer (λ= 517 nm).The absorption inhibitory percentage of DPPH was used to quantify antioxidant activity, and the radical scavenging activity (% RSA) was calculated using the following formula: x 100

Antimicrobial Activity Assay
The antibacterial activity of each crude extract was examined using the disc diffusion method against Bacillus subtilis, Eschericia coli, Micrococcus luteus, Pseudomonas aeruginosa, and Candida tropicalis.The bacterial pathogens were refreshed on Nutrient Agar (NA), while fungal pathogens on Potato Dextrose Agar (PDA) for 24 hours before the assay.The pathogens were then inoculated using a sterile cotton swab into new Mueller-Hinton Agar (MHA) and Potato Dextrose Agar (PDA).Afterward, 10 µL crude extract solution was injected into a paper disc (6 mm) and placed onto the MHA and PDA medium then incubated for 24 hours at 32 °C.Chloramphenicol and nystatin were used as positive control.Antimicrobial activity was done with two replications.The clear zone around the paper disc indicates the presence of antibacterial and antifungal activity [10].

Results and discussion
The fungal isolate SUCCF0001 was characterized as small white colonies and produced exudate and orange-coloured soluble pigment.Based on its macroscopic morphology, the isolate had white, and round with a raised centre (Figure 1).Mugesh et al. [16] stated that sucrose and trace elements affect the growth and pigment production, imply that nutrient content are important for fungal pigments production and different growth medium might produce slightly different pigment colour.The fungal isolate generated extracellular pigment on the PDA medium, which is known to have biological activities such as antioxidant and antimicrobial [13,3].Several studies have reported fungal pigment biological activities.Sibero et al. stated that fungal pigment had antibacterial activity against multidrugresistant (MDR) human pathogen such as ESBL Escherichia coli, S. enterica ser.Typhi, S. haemolyticus, methicillin-resistant Staphylococcus aureus (MRSA), and Klebsiella pneumoniae [4,5,11].Ji and Kim [18] also reported that the pigment produced by Serratia sp.PDGS 120915 had antibacterial activity against Staphylococcus, Bacillus, Enterococcus, and Streptococcus.In this study, fungal isolate SUCCF0001 was cultivated in several culture media.Semi-solid media namely MEA and PDA, and two different liquids media namely MEB and PDB.The objective of this approach was to determine the best media for antioxidant and antibacterial compound production.The result of metabolite profiling using Thin Layer Chromatography (TLC) plate is presented by Figure 2 and Table 1.The components of bioactive compounds contained in the crude extract were characterized using thin layer chromatography (TLC).The subjected crude extracts on TLC plates: MEA (Malt Extract Agar), MMB (Malt Extract Broth Mycelium), MEB (Malt Extract Broth), PDA (Potato Dextrose Agar), MPB (Potato Dextrose Broth Mycelium), PDB (Potato Dextrose Broth) and β-Car (β-Carotene) as a pigment marker was shown on Figure 2a-b.Detection of several targeted compounds using the TLC plate was carried out by DPPH and Vanillin-H2SO4 spraying reagents and dipping the TLC plates for FeCl3.A basic visualization was conducted by observation under UV 254 nm and 365 nm.Alen et al. [19] stated that if the same spots were able to observed under UV 254 nm and 365 nm indicated that the compounds at least had two conjugated double bonds under UV 254 nm, while under UV 365 nm showed that the compounds have a longer conjugated double bonds (chromophore) and an auxochrome group.Based on TLC analysis, the crude extracts contained antioxidant, phenol and terpenoid compounds (Figure 2c-e).According to several studies it is known that fungal pigment containing phenolic, flavonoid, lipids, terpenes, carotenoids, polyketydes, anthraquinones, hydroxyanthraquinone, azaphilones and naphthoquinones [13,14,15,18].Hence, these crude extracts were suspected to have the ability as an antioxidant and antimicrobial agent due to its compound content, since terpenoids and phenolic compounds are natural antioxidants and antimicrobial agents [14][15][16].Thin Layer Chromatography (TLC) profiling for antioxidant detection of fungal pigment crude extract has been done.Following spraying with DPPH reagent, the results showed positive free radical scavenging activity on TLC plates, as shown by a yellow spot on a purple background (Figure 2c) [12].TLC profiling for phenols detection showed that after the TLC plates were dipped in FeCl3 reagent and heated at 110 o C indicated a positive results because a grey to black colour was formed (Figure 2d) [19].The TLC profiling for terpenoids detection gave the results after the TLC plates were sprayed with Vanillin-H2SO4 reagent and heated at 110 o C. The formation of blue to purple or brown color indicates a positive result (Figure 2e) [22].In the terpenoid and phenol detection, the result showed that several crude extracts contained spots of the same Rf value.These results indicated that fungi might produce similar or even the same terpenoid and phenol compounds [23].The fungal pigment was suspected to contain -carotene due to its color, hence Thin Layer Chromatography (TLC) profiling was performed using -carotene as a pigment standard.However, the result of fungal pigment characterization using TLC concluded that the pigment was not a -carotene, because the TLC profiling gave different Rf value (spots) between the marker (pigment standard) and the fungal pigment (samples) (Figure 2a-b).Hence, further investigation to identify this pigment is very interesting to be conducted.According to Lin and Xu, fungal pigments can be grouped into carotenoids (-carotene, astaxanthin, lycopene, canthaxanthin, and zeaxanthin), melanin (1,8-dihydroxynaphthalene, eumelanin, and pyomelanin), polyketides (anthraquinones and naphthoquinones), azaphilones (monascorubrin, monacolin K, monascin and ankaflavin) [24].
Antioxidants have an important role to health maintenance because it reduces the risk of chronic diseases such as cancer and heart disease.Antioxidant was known for its ability to stabilized free radicals which degrading the healthy cells [25].Following the detection of antioxidant properties using TLC, we performed the antioxidant assay to determine the antioxidant activity (radical scavenging activity) of each crude extract using 2,2-diphenyl-1-picrylhydrazyl (DPPH) method.The ability of the crude extracts to donate electrons to the radical of 2,2-diphenyl-1-picrylhydrazyl (DPPH) was used to determine their antioxidant activity.Unfortunately, only one out of six crude extracts gave the significant result as a prospective antioxidant agent which is PDB crude extract.The scavenging activity was 61.26 % ± 3.24 (Figure 3).Gajalakshmi et al. [26] stated that fungi with orange-coloured pigment had lower antioxidant activity compared to others with different colour, such as red-coloured pigment and yellowcoloured pigment.The crude pigment extracts used in this study contains phenols and terpenes.In accordance to Zeb [21], Achika et al. [27], and Wang et al. [28] which stated that phenols and terpenes contributed in antioxidant activity.Therefore, the antioxidant activity (radical scavenging activity) may result from the coexistence of phenolic and terpenoid-type compounds [16,20,21].The DPPH assay measures an antioxidant's ability to scavenge free radicals, while the β-carotene assay demonstrates the antioxidant's protective properties [29].Another bioassay was conducted to evaluate the antimicrobial activity of this fungal pigment.The result of this bioassay is shown by Figure 4 and Table 2. Regrettably, the pigment extract from fungus SUCCF001 only exhibited a weak antimicrobial property to B. subtilis with diameter 2.9 mm.According to Trianto et al. [31], this diameter of inhibition zone is categorized as weak activity.Although several records stated pigments from marine fungi had a strong antimicrobial activity [4][5][6], our study got a reverse result.It is north worthy to be considered that all pathogens in this study were clinical isolates and noted as multidrug-resistant organisms (MDRO).It is represented by the absence of inhibition zones on the control positive drugs (Figure 4 and Table 2).Previous study was done by Sibero et al. [32] also denounced a weak antibacterial activity of yellow pigmented marine sponge-associated Trichoderma parareesei against MDR bacteria and no antifungal activity against MDR dermatophytes.Besides drug-resistance profile of the pathogens, other factors were also considered as the reason of the absence of antimicrobial activity from this fungus such as unsuitable media for metabolite production and solvent's polarity [19,20,21].

Conclusion
The characterization of secondary metabolites profile using Thin Layer Chromatography (TLC) showed that each crude extract contained antioxidant, terpenoid and phenol compounds.It is also highlighted that the orange pigment from this fungus was not -carotene.Only PDB crude extract had radical scavenging activity (RSA) > 50%.In addition, MEB crude extract demonstrated weak antibacterial activity against Bacillus subtilis.Therefore, only PDB and MEB crude extracts will be continued for our further study.

Figure 2 .
Figure 2. Visualization of fungal metabolites & β-carotene standard on TLC plate a) under UV 254 nm, b) under UV 365 nm, c) addition of DPPH reagent, d) addition of FeCl3, e) addition of Vanillin-H2SO4 with heating at 110 C.