Unraveling the Therapeutic Potential of Antifungal Metabolites of Aspergillus giganteus on Human Fungal Pathogen

Abstract

Fungal infections represent a worldwide risk to the health of animals, humans, and wildlife. Among the pathogenic fungal strains, Aspergillus flavus plays a key role in the onset of invasive aspergillosis (IA). Over a while, pathogenic fungi develop resistance to all licensed systemic antifungal drugs. The resistance development in fungal pathogens is attributed to the improper usage of drugs and the inference of the pathogen's cellular mechanisms. This concern could be resolved by the use of antifungal metabolites. Antifungal metabolites derived from Aspergillus giganteus are of profound interest in recent times due to their therapeutic potential. This particular antifungal protein (AFP) holds substantial promise in breaking the virulence mechanism in pathogenic fungi. In light of these circumstances, the study examined how AFP from A. giganteus affects A. flavus, the pathogen responsible for aspergillosis. The co-culturing technique firmly drew the potential of antifungal metabolites to counteract the effects of fungal agonists. A reduction of approximately 91% in the growth of pathogenic A. flavus treated with culture filtrates of antagonist fungi confirms the antagonistic effect of the culture filtrate metabolites on A. flavus viability and indicates that these metabolites are extracellular. The presence of shrunken and damaged spores in the SEM images of A. flavus treated with culture filtrates is evident in supporting its impact on the cellular membrane of the pathogenic bacteria. The MIC and MFC of culture filtrates were found to be 125 μg/ml and 250 μg/ml. Furthermore, the free radical scavenging activity of antifungal metabolites emphasizes its significant antioxidant potential. Finally, the findings of cytotoxicity studies confirmed the druggability of antifungal metabolites of A. giganteus. Thus, the study strongly endorses the antagonistic potential of AFP against A. flavus, suggesting that this metabolite could be developed into a novel treatment for invasive aspergillosis caused by A. flavus.

Introduction

Fungal diseases have an indispensable global impact in recent times, presenting a formidable challenge to human health, particularly among high-risk individuals with compromised immune systems. The epidemiology of these infections varies across different regions, influenced by various factors such as the population at risk, socioeconomic conditions, and the prevalence of specific fungi which are shaped geographical and ecological characteristics [1, 2].

Consequently, the impact of fungal diseases on morbidity and mortality remains substantial. Aspergillus spp, Cryptococcus, Candida spp, and Pneumocystis spp are the predominant fungi responsible for over 90% of fatalities associated with fungal infections [3–5]. Notably, Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Aspergillus nidulans, and Aspergillus terreus are recognized as significant opportunistic pathogens in humans. They are implicated in various human diseases, spanning from localized infections and allergic reactions to more severe conditions such as bronchopulmonary infections, bronchitis, and the potentially life-threatening invasive aspergillosis (IA) [6, 7].

Aspergillus flavus is a widespread mold that is a concern in medical, veterinary, and agricultural fields. It is imperative in the progression of immediate cutaneous aspergillosis. Additionally, A. flavus is noteworthy for its capacity to induce disease in both animals and crops, by the production of carcinogenic mycotoxins [7, 8]. Globally, A. flavus is responsible for approximately 10% of bronchopulmonary aspergillosis cases. Additionally, a study conducted across multiple sites on invasive mold infections in Indian intensive care units revealed a notably high prevalence of A. flavus among the patients affected. This underscores the significant role of A. flavus in causing respiratory infections, particularly in clinical settings [9]. The primary treatment for IA typically starts with antifungal drugs, with azoles as a common choice. Voriconazole, in particular, emerges as the preferred medication for patients with aspergillosis in later stages. While the metabolites of these drugs show promise in treating invasive infections, concerns over the adverse effects of synthetic medicines have led to a discontinuation of their use in certain medical contexts [10–12].

Conversely, the emergence of resistance in A. flavus against these medications is a developing concern. Hence, it is of paramount importance to find an alternative metabolite to counteract fungal infections. A wide array and abundance of chemotherapeutic compounds discovered in microorganisms could be exploited to resolve the concern at this critical moment. Metabolites synthesized by fungi are recognized for their ability to generate numerous proteins with intriguing biological effects. Antifungal proteins (AFPs) from fungi gain interest due to their potential application in combating fungal pathogens [13–16].

AFP from Aspergillus giganteus is particularly notable as it selectively targets filamentous fungal growth while leaving mammalian, plant, and bacterial cells unharmed. Small, positively charged proteins from the Antifungal Protein (AFP) family, which are released by various ascomycete fungi, represent potential candidates for inhibiting fungal spore germination [17–19]. The structural makeup of the AFP has been extensively studied. It exhibits a compact β-barrel structure formed by five tightly twisted antiparallel β-strands. Though the structural information and the antifungal potential of AFP have been studied against phytopathogens, its application to treat the invasive infections in humans remains untouched in the field of medicine [20]. In addition to this, the mechanistic action of AFP on pathogenic fungi is yet to be unrevealed. To better understand how the antifungal metabolite works, the present research work aims to delve into action mechanism of AFP against aspergillosis causing A. flavus. Thus the present study was hypothesized with the query whether the AFP has an pathless eventuality to suppress the excrescence of A. flavus. Hence, the antifungal efficacy and the mechanism of AFP targeting pathogenic Aspergillus were investigated and reported.

Materials and Methods

Test Microorganisms and Maintenance

Aspergillus giganteus MTCC 8408 was procured from MTCC—IMTECH Chandigarh. The pathogenic fungus Aspergillus flavus was acquired from PSGIMS in Coimbatore. The obtained fungal strains were subcultured and kept under suitable culture conditions.

Extraction of Culture Filtrates of Aspergillus giganteus

The cell free suspensions of A. giganteus, were cultured at optimized culture condition at 28 °C with continuous agitation at 170 rpm for 72 h. The supernatant was set free from spores, by removing the mat formed by the fungal strain and the supernatant of the culture filtrate was separated with a 0.22 μm membrane filter. The supernatant devoid of cellular material was used for further experiments.

Evaluation of Antagonistic Efficacy of Culture Filtrates of Aspergillus giganteus

The antagonistic potential of the cell-free cell free suspensions of A. giganteus against A. flavus was estimated by co-culturing the agonist and antagonist in the czapek yeast extract medium. The czapek yeast extract medium with the suitable pH, temperature, carbon, and nitrogen source was prepared accordingly. The prepared media were over-layered with 100 μl of filtrates of Aspergillus giganteus and the over-layered plates were inoculated with 0.5 cm mycelia growth discs of a week old Aspergillus flavus and kept at 28 °C for 4 days for incubation. The development of A. flavus in the overlay culture was observed and compared with that of A. flavus treated with antifungal metabolites, and the rate of growth inhibition was measured [21].

Efficacy of Extracted Antifungal Metabolites on A. flavus

The compound’s locations provide valuable insights into their functions within an organism. The sub cellular location of the metabolite released from Aspergillus giganteus was identified by centrifuging the suspension culture at 10000 rpm for 20 min and the supernatant was carefully separated from the pellet. The effectiveness of the antifungal metabolites in both the supernatant and pellet fractions against Aspergillus flavus was evaluated using an overlay assay.

Czapek yeast extract medium with the optimized culture conditions was prepared, then over-layered with 100 μl of the supernatant and pellets of filtrates of antagonist pathogen. The overlayered plates were co cultured with 0.5 cm mycelia growth discs of a week old culture of Aspergillus flavus and incubated at 28 °C for 4 days. The radial expansion of Aspergillus flavus in standard plates and antifungal metabolite treated plates was compared and the growth was measured. The nature of the antimycotic agent in the filtrates of A. giganteus was confirmed by calculating the percentage of growth inhibition on A. flavus in both supernatant and pellet over layered plates. The level of damage driven on Aspergillus flavus by the antifungal metabolite was further confirmed by hyphal interaction assay [22].

Effect of Antifungal Metabolites on the Cell Morphology of A. flavus

Microscopic Analysis of Mycelia Morphology

A. flavus was exposed to the antifungal metabolites of Aspergillus giganteus for 24 h at 28 °C. The impact of these metabolites on the hyphae of A. flavus was then examined microscopically using Lacto phenol cotton blue staining.

Analysis of Cell Membrane Damage Through SEM

The geometrical variation in the membranes of the pathogenic fungi A. flavus after exposure to antifungal metabolites in the filtrates was validated by SEM analysis.

Determination of The Optimal Dosage Level of Antifungal Metabolite of Aspergillus giganteus

To finalize the optimal dosage level of antifungal metabolites to treat A. flavus, MIC, and MFC of AFP were calculated. Inocula were prepared by the dispersion of in sterile 0.85% NaCl solution. In a 96-well plate, Czapek Yeast Extract broth containing antifungal metabolites ranging from 1000 to 62.5 μg/ml was added, along with freshly isolated pathogenic fungal cells, and incubated at 28 °C for 24–48 h to monitor pathogen development in the presence antifungal metabolites. Additionally, a negative control without AFP was included to assess the impact of AFP on the cell viability of the pathogenic strain [12].

Investigation on the Cell Viability of A. flavus Treated with Antifungal Metabolites

UV–Visible Spectroscopic Analysis

UV–Vis spectroscopy is a widely used technique for estimating cell concentrations in a solution and for quantitatively analyzing crucial cellular constituents in the culture broth. Thus the viability of A. flavus in the presence of metabolites was evaluated by utilizing UV visible spectroscopy by slightly altering the methodology discussed in Park et al. 2012 briefly, the inocula of the A. flavus prepared by culturing it in a czapek medium for 24 h with and without the addition of filtrates of A. giganteus. The inoculated culture was treated at 28 °C for 24–48 h. The UV–Vis spectra of the fungal cultures were measured with a UV–Vis spectrophotometer over a wavelength range of 200–900 nm (Shimadzu, Japan). The control spectrum was obtained by measuring the suspending medium used to prepare the initial sample, to neglect any homogeneity in the medium.

Resazurin Reduction Assay

The impact of antifungal metabolites from A. giganteus on the viability of A. flavus cells was assessed using a Resazurin reduction assay. This assay leverages the resazurin reduction capability of live cells to determine fungal viability in the presence of antagonistic metabolites. In a 96-well plate, 20 μl of resazurin dye was incorporated into the czapek yeast extract broth containing both untreated and treated A. flavus cultures, and held at ambient temperature. The reduction of resazurin by live cells was observed at 600 nm. The Cell survival rate was subsequently assessed by the formula provided.

$$\%\text{Viability}=\left(\left({\text{MeanOD}}_{\text{sample}}\right){\text{/MeanOD}}_{\text{blank}}\right)\times 100$$

Druggability Profiling of Antifungal Metabolites from Aspergillus giganteus

The cytotoxicity assay determined the druggability profile of the antifungal metabolites by testing antifungal compounds against erythrocytes. The hemolytic effect of antifungal compounds was assessed using a suspension containing 4% of goat red blood cells (gRBCs). This was done by calculating the percentage of hemolysis in blood cells in presence of 500–250 μg of the antifungal compound. The gRBCs were sterilized with phosphate-buffered saline and 100 μl aliquots were added to microtiter plates, followed by 100 μl of the antifungal compound in phosphate-buffered saline. The mixtures were held at 37 °C for an hour and spinned at 1500 rpm for 10 min. Part of samples were subsequently moved to another microtiter plate for measurement with an ELISA reader. Hemolysis levels were measured with PBS and 0.1% Triton X-100 as standards for 0% and 100% hemolysis, respectively. The hemolytic potential was determined by utilizing the subsequent equation [20, 23].

$$\text{Percentage}\text{Hemolysis =}\frac{\left[\text{Abs}414\text{nm}\text{in}\text{the}\text{compound}\text{solution}-\text{Abs}414\text{nm}\text{in}\text{PBS}\right]}{\left[\text{Abs}414\text{nm}\text{in}0.1\%\text{Trition}\times 100-\text{Abs}414\text{nm}\text{in}\text{PBS}\right]}\times 100$$

Evaluation of the Anti Radical Activity of Metabolites in the Culture Filtrates

DPPH Assay

The antioxidant capacity of an organic molecule is assessed based on its ability to donate hydrogen atoms or electrons to the DPPH radical. This reaction converts the radical into a stable, diamagnetic form. The interaction of antifungal metabolites with stable DPPH free radicals demonstrates their free radical scavenging ability. A color change in the DPPH solution from deep violet to colorless or pale yellow indicates the presence of antioxidant activity. Reactive species neutralization capability of the antifungal metabolites from the filtrates of A. giganteus was assessed using the DPPH assay, as stated by Chang et al. (2001). The color change of the DPPH by the action of the antioxidant solution was observed at 517 nm. In contrast, ascorbic acid served as the reference compound for the experiment [24].

FRAP Assay

The antimicrobial metabolite’s Fe (III) reducing ability (FRAP) was determined by adopting the procedure, as stated in Gohari et al. 2011. To summarize, a mixture of 3.6 ml of FRAP solution and 0.4 ml of dis.H₂O were kept at 37 °C for 5 min. Afterward, the reaction solution was combined with varying levels of antifungal metabolites and treated at 37 °C for another 10 min. Eventually, optical density of the solution mixture was observed at a wavelength of 593 nm [25].

Statistical Analysis

Antimicrobial and antioxidant research was repeated three times, with the findings reported as mean ± standard deviation (SD). To assess data homogeneity, SPSS version 20.1 was used. One-way ANOVA was utilized to measure the major disparities between groups, with a Statistical significance of p ≤ 0.05.

Results and Discussion

Aspergillus giganteus cell-free filtrates were prepared by removing the mat formed on the surface of the broth. The supernatant below the mat was passed through a 0.22 μm membrane filter to remove all fungal spores. The prepared culture filtrates (CFS) were further used to understand the nature and quantify the antifungal compounds released in the suspension culture.

Evaluation of Antagonistic Efficacy of Antifungal Metabolites in the Culture Filtrate

The presence of filtrates of A. giganteus in the overlaid plates has resulted in a 91% growth inhibition in Aspergillus flavus (Fig. 1). The changes in the structure of the vegetative organelles of A. flavus treated with CFS of antagonist were further confirmed through the hyphal interaction assay.

Fig. 1.

Overlay assay of Aspergillus flavus with culture filtrates of Aspergillus giganteus in optimized culture conditions

Similarly, the P.pentosaceus showed a substantial (p < 0.05) antifungal impact on Aspergillus niger and Aspergillus flavus in comparison with control samples [26]. The yeast and LABs antimicrobial on A. westerdijkiae at different pH values has confirmed the effect of P. fermentans LPBYB13 and Lactbrevis LPBB03 on the growth of A. westerdijkiae [27, 28].

Efficacy of Extracted Antifungal Metabolites on A. flavus

The sub cellular location of the antifungal metabolite in Aspergillus giganteus is confirmed by the overlay assay. The inhibition potential of supernatant and pellet on A. flavus growth was reviewed and the findings are shown in Table 1. The supernatant from the CFS showed significantly greater inhibition of pathogenic fungi compared to the pellet. Specifically, the supernatant exhibited an inhibition rate of 84.6%, (p < 0.001) whereas the pellet only exhibited 30.8% (p < 0.001) inhibition. These results validate that the antifungal compound accountable for the antagonistic behavior of Aspergillus giganteus operates extracellular.

Table 1.

Inhibitory potential of extracted antifungal metabolites of Aspergillus giganteus on Aspergillus flavus

S. No	Culture filtrate	Inhibition in cm	%inhibition						Mean	Standard deviation
		C	T1	T2	T3	T1	T2	T3
1	Supernatant	4	0.5	0.8	0.5	87	80	87	84.6	84.6 ± 4.04
2	Pellet	4	3.0	3.8	3.3	25	25	30	30.8	30.8 ± 6.3

Results are expressed in Mean ± Standard deviation (p < 0.001)

Impact of Antifungal Metabolites on the Mycelia Morphology of A. flavus

To analyze the effect of antifungal metabolites from filtrates on A. flavus we have evaluated the mycelia growth of A. flavus on czapek plates amended with a series of different concentrations of antifungal metabolites. The results revealed that the treated A. flavus had elongated hyphae, and extended mycelium compared to that of control fungal strains. Indeed, The hyphal tips appeared swollen, which suggests that the orientation of the hyphal apex was changed, as illustrated in Fig. 2. These findings confirm, that the antifungal metabolite from Aspergillus giganteus has a discernible antifungal effect Aspergillus flavus growth by altering its hyphal morphology.

Fig. 2.

Hyphal morphology of Aspergillus flavus in presence and absence of antifungal metabolites of Aspergillus giganteus

Several studies have already supported the hyphal interaction assay in the field determining of antagonistic efficacy of several metabolites. Bacillo amyoliquefacians BAS23 has shown a prominent effect on various pathogenic fungi. While culturing Trichoderma harzianum and Aspergillus piperis with phytopathogenic fungi, the antagonistic fungi affected the hyphae of the pathogenic fungi, resulting in irregular morphology compared to the control plates without antagonistic fungi [29, 30].

Scanning Electron Microscopic Analysis of Cell Membrane Damage of A. flavus

The morphological changes in the Aspergillus flavus cellular membrane by the action of an antifungal metabolite of Aspergillus giganteus were assessed using a scanning electron microscope. Results of SEM analysis supports the fungicidal effect of antifungal metabolites on A. flavus cellular membrane and conidiophore characteristics. The morphological changes of Aspergillus flavus spores are given in Fig. 3. The change in conidia sizes of treated A. flavus of 3.2–4.6 nm, spore degranulation, and modification in hyphae structures further confirm the fungicidal effect of the metabolites on the pathogens. Untreated Aspergillus flavus had fine spores and elongated hyphae, whereas in the presence of Aspergillus giganteus, the spores shrank and the hyphae completely collapsed. Furthermore, a major difference in cytoplasmic content and membrane integrity was observed. This highlighted the suppressive capability of Aspergillus giganteus against Aspergillus flavus. These findings lay the strong impulse about the target effect produced by the antifungal metabolites in Aspergillus giganteus culture filtrate.

Fig. 3.

Morphological analysis of Aspergillus flavus treated treated with antifungal metabolites of Aspergillus giganteus using SEM

Several experiments have utilized SEM to analyze the structural morphology of fungi. Hdep film was subjected to SEM analysis. SEM analysis confirmed that fungi had formed a coating applied to the surface of the HDPE. Later the fungi were removed, the surface exhibited physical pitting and erosion. Similarly, SEM images revealed the alterations in the exterior topography of HDPE films after being exposed to fungal strains [31–33].

Determination of the Concentration of Antifungal Metabolite of Aspergillus giganteus

Minimum Inhibitory Concentration (MIC)

MIC was considered to be the minimal inhibitory concentration of antagonistic substances to completely reduce the growth of pathogenic microorganisms. The micro broth dilution technique stands as the gold standard method for assessing MIC values of antifungal compounds against Aspergillus flavus, gauging their potency in inhibiting its growth. The effect of antifungal culture filtrate on the pathogenic fungi at different concentrations is shown in Table 2.

Table 2.

MIC of antifungal metabolites of Aspergillus giganteus to treat Aspergillus flavus

S.No	Concentration of antifungal compounds in the culture filtrates (μg)	Growth of the pathogenic organism
1	0 (Control)	+
2	500	−
3	250	−
4	125	+
5	62.5	+
6	31.25	+

“ + ” indicates the growth of the fungal pathogen

“−” indicates the inhibition of pathogenic fungal growth by antagonistic fungal culture filtrates

Control = Inoculation of pathogenic fungi alone in the czapek yeast extract medium

The results indicated that no growth was found in the Aspergillus flavus treated with CFS containing less than 125 μg of antifungal metabolites. Thus 125 μg/ml of antifungal metabolites in the CFS is found to be its MIC to initiate the inhibition of pathogenic Aspergillus flavus.

Minimum Fungicidal Concentration (MFC)

MFC is the least amount of fungicidal concentration required to prevent 98 to 99.9% of fungal pathogens. The fungicidal effect of antifungal metabolites in A. giganteus filtrates was investigated (Fig. 4). Among the different concentrations examined, 250 μg/ml of antifungal metabolite in the filtrates is considered to be the minimum fungicidal concentration required to inhibit the complete growth of Aspergillus flavus.

Fig. 4.

Effect of antifungal metabolites of Aspergillus giganteus on Aspergillus flavus at minimum fungicidal conceration

The findings align with those of other studies. A.campestris essential oil inhibited Fusarium graminearum with MIC and MFC values as 1.25 µL/mL (v/v). The oil demonstrated a strong inhibitory effect against Fusarium moniliforme, Fusarium culmorum, Penicillium expansum, Aspergillus flavus, Aspergillus ochraceus, and Aspergillus parasiticus, with a minimum inhibitory concentration (MIC) of 2.5 μl/ml (v/v). It also showed significant fungicidal activity against these fungi, although it was less effective against Penicillium citrinum, Penicillium viridicatum, and Aspergillus niger, with a minimum fungicidal concentration (MFC) greater than 20 µL/ml.[34].

The minimum amount of CMEO required to demonstrate antifungal activity against Fusarium graminearum was assessed using the microtiter dilution method in a 96-well microtiter plate. The MIC and MFC of CMEO against F. graminearum were determined to be 421.7 ± 27.14 ppm and 618.3 ± 79.35 ppm, respectively [35].

Impact of Antifungal Metabolites of A. giganteus on the Cell Viability of A. flavus

The UV–visible spectroscopic analysis evaluated the impact of the antifungal metabolites on the cells of A. flavus. The UV spectra of treated and untreated A. flavus cultures are shown in Fig. 5. A clear shift in the spectra of the A. flavus CFS treated with antifungal metabolites from 300 to 320 nm was observed. Thus the shifts of peaks in the spectra of treated A. flavus pathogens in comparison with untreated A. flavus spectra sheds strong evidence on the interaction of antifungal metabolites to the cell membrane of A. flavus. It is also considered that the antagonist effect of antifungal metabolites has resulted in the leakage of cellular metabolites. This interaction of antifungal metabolites to the cell membrane in turn affects the cell viability of A. flavus. Additionally, cell viability was assessed using the Resazurin reduction assay. The 0.1% and 7% of cell viability of A. flavus in the presence metabolites at its MIC level was observed respectively (Fig. 6). These results further reiterate the effect of antifungal metabolites from Aspergillus giganteus.

Fig. 5.

UV–visible absorption spectra of untreated and treated A. flavus in the culture medium

Fig. 6.

Effect of antifungal metabolites on the cell viability of A. flavus by resazurin reduction assay

Evaluation of Antioxidant Activity of Antifungal Metabolites in the CFS of A. giganteus

The antioxidant potential of antifungal metabolites in the CFS of A. giganteus was assessed. The scavenging efficiency rate was evaluated using the following formula.

$$\%\text{Scavenging}\text{Activity =}\left(\left({\text{A}}_{\text{b}}-{\text{A}}_{\text{s}}\right){\text{/A}}_b\right)\times 100$$

A_b is the blank absorbance and A_s is the sample absorbance. Antifungal metabolites showed significant antioxidant activity with a 17 to 77% (p < 0.001) reduction rate in the DPPH assay. The ability of antifungal metabolites to reduce Fe3 + to Fe2 + was assessed by FRAP assay. The antifungal metabolites exhibited a significant radical scavenging activity, with values between 29 and 72% (p < 0.001) in the FRAP assay. The standard drug, ascorbic acid, demonstrated significant inhibition of the radical scavenging activity of about 97% (p < 0.001) and 86% (p < 0.001) for DPPH and Fe3 + respectively. The results shown in Figs. 7 and 8 confirm that the antifungal metabolites possess a significant antioxidant potential. Thus the results confirm that the antifungal metabolites hold a strong antioxidant potential.

Fig. 7.

DPPH scavenging activity of antifungal metabolites of Aspergillus giganteus. Data are represented as mean ± SD (n = 3) p < 0.001

Fig. 8.

FRAP scavenging activity of antifungal metabolites of Aspergillus giganteus. Data are represented as mean ± SD (n = 3) p < 0.001

Druggability Profiling of Antifungal Metabolites from Aspergillus giganteus

The Druggability of antimycotic metabolites in CFS of A. giganteus was investigated by hemolytic assay with goat red blood cells. The percentage of hemolysis of goat red blood cell suspensions exposed to five different concentrations of antifungal compounds present in the filtrates (50–250 μg) was measured. The percentage of hemolysis observed is depicted in Table 3.

Table 3.

Hemolytic percentage of antifungal metabolites of Aspergillus giganteus on goat red blood cells

S. No	Concentration of antifungal compounds (μg)	Percentage of hemolysis			Mean	Standard deviation
		T1	T2	T3
1	50	8.1	9.1	8.4	8.5	8.5 ± 0.41
2	100	15.2	15.2	15.5	15.3	15.3 ± 0.14
3	150	17.6	16.6	17.0	17.1	17.1 ± 0.41
4	200	22.3	21.5	23.0	22.6	22.6 ± 0.61
5	250	25.0	25.4	25.0	25.1	25.1 ± 0.18

Results are expressed in Mean ± Standard deviation (p < 0.001)

The results of the hemolytic assay of antifungal metabolite-treated goat red blood cells revealed that the antifungal metabolite is not harmful with no significant cytotoxicity within the range of 50–250 μg (p < 0.001) of antifungal compounds to the gRBCs. This demonstrated the effectiveness and safety of using antifungal compounds derived from Aspergillus giganteus to treat fungal infections, showing minimal side effects. The results correlate with the other results, namely antagonistic fungal proteins and peptides of Penicillium expansum where none of the proteins showed hemolytic activity in the concentration of 1–100 μM. Different extracts of Periophthalmodon schlosseri were innocuous with human and chicken red blood cells with no cytotoxic effect [36–38].

Conclusion

Fungal infections appear to be on the rise all over the world. With a focus on fungi virulence mechanisms and the development of effective strategies for overcoming and preventing fungal infection, the need for new antifungal drugs has spiked in recent times. The present findings revealed the nature and antagonistic potential of the antimycotic compounds from Aspergillus giganteus. The inhibitory effects of antifungal metabolites on pathogenic Aspergillus flavus confirm the nature of the metabolites. The morphological changes in the damaged cellular components of the culture filtrate-treated A. flavus adds strong evidence to the antagonistic effect of antifungal metabolites on pathogenic fungi. Furthermore, results of the hemolytic assay of antifungal metabolites ensure its safety for human cells. Conclusively, our results state that the Aspergillus flavus seemed to be completely shattered on antifungal metabolite exposure, showing fully damaged and irregular clumping of the cells. Our findings are in good agreement with those from other groups. The mechanistic studies to reveal the molecular changes undergone by the pathogenic microbes on the exposure of antifungal metabolites from Aspergillus giganteus could be done in future studies.

Author Contributions

Karthiga Sivarajan: Conceptualization, Writing—original draft, Ramya Ravindhiram: Methodology, Jothi Nayaki Sekar—reviewing and Editing, Kavitha Dhandapani—Conceptualization, Supervision, Validation, Project administration.

Funding

No funds, grants, or other support were received for the paperwork.

Declarations

Conflict of interest

The authors declare that they have no competing interests.

Ethical Approval and Consent to participate

Not applicable.

Consent for Publication

Not applicable.