Biodiversity of endosymbiont fungi associated with a marine sponge Lamellodysidea herbacea and their potential as antioxidant producers

Abstract

This study aims to isolate endosymbiontic fungi from the marine sponge Lamellodysidea herbacea and to explore their antioxidant potential. Marine-derived fungi, with their vast biodiversity, are considered a promising source of novel antioxidants which can replace synthetic ones. Marine sponges have previously reported bioactive properties that could ameliorate oxidative stress, particularly their associated fungi, producing high-frequency bioactive molecules (adaptogenic molecules) in response to stressors. 19 endosymbiont fungi associated with marine sponges were isolated, and their extracts were evaluated for their antioxidant capacities. Extract of an endosymbiont fungus, isolate SPG6, identified as Alternaria destruens, through surface electron microscopy (SEM) and ITS gene sequencing, showed broad range antioxidant activities (EC₅₀ values) (free radical scavenging 32.54 mg L⁻¹, Hydroxyl radical scavenging activity < 0.078 g L⁻¹, total reducing power 0.114 g L⁻¹, Chelating power 0.262 g L⁻¹, H₂O₂ scavenging activity < 0.078 g L⁻¹, and Superoxide radical scavenging activity > 5.0 g L⁻¹). The extract of isolate SPG6 was fractioned and analyzed through GC–MS. Marine sponge-associated endosymbiont fungi are a rich source of antioxidant molecules.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-024-03972-1.

Introduction

Extensive and intense exposure to pro-oxidant factors, viz. ionizing radiations, hypoxia, processed food, a range of chemicals, etc., induce oxidative stress through excessive production of reactive oxygen species (ROS) beyond the antioxidant defense capacity (Kamatou & Viljoen 2010; Sun et al. 2004). ROS are also an influential player in the pathophysiology of aging, inflammation, cardiovascular problems, neurodegenerative disease, diabetes, cancer, etc. (Sharifi-Rad et al. 2020; Kamatou & Viljoen 2010; Sun et al. 2004). Moreover, synthetic antioxidants viz. Amifostine, 5-aminosalicylic acid, etc., are expensive and have adverse effects (Kuruba & Gollapalli 2018). However, due to the high rediscovery rate of natural antioxidants from the sources used by the pharmaceutical sector, the scientific community has recently amplified its interest in the blue economy and has begun to look toward lesser-known areas like the deep sea (greater than 25 m), as novel sources of drug compounds (Sajeevan 2020; Proksch et al. 2010).

The exploration of endophytic fungi within marine sponges and their endophytic potential represent a burgeoning field of research with significant knowledge gaps. While the marine environment offers a vast and largely unexplored reservoir of biological diversity, the understanding of endophytic fungi inhabiting marine sponges is still in its infancy. Identification, isolation, and characterization of these endophytes present unique challenges due to the complexity of the marine ecosystem. The research gap lies in the limited number of studies specifically focusing on unraveling the diversity, ecological roles, and biotechnological applications of endophytic fungi associated with marine sponges. Furthermore, there is a lack of comprehensive investigations into the mechanisms underlying the symbiotic relationships between these fungi and their host sponges and their potential to produce bioactive compounds with pharmaceutical, agricultural, or industrial significance. Bridging these gaps is essential for unlocking the full potential of marine sponge-associated endophytic fungi and harnessing their applications in various fields.

In the marine ecosystem, due to fluctuating physicochemical conditions, anthropogenic activities and climate change, various stressors, viz., sedimentation, eutrophication, food shortage, and the oxygen minimum zone are expanding. They lead to escape/massive mortality of motile/sessile (coral and sponges) species (Cerrano et al. 2000; Wulff 2006; Bell et al. 2013; Schuster et al. 2021). Elevated sedimentation also causes closure of the oscula, a reduction in pumping, and a respiratory rate that could lead to hypoxia (Strehlow 2017). During the adaptation process, the tropical marine environment also reported a significant rise in the abundance of L. herbacea with negligible toxic effects. This is due to its ability to alter respiratory rate to fulfill metabolic energy demand for mucus production and to prevent clogging in the aquiferous system (Schuster et al. 2021; Aly et al. 2008; Proksch et al. 2008). Such an adaptive trait of L. herbacea could indicate a hidden adaptogenic source in it.

Along with this, marine microorganisms have evolved with distinct metabolic and genetic adaptations, thus contributing significantly to the stability of holobiont (Devi et al. 2011; Proksch et al. 2003; Thirunavukkarasu et al. 2013). They have produced an incredible diversity of secondary metabolites to cope with the variety of ecological niches that characterize the ocean environment (Bugni and Ireland 2004; Chen et al. 2016; Tan and Zou 2001; Blunt et al. 2016; Perdicaris et al. 2013; Vitale et al. 2020; Sajeevan 2020). Also, the mainstay of drug discovery has been microbial natural products because of their bioactive properties, viz., free radical scavenging capacity, neurogenic activity, anticancer activity, kinase inhibition, antimicrobial activity, etc. (Hasan et al. 2015; Mohan et al. 2016). Up to 40% of sponges' biomass is composed of microbial populations. The tropical marine sponge L. herbacea is associated with microbes, such as Oscillatoria spongeliae, Lyngbya majuscule, Vibrio sp., or Proteobacteria, which produce a variety of beneficial chemotypes, including chlorinated diketopiperazines, dihydrodysamide C, didechlorodihydrodysamide C, and brominated diphenyl ethers (Elyakov et al. 1991; Flowers et al. 1998; Scheuermayer et al. 2006; Unson & Faulkner 1993).

L. herbacea has not yet been targeted for endosymbiont fungi and their curative potential. Therefore, the present study investigates the biodiversity of the endosymbiont fungus of marine sponge, L. herbacea, and its potential as an antioxidant producer. Among the isolated endosymbionts, SPG 6 (A. destruens) was studied in detail.

Materials and methods

Collection and isolation of endosymbiont fungi

The collection and isolation of endosymbiont fungi from sponge (Lamellodysidea herbacea) tissue was previously done by Katoch et al. (2014, 2023) and Strobel and Daisy (2003).

Identification of endosymbiontic fungi

All isolated endosymbiontic fungi identified through morphological and microscopic techniques but only a promising strain with antioxidant activity was identified using ITS sequence.

The morphological traits, such as colony size, texture, and color, as well as microscopic traits, such as hyphae, conidiophores, and conidia, were used to identify the fungi. For identification, isolates of endosymbiont fungi were cultivated for 8–10 days at 26 °C with a 12-h photoperiod on Malt extract and Synthetischer Nahrstoffarmer Agar (SNA: KH₂PO₄ 1.0 g, KNO₃ 1.0 g, MgSO₄ 0.5 g, KCI 0.5 g, glucose 0.2 g, sucrose 0.2 g and agar 15 g for 1L).

The molecular identification of promising strain with higher antioxidant activity was accomplished through ITS-based rDNA sequencing. The fungal biomass was used for genomic DNA isolation (Raeder & Broda 1985). ITS1 (5′-TCCGTAGGTGAACCTTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′), universal primers described by White et al. (1990), were used to amplify the ITS1-5.8S-ITS2 region (500–600 bp). 1–10 ng of DNA, 1 × PCR buffer (15 mM MgCl₂), 200 mM of each dNTP, both primers (10 pmol, IDT, Belgium), and Taq DNA polymerase (1U) were added to a 20 µL PCR reaction (Promega, US). The PCR was performed under the following conditions: initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 1 min, elongation at 72 °C for 1 min, and extension at 72 °C for 10 min. The cycling conditions were 5 min at 94 °C. The sequencing reaction (10 µL) contained the following ingredients: purified PCR product, forward/reserve primer (3.2 pmol), and Big Dye Terminator sequencing mix (8 µL). The elution of the PCR product was done using a Gel Extraction Kit (Qiagen, USA). On an automated sequencing system, samples were sequenced (Applied Biosystems). The resultant sequence was submitted to the Gene bank (OR915497). For a conclusive identification, it was compared with the nucleotide database of the US National Centre for Biotechnology Information (NCBI) using blastn tool (Altschul et al. 1997). Alignment Explorer's CLUSTALW option was used to analyze the ITS region of isolate SPG6 from this study and sequences from closely related fungi using the MUSCLE (UPGMA algorithm), maximum composite likelihood, and neighbor-joining methods, respectively (Tamura et al. 2007). The bootstrap technique (1000 repetitions) was used to infer the phylogenetic relationship.

Scanning electron microscopy (SEM)

The isolates of endosymbiont fungi were prepared for SEM using conventional methods (Ho et al. 1999). The sample was initially fixed with glutaraldehyde (2.5% in phosphate buffer, pH 7.3) at 4 °C for 4 h, followed by three washes. Then, in the same buffer at 4 °C for 1 h, they were fixed with 1% (w/v) osmium tetroxide (OsO₄). The specimens were dehydrated using increasing acetone concentrations (50, 70, 80, 90, 95, and 100%) for 15 to 20 min for each solution. They were then transferred to a solution of iso-amyl acetate and acetone (v/v 1:1) for 30 min and later in pure iso-amyl acetate for 1 h. After that, the specimens were dried and dehydrated using a critical point dryer (Balzer’s union), coated with gold–palladium and liquid CO₂. The samples were then examined with an ASID at a 40 kV electron microscope (JOEL JSM 6400, Japan).

Fermentation and extraction

Two actively developing fungal blocks (1 cm) were inoculated at 27 ± 2 °C and 180 rpm to cultivate endosymbionts in an Erlenmeyer flask (1000 mL). The culture was extracted using HPLC grade ethyl acetate (four times) after 10 days. The vacuum was used to dry the extracts. For the determination of the antioxidant capacity, stock solutions (10 mg mL⁻¹) of the extracts were made as per assay (Katoch et al. 2023).

Antioxidant activities

Free radical scavenging activity

The fungal extract’s scavenging ability was determined using α, α-diphenyl-β-picrylhydrazyl (DPPH) assay with some modification (Hulikere et al. 2016; Prieto 2012). The stock solution of DPPH was prepared with 0.2 mM DPPH in methanol. In the test and control wells, 100 µL DPPH (0.2 mM) was added, incubated in the dark at room temperature (RT), and absorbance was taken at 517 nm after 30 min. As a positive and negative control, ascorbic acid and dimethyl sulfoxide were used. All the tests were performed thrice. A percentage for scavenging free radicals was calculated using the formula given below. The EC₅₀ value was interpolated by plotting a non-linear regression curve.

$$\text{Free radical scavenging (\%)}=\frac{\left(\text{Control absorbance}-\text{Extract absorbance}\right)}{\text{Control absorbance}}\times 100$$

Hydroxyl (OH) radical scavenging  activity

OH, inhibition ability of extracts was analyzed using Li et al. (2011) method with little modifications. The reaction mixture composed of 80 µL of extract solution (of different concentrations viz. 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, and 5 g L⁻¹ in water), 80 µL phosphate buffer solution (0.1 M, pH 7.4), 40 µL of 1, 10-phenanthroline (0.75 mM), 40 µL of ferrous sulfate (7.5 mM), and 40 µL of H₂O₂ (0.3%) was added and diluted to 300 µL with distilled water. For blank and positive control, phosphate buffer and ascorbic acid were used. After incubation for 30 min at 37 °C, the absorbance was measured at 510 nm. The following formula was used to calculate the ability to inhibit the OH radical.

$$\text{OH scavenging activity (\% )}=[1-(P1-P2)/P0]\times 100$$

P0 = control absorbance (water), P1 = sample absorbance, and P2 = absorbance of the sample with distilled water instead of FeSO₄ solution under identical conditions.

Superoxide anion radical scavenging activity

The extract’s activity was done according to PMS (Phenazine methosulfate)-NADH (Nicotinamide adenine dinucleotide)-NBT (Nitroblue tetrazolium chloride) method with few modifications (Keshari et al. 2018). Initially, the reaction mixture is made up of varying concentrations (0.03125, 0.0625, 0.125,  0.25, 0.5, 1, and 2 g L⁻¹ of extracts (10 µL), 20 µL of NBT (1 mM), 20 µL of PMS (0.1 mM), and 40 µL of Phosphate buffer solution (pH 7.4, 0.1 M) and an equal volume of distilled water. By adding 20 µL NADH (2 mM) to the above solution, the reaction was initiated. At room temperature, the reaction continued for 10 min and absorbance was measured at 570 nm. Ascorbic acid was employed as the positive control, and water acted as the blank. The following formula calculated the superoxide radical scavenging activity:

$$\text{Superoxide anion radicle scavenging activity}(\%)=\frac{Ac-Ab}{\mathit{Ac}}\times 100$$

where Ac = Control absorbance, Ab = Test sample absorbance.

Chelating power on ferrous (Fe²⁺) ions

Endosymbiont extract’s ability to chelate ferrous ions was evaluated using Guleria et al. (2011) method with some modifications. Briefly, 200 µl of extracts with different concentrations was mixed with 740 µl of methanol and 20 µl of 2 mM FeCl₂. To initiate the reaction, 40 µl of ferrozine was added and was kept at RT for 10 min. At 562 nm, absorbance was measured. EDTA (Ethylenediaminetetraacetic acid) was taken as the positive control. The ferrozine–Fe^2 + complex formation ratio was determined using

$$\%\text{inhibition}=\frac{Tc-Ts}{\mathit{Tc}}\times 100$$

Tc = Absorbance of control, Ts = Absorbance of the test sample.

Total reduction ability

The reducing power of extracts was evaluated in a 96-well plate by Guleria et al. (2011) with few modifications. 40 µL of extracts with different concentrations ranging from 0.0312, 0.0625, 0.125, 0.25, 0.5, and 2 g L⁻¹ was mixed with 0.2 M 50 µL phosphate buffer solution (6.6 pH), and 50 µL potassium ferricyanide (K₃Fe[CN]₆) and kept at 50 °C for 30 min. Then, 50 µL 10% trichloroacetic acid was added and incubated at room temperature (10 min) and followed by addition of 10 µL of ferric chloride (0.1%). Finally, 50 µL distilled water was added to each well and incubated at room temperature, and absorbance was measured at 700 nm. A sample without extract was taken as the negative control. Ascorbic acid was used as a positive control. A percentage of total reduction ability was calculated as per the equation given below. The EC₅₀ was calculated from the graph.

$$\text{Reducing ability}=A1-A0$$

where A1 is the absorbance of extract, A0 is the absorbance of deionized water instead of ferric chloride.

Hydrogen peroxide radical scavenging potential

This method was based on Keshari et al. (2018) with a few modifications. Homogenized 100 µl of fungal extracts (with different doses of 0.0312, 0.0625, 0.125, 2.5, 0.5, and 2 g L⁻¹), 300 µl phosphate buffer (50 mM, pH,7.4) and 600 µl H₂O₂ (2 mM in 50 mM phosphate buffer) were mixed and incubated for 10 min. The absorbance was measured at 230 nm using a UV–Vis spectrophotometer. The H₂O₂ scavenging ability was calculated using the formula.

$$\text{scavenging}\left(\%\right)=\frac{\left(\text{Control absorbance}-\text{Extract absorbance}\right)}{\text{Control absorbance}}\times 100$$

The correlation between antioxidant activities is presented by Pearson correlation coefficient. Results were considered statistically significant when P-values were below 0.05.

Cytotoxic effect of endosymbiont fungi

We accessed the cytotoxicity of fungal extracts using an MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay (Pathania et al. 2013) based on colorimetry. We bought cell lines from National Centre for Cell Sciences (NCCS), Pune, India, including HCT-116 (colorectal carcinoma) and HT-1080 (fibrosarcoma), and cultured them in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal calf serum (FCS) and penicillin (100U) by incubating in a CO₂ incubator (Thermo Electron Corporation, USA), at 37 °C with 98% humidity. 200 µl of cell suspension (10⁵ cells/mL) was added to different dilutions of fungal extracts (10–100 µg/mL), and positive (5-FU, paclitaxel, adriamycin) and negative controls (Dimethyl sulfoxide) and then incubated for 48 h. After incubation, we discarded the plate’s contents and added fresh medium with MTT (100 µg/mL) for 3 h. Later, plates were washed, and DMSO was added. We recorded OD using an ELISA reader (Thermo Labs, USA) at 540 nm with the reference wavelength of 620 nm and calculated percent growth inhibition. CurveFit software was used to analyze the IC₅₀ value.

GC/MS analysis

Analysis of volatile compounds in extract of SPG6 was accomplished with Varian mass spectrometer 4000 and a GC 3800 equipped with a CP-Sil-8 capillary column (30 m × 0.32 mm × 0.25 µm film thickness). The analytical condition was as follows: column temperature was programmed: 60 °C (5 min), from 60 °C to 250 °C with an increase of 3 °C/min, and 7 min hold. Temperatures for the injector and detector were set at 280 °C. 1 mL/min of ultra-pure helium gas was employed as the carrier gas. An electron ionization device with a 70-eV energy level was employed for GC/MS. Sample (2 µL of stock solution: 10 g L⁻¹prepared in ethyl acetate) was injected automatically in split mode. Mass spectra from 50 to 300 amu were taken at one scan per second.

The NIST library (National Institute of Standards and Technology) database for the initial identification of VOCs. Thus, all VOCs presented in this research follow the NIST database's chemical nomenclature. This report listed and explained only compounds with a quality match score higher than 70%. Peak regions for all other undetermined components were combined and added for the extract. The relative peak area derived from GC/MS analysis was used as the first approximation in the quantitative analysis of each component discovered in fungal culture. GC/MS analyses were conducted three times, and mean values were provided.

Statistical analysis

Graph Pad Prism 7 software was used to analyze antioxidant activity data using One-way ANOVA and Dunnett’s multiple comparisons tests and to establish co-relation between the antioxidant activities.

Results

Isolation of endosymbiont

Nineteen endosymbiont fungi were isolated from the collected marine sponge, L. herbacea (Katoch et al. 2023). Eleven, six, and two fungi were isolated on Malt, Martin, and Malt Yeast Peptone Glucose (MYPG) medium, respectively. The highest diversity was recorded on Malt medium, which includes genera Aspergillus (8), Talaromyces sp. (1) Alternaria (1), Bipolaris sp. (1). On Martin medium, two genera viz. Aspergillus sp. (4), Alternaria spp. (2) were isolated, whereas on MYPG, only Aspergillus sp. (2) was isolated. It was observed, Genera Aspergillus was dominant. It includes three species viz. fumigatus, flavus, one was unidentified.

Identification of isolate SPG6

The aerial mass color of isolate SPG6 was whitish brown, while the back side looked brownish black (Supplementary file S1).

Microscopic identification of isolate SPG6

The conidia were in muriform shape and dark brown in color. The length of their conidia varied. The conidia had 2–6 cells (Fig. 1).

Fig. 1.

A Conidia over PDA B Conidiophores and muriform Conidia of the isolate SPG6 under SEM

Scanning electron microscopy (SEM)

Conidia and conidiophore morphological traits were observed in more detail using SEM. According to SEM studies, the fungus produced numerous smooth muriform conidia singly on short conidiophores (Fig. 1B). Conidiophores were singular and either straight or slightly bent. Hyphae had smooth, thin-walled, septate, and hyaline surfaces. They showed morphological characteristics with Alternaria spp.

Molecular identification and phylogenetic analysis

The ITS gene sequence data showed the highest sequence similarity (100%) with several Alternaria destruens strains, where the maximum value was shown for Alternaria destruens strain ATCC 46561, followed by Alternaria destruens strain ATCC 6663 and A. destruens strain ATCC MYA-4642 (Table 1).

Table 1.

Comparison of the ITS-5.8S-ITS2 region sequence of isolate SPG6 among strains of Alternaria spp

Species	Strain	Similarity (%)	Total score	GenBank accession number
Alternaria destruens	ATCC 204363	99.81	979	NR_137143.1
Alternaria eichhorniae	ATCC 22255	99.25	963	NR_111832.1
Alternaria terricola	CBS 202.67	95.66	843	MH858945.1
Alternaria multirostrata	CBS 712.68	93.72	822	MH859206.1
Alternaria chartarum	ATCC 18044	95.36	811	NR_130656.1

The phylogenetic location of isolate SPG6 is manifested in Fig. 2. The tree is divided into 3 clusters predominantly. Isolate SPG6 is taxonomically positioned in a cluster with a strain of A. destruens, and A. eichhorniae.

Fig. 2.

Phylogenetic tree of the isolate SPG6 constructed based on the nucleotide sequence of ITS by neighbor-joining (NJ) method using molecular evolutionary genetic analysis (MEGA) software version 4.0

Free radical scavenging activity

The free radical scavenging activity of extracts is represented in Table 2. The highest activity was observed in isolate SPG6 and SPG19 (EC₅₀ of 32.54 ± 0.14 mg L⁻¹: and 33.53 mg L⁻¹). Isolate SPG5(02) extract (EC₅₀ of 99.377 ± 1.35 mg L⁻¹) showed moderate free radical scavenging effects. The comparison of the extract’s DPPH radical scavenging effects indicates that the crude extract of a few Aspergillus and Alternaria isolates was better. Hence, they could be the reservoir of natural antioxidant compounds. None of the extracts showed free radical scavenging activity as strong (EC₅₀ 2.32 ± 0.05) as ascorbic acid (positive control).

Table 2.

Free radical-scavenging activity (DPPH) (EC₅₀ mg L⁻¹) of the extracts prepared from endosymbiont fungi isolated from Lamellodysidea herbacea

Isolates	Free radical-scavenging activity
SPG 3(a)	132.33 ± 0.25a
SPG 05/01	121.42 ± 0.23a
SPG 5(2)	99.377 ± 0.54a
SPG 6	32.54 ± 0.25a
SPG 7(2)	> 250 ± 0.87a
SPG11	173.09 ± 0.45a
SPG 12	221.42 ± 0.45a
SPG 13	192.4 ± 0.36a
SPG 14	> 250 ± 0.54a
SPG 15	105.58 ± 0.56a
SPG 16	> 250 ± 0.55a
SPG 17	232.69 ± 0.48a
SPG 18	118.75 ± 0.57a
SPG 19	33.53 ± 0.64a
SPG21	135.76 ± 0.56a
SDHY01/01	> 250 ± 0.45a
SDHY 01/02	101.77 ± 0.33a
SDHY 04/1	> 250 ± 0.45a
SDHY 04/02	> 250 ± 0.12a
Ascorbic acid	2.32 ± 0.05

^ap < 0.001

Hydroxyl radical scavenging potential

The scavenging potential of endosymbiont extracts is presented in Table 3. The highest activity (EC₅₀ of < 0.078 ± 0.14 g L⁻¹) was observed in nine endosymbiont extracts. Isolate SPG 12 extract EC₅₀ of 0.095 ± 1.35 g L⁻¹) showed a moderate hydroxyl radical scavenging effect. Eleven extracts (57%) showed stronger hydroxyl radical scavenging activity than ascorbic acid (EC₅₀ 0.145 ± 0.003, positive control).

Table 3.

Antioxidant activities (EC₅₀ g L⁻¹) of the extracts prepared from endophytic fungi isolated from Lamellodysidea herbacea

Isolates	Hydroxyl radical scavenging activity	Total reducing power	Chelating power	H2O2 scavenging activity	Superoxide radical scavenging activity
SPG 3(a)	< 0.078 ± 0.45a	1.839 ± 0.96b	> 5.0 ± 0.65a	1.987 ± 0.98b	> 5.0 ± 0.62d
SPG 05/01	1.803 ± 0.12a	> 2.0 ± 045b	> 5.0 ± 0.25a	< 0.078 ± 0.12d	> 5.0 ± 0.45d
SPG 5(2)	0.83 ± 0.32a	> 2.0 ± 0.65b	> 5.0 ± 0.45a	0.410 ± 0.25d	> 5.0 ± 0.96d
SPG 6	< 0.078 ± 0.85a	0.114 ± 0.12d	0.262 ± 0.2d	< 0.078 ± 0.45d	> 5.0 ± 0.25d
SPG 7(2)	> 5.0 ± 0.98a	1.509 ± 0.60c	> 5.0 ± 0.45a	2.562 ± 0.35a	> 5.0 ± 0.15d
SPG11	< 0.078 ± 0.12a	> 2.0 ± 0.25b	1.186 ± 0.4d	1.621 ± 0.15c	3.623 ± 0.45d
SPG 12	0.095 ± 0.64a	1.305 ± 0.85d	> 5.0 ± 0.15a	1.403 ± 0.85d	4.548 ± 0.95d
SPG 13	< 0.078 ± 0.14a	0.489 ± 0.60d	> 5.0 ± 0.25a	0.355 ± 0.65d	> 5.0 ± 0.45d
SPG 14	0.197 ± 0.62a	> 2.0 ± 0.45b	1.736 ± 0.45a	0.050 ± 0.75d	5.443 ± 0.75d
SPG 15	< 0.078 ± 0.85a	1.739 ± 0.15c	> 5.0 ± 0.12a	0.625 ± 0.12d	> 5.0 ± 0.52d
SPG 16	< 0.078 ± 0.12a	0.498 ± 0.25d	> 5.0 ± 0.14a	< 0.078 ± 0.65d	7.143 ± 0.96b
SPG 17	< 0.078 ± 0.62a	0.976 ± 0.25d	> 5.0 ± 0.12a	< 0.078 ± 0.15d	6.673 ± 0.48c
SPG 18	< 0.078 ± 0.12a	0.607 ± 0.25d	< 0.078 ± 0.25d	< 0.078 ± 0.85d	1.809 ± 0.74a
SPG 19	< 0.078 ± 0.25a	> 2.0 ± 0.65b	0.253 ± 0.98d	< 0.078 ± 0.12d	3.196 ± 0.89c
SPG21	0.308 ± 0.62a	> 2.0 ± 0.85b	> 5.0 ± 0.52a	1.069 ± 0.36d	4.995 ± 0.65d
SDHY01/01	0.283 ± 0.85a	> 2.0 ± 0.25b	> 5.0 ± 0.85a	0.078 ± 0.95d	> 5.0 ± 0.32d
SDHY 01/02	0.112 ± 0.45a	0.248 ± 0.26d	2.349 ± 0.64a	> 5 ± 0.45a	> 5.0 ± 0.35d
SDHY 04/1	> 5.0 ± 60a	> 2.0 ± 0.65b	> 5.0 ± 0.24a	> 5 ± 0.75a	6.429 ± 0.65d
SDHY 04/02	0.389 ± 0.56a	> 2.0 ± 0.85b	> 5.0 ± 0.96a	> 5 ± 0.12a	> 5.0 ± 0.32d
Ascorbic acid/EDTA	145 ± 0.003	0.114 ± 0.34	0.219 ± 0.32	< 0.078 ± 0.86	> 5.0 ± 0.84

(a = p < 0.001, b = p < 0.01, c = p < 0.05, d = No significance)

Chelating power on ferrous (Fe²⁺⁾ ions

It is evident from Table 3 that isolate SPG18 was active (EC₅₀ < 0.078 ± 0.35 g L⁻¹) in the chelating assay. It showed better activity than the positive control, EDTA (EC₅₀ 0.219 ± 0.53 g L⁻¹). Isolate SPG 6 and isolate SPG19 extracts (EC₅₀ 0.262 ± 0.36 g L⁻¹, 0.253 ± 1.25 g L⁻¹) also showed a moderate chelating effect.

Superoxide anion radical scavenging activity

The superoxide anion radical scavenging power of endosymbiont extracts is shown in Table 3. Only a single extract, i.e., isolate SPG6, showed the highest activity (EC₅₀ 1.809 ± 0.24 g L⁻¹). It offered better activity than the positive control, Ascorbic acid (EC₅₀ < 5.0 ± 0.24 g L⁻¹).

Total reduction ability

The ferricyanide reducing technique was used to calculate the overall reduction potential of extracts. The reduction of Fe³⁺ complexes to Fe²⁺ forms occurs when antioxidants are present in samples. Only a single extract, i.e., of isolate SPG6, was as active as ascorbic acid, a well-known antioxidant, with EC₅₀ 0.114 ± 0.64 g L⁻¹. SDHY 01/02 extract (EC₅₀ of 0.248 ± 1.35 g L⁻¹) showed a moderate level of reducing effect (Table 3).

Hydrogen peroxide radical scavenging activity

It is evident from Table 3 that the highest H₂O₂ scavenging activity (EC₅₀ of < 0.078 ± 0.14 g L⁻¹) was observed in seven endosymbiont extracts and ascorbic acid (positive control). Hence, seven extracts were as potent as a positive control in terms of hydrogen peroxide radical scavenging activity. Isolate SPG 14 extract (EC₅₀ of 0.095 ± 1.35 g L⁻¹) showed a moderate H₂O₂ radical scavenging effect Table 4.

Table 4.

Volatile organic constituents in diethyl ether fraction of isolate SPG6 extract as determined by GC/MS

S. No.	RT	Compound name	Area percentage	Match percentage
1	29.908	Dimethyl palmitamine	23.54	95.1
2	30.887	n-Hexadecanoic acid	3.758	82.4
3	31.202	Dimethylethylcetylammonium bromide	49.95	95.3
4	33.410	Octadecanoic acid, methyl ester	1.77	77.2
5	33.665	9,12-octadecadienoic acid (Z,Z)-	11.65	84.6

Correlation between the different antioxidant/ scavenging activities is highlighted in Table 5.

Table 5.

Pearson’s correlation coefficient analysis of antioxidant activities of endophytes associated with sponge

Correlation coefficient/p value	Free radical activity	Hydroxyl radical activity	Total reducing	Chelating	H2O2	Superoxide
Free radical activity		0.130	0.172	0.004*	0.216	0.052*
Hydroxyl radical activity	0.349		0.240	0.173	0.046*	0.314
Total reducing	0.317	0.275		0.105	0.552	0.820
Chelating	0.610	0.316	0.373		0.341	0.012*
H2O2	0.288	0.449	0.141	0.224		0.534
Superoxide	0.439	0.236	− 0.054	0.547	0.147

Bold text with * depicts highly significant p values; Rest Bold text depicts high correlation coefficient

Cytotoxic effect of isolate SPG6 extract

We accessed the cytotoxic effect of SPG6 extract on HCT-116 (colorectal carcinoma) and HT-1080 (fibrosarcoma) cell lines using an MTT assay (Fig. 3). At 100 µg/ml concentration, isolate SPG-6 extract has shown 10% and 8% growth inhibition against HCT-116 and HT-1080 cell lines, respectively.

Fig. 3.

Cytotoxic effect of endosymbiont isolate SPG6 extract against HCT-116 and HT-1080 cells lines

GC/MS analysis

According to GS–MS analysis, five components were found in the diethyl ether fraction (Table 4). The main constituents were octadecenoic acid, methyl ester (77.2%); 9,12-Octadecadienoic acid (Z,Z)- (84.6%), Dimethyl palmitamine (95.1%), n-Hexadecanoic acid (82.4%), and Dimethylethylcetylammonium bromide (95.3%) (Supplementary file S2).

Discussion

Psychosocial and climatic stress augments the level of free radicals and mediates different medical problems viz. aging, cognitive problems, diabetes, cardiovascular diseases, etc. (Fang et al. 2002; Fridovich 1999; Hermes et al. 2001). Furthermore, acute and continuous stress causes a metabolic imbalance and generates exogenous and endogenous xenobiotics. Transformation of xenobiotics to quinone and then to semiquinone via cytochrome P450 reductase leads to ROS-induced oxidative stress (Kumar et al. 2019). Natural antioxidants play a crucial role to alleviate the oxidative damage in the cellular milieu for promoting health (Kumar et al. 2015). Sponges harbor a diverse variety of bioactive molecules (Faisal et al. 2021). It is widely known that marine sponges sustain a large community of microorganisms (up to 50–60% of the holobiont biomass). Endosymbiont fungi of the holobiont produce high-frequency adaptogenic molecules with distinct structures and functions (Hasan et al. 2015; Sajeevan 2020). The marine fungal endosymbiont of L. herbacea might be a novel source of naturally occurring antioxidants. Therefore, in the current research, sponge-derived fungi were isolated from the marine sponge L. herbacea.

Research on the ecology and diversity of marine sponges associated endosymbiont fungi are rather very less in comparison to those targeting these fungi's metabolic versatility (Suryanarayanan 2012). Microscopic visualization of the isolated endosymbiont revealed that the genera Aspergillus, Alternaria, Talaromyces, and Bipolaris constitute the main components of the marine sponge L. herbacea’s fungal microbiome. Thus, based on morphological differences, the endosymbiont fungi associated with marine sponges L. herbacea have moderate species diversity.

Extracts/fractions of endosymbiont fungi are a mixture of molecules with distinct antioxidant capacities (Hou et al. 2003; Mensor et al. 2001). Distinct antioxidant capacities of an extract in different assays might be due to the redox potential and electrostatic interactions (chemotype: radical) among a diverse range of molecules present in the crude extract (Kumar et al. 2014). Thus, in the present investigation, we have performed different antioxidant tests viz. DPPH, OH radical scavenging assay, metal chelating assay, and reducing power assay were used to conclude a precise perception of the capabilities of marine fungal endosymbionts to ameliorate oxidative stress.

The DPPH analysis has been widely used to evaluate the antioxidant potential (Sánchez-Moreno 2002). In the present study, the highest DPPH radical scavenging effect was observed in extracts of isolates SPG6 and SPG19. Compared to Pestalotiopsis sp. fungi (EC₅₀ of 34.85 mg L⁻¹) isolated from the mangrove plants Rhizophora stylosa and R. mucronata, the SPG6 extract used in the current investigation had a better capacity to scavenge free radicals (Zhou et al. 2018). Chen et al. (2016) reported that the Aspergillus sydowii isolate from a sponge showed antioxidant (DPPH) potential. Abdel-Monem et al. (2013) reported that G. dankaliensis marine-associated fungus showed 59.28% activity against DPPH radicals compared to ascorbic acid (61.83%).

Hydroxyl radicals are highly reactive oxygen species in the cellular environment, which can damage various biomolecules and other organic substrates (Jirovetz et al. 2006). This radical can break the DNA strand, resulting in mutagenesis and carcinogenesis (Hochstein & Atallah 1988). Aldehydes and dimers are oxidation products produced due to phenolic compound’s scavenging ability against hydroxyl radicals (Halliwell & Gutteridge 1990). In the present study, on comparing the OH radical scavenging potential indicates that crude extracts of most Aspergillus spp. and Alternaria spp. isolates have better potential than others. The endosymbiont extract’s ability to quench hydroxyl radicals appears capable of inhibiting lipid peroxidation and hydroxyl radical-induced DNA damage (Borra et al. 2014). The hydroxyl radical scavenging ability of the endosymbiont fungal extract of the present study was better than that of endophytic fungi derived from the Ocimum sanctum (EC₅₀ of 298.6–362.76 g L⁻¹) (Madagundi et al. 2013). Muthiyan et al. (2018) reported sponge associated fungal extract showed more than 50% inhibition (half inhibitory concentration) in hydroxyl and superoxide anion radical scavenging assay at a concentration of 50 µg/mL.

Iron is one of the fundamental elements of life, being necessary for numerous physiological functions, including oxygen transport, breathing, and the action of numerous enzymes. Additionally, it is a highly reactive metal that induces an oxidative change in lipids, proteins, and other cellular components. Thus, the metal chelating ability is another important mechanism used in our evaluation of antioxidant potential. From our study, 31% of the extracts exhibited metal chelating ability. The comparison of the metal chelating capabilities of endosymbiont extracts indicates that extracts of a few Aspergillus and Alternaria isolates have better potential than others. The metal chelating ability of the endosymbiont extract in the present study was stronger than that of Penicillium citrinum, an endosymbiont fungus derived from seaweed (Sargassum wightii) (Hulikere et al. 2016).

Reducing ability has been used to evaluate the antioxidant activity of natural products (Zhang et al. 2014). The reduction of Fe³⁺ complexes to Fe²⁺ forms occurs when antioxidants are present in samples. In this study, isolate SPG6 was as active as ascorbic acid, a well-known antioxidant, with EC₅₀ 114 g L⁻¹, and SDHY 01/02 extract (EC₅₀ of 248 ± 1.35 g L⁻¹) also showed a moderate reducing effect. Therefore, these extracts have a higher ability to reduce Fe³⁺.

Normally, a high intra-mitochondrial ATP/ADP ratio induces ATP-associated allosteric inhibition of COX to decrease the O₂ uptake and keep the membrane potential low (Ramzan et al. 2020). Abiotic stresses, viz. hypoxia, cold, osmotic stress, salt, mechanical stimuli, etc., rapidly increase cytosolic Ca²⁺ that activates the mitochondrial phosphatase, which in turn dephosphorylates the COX. This causes an increase in membrane potential that leads to excessive ROS production (Ramzan et al. 2020). Even after the absence of hypoxia inducible factor, holobionts are highly stable to hypoxia/anoxia (sedimentation-induced fluctuation in respiratory rate) as well as other stressors (Schuster et al. 2021). Therefore, enriched antioxidant capacities and potent molecules within the respective fungal endosymbiont, viz. isolate SPG6, isolate SPG18 etc., could also be a part of the adaptive response for the stability of L. herbacea. These radical scavenging capacities also appear to intensify the antioxidant defenses that could be a necessity of photosynthetically induced oxidative stress to maintain the stability of holobiont during high sedimentation rates (Taylor et al. 2007).

The present study showed that the endosymbiont fungi associated with marine sponges L. herbacea have high antioxidant potential. The best promising antioxidant activity was shown by SPG6, which was identified as an isolate of Alternaria destruens. Thirunavukkarasu et al. (2012) reported that marine-derived fungi associated with sponges are good sources of antioxidants. Similarly, Suryanarayanan (2012) also reported that sponge-associated Aspergillus spp. isolates showed antioxidant properties.

Correlations between the different antioxidant/scavenging activities were established (Table 5). The best positive correlation was found between chelating and free radical scavenging activity of the endophytes in terms of correlation coefficient at p value < 0.005. Further, hydrogen peroxide radical scavenging activity of the endophytes was also moderately correlated with hydroxyl radical activity. Free radical scavenging activity/chelating activity were found correlated with superoxide radical scavenging activity. The result is in line with previous studies (Ng et al. 2023), although there are no previous records of volatile compound’s content correlation with antioxidant activity.

The bob of the antioxidants seems to oscillate from normal panacea to toxicity. The MTT assay is less sensitive to minor changes (Nibret et al. 2021). So the MTT assay was used to confirm the cytotoxic effect. Generally, cancer cells have defects in the DNA repair mechanism and genomic instability that can produce more sensitivity to DNA-specific cytotoxic agents than normal cells (Haririan et al. 2010; Helleday et al. 2008; Luo et al. 2009). HCT-116 and HT-1080 cells lines have short doubling times (17.4 h and 26 h, respectively) and are among the sensitive cancer cell lines that could be more vulnerable to the cytotoxic agent (Abalos et al. 2021; Al-Oqail et al. 2016; Kazakova et al. 2021; Kim et al. 2008; Silva et al. 2018; Wang et al. 2013, 2020). Consequently, we have performed the cytotoxic study in HCT-116 and HT-1080 cells lines for isolate SPG6. At 100 µg/ml concentration, isolate SPG6 extract has shown 10% and 8% growth inhibition against HCT-116 and HT-1080 cell lines, respectively. If viability of cells is ≥ 90% (as compared to control wells), then the drug concentration under investigation is considered as non-cytotoxic (Basaka et al. 2016). Moreover, according to guideline of National Cancer Institute (NCI), crude extract that did not show cytotoxicity up to concentration of 100 µg/ml, can be considered nontoxic. (Ashraf et al. 2013; http://www.cancer.gov Accessed May 29, 2014). As compared to control wells, GI₅₀ (concentration that shows 50% growth inhibition) of isolate SPG6 is above and far away from 100 µg/ml of extract. Therefore, the isolate SPG6 can be considered as non-toxic per the NCI guideline.

GS–MS analysis is used to detect the antioxidant molecules that could ameliorate the free radical-mediated disease. It revealed that diethyl ether fraction of SPG6 extract contained constituents, such as Octadecenoic acid, methyl ester; 9,12-Octadecadienoic acid (Z,Z)-, Dimethyl palmitamine, n-Hexadecanoic acid, and Dimethylethylcetylammonium bromide. n-Hexadecanoic acid is famous for their antioxidant potential (Abubakar & Majinda 2016). Evidences of n-Hexadecanoic (synonym—Palmitic acid) acid to alleviate oxidative stress, free radical (hydroxyl radical, singlet oxygen, hydrogen peroxide, etc.)-mediated hypercholesterolemia and hemolysis have already been reported (Krishnamoorthy & Subramaniam 2014). Besides antioxidant, hypocholesterolaemia, antihaemolytic, anti-arthritis, and anticancer capacities, 9,12-octadecadienoic acid has also been reported for its evidences to ameliorate oxidative stress-mediated inflammatory, histaminic, and eczemic conditions (Krishnamoorthy & Subramaniam 2014). Moreover, octadecanoic acid, methyl ester is also an anti-inflammatory antioxidant (Mazumder et al. 2020; Siswadi & Saragih 2021). n-Hexadecanoic acid, along with other compounds, inhibits phospholipase A(2), an enzyme responsible for inflammation (due to the release of fatty acids by hydrolyzing the ester bonds in membrane phospholipids) (Aparna et al. 2012). Other bioactive compounds with acidic hydroxyl and amine groups could also be responsible for the antioxidant capacities of SPG6 (Palanichamy et al. 2018). These compounds can be detected and isolated in future.

Therefore, the study confirmed that isolate SPG6 has potent antioxidant capacities, which might be because of a diverse group of bioactive molecules that could help ameliorate oxidative stress. Future direction includes optimization of mass culture of marine SPG6 for adaptogenic/antioxidant molecules in ex-situ. However, the safe use of SP6 further needs in vivo biocompatibility study.

Conclusion

In the present work, we illustrate that sponge-associated endosymbiont fungi are an abundant source of bioactive compounds. Further research is required to identify and characterize the principal compounds responsible for these activities. However, this is the first report of Alternaria destruens, an endosymbiont of marine sponge, Lamellodysidea herbacea, along with their antioxidant potential. This study will pave the path for additional revolutionary research from previously unknown marine organisms to endow the pharmaceutical industry with new bio-resources.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

The research idea belongs to MK. MA isolated the endosymbiont, EB carried out the fermentation part and partly identified the fungus, while GS performed antioxidant activities of endosymbiotic extracts under the supervision of MK. APG performed the GC–MS. SK was involved in the cytotoxicity assay. The manuscript was written by the team (GS, PR, and JK) and edited and reviewed by JK & MK.

Funding

The authors have no financial or proprietary interests in any material discussed in this article.

Data availability

This published article and its supporting information files contain all of the data created or analyzed during this investigation.

Declarations

Conflict of interest

The authors declare no competing interests.

Research involving human participants and/or animals

Not applicable.

Informed consent

Not applicable.