Assessment of the photoprotective potential and structural characterization of secondary metabolites of Antarctic fungus Arthrinium sp

doi:10.21203/rs.3.rs-3352625/v1

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Assessment of the photoprotective potential and structural characterization of secondary metabolites of Antarctic fungus Arthrinium sp

https://doi.org/10.21203/rs.3.rs-3352625/v1

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published 23 Dec, 2023

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Interest in Antarctic fungi has been growing due to their ability to survive in the harsh environment, suggesting the presence of compounds for human use. Due to the damaging effects of sunlight, sunscreen use has been growing but, scientific evidence has pointed to the toxic potential of UV filters for human and environment, leading to a ban the use of UV filters (avobenzone and oxybenzone) in some locations. So, this work evaluated the photoprotective potential of molecules from sustainable marine sources (seaweed-derived fungi), aiming to discover natural, efficient, and safer UV filter alternatives. Thus, photoprotective and antioxidant activity of metabolites from Arthrinium sp. was investigated. Six compounds (1-6) were isolated: 3-Hydroxybenzyl alcohol (1), (-)-orthosporin (2), norlichexanthone (3), anomalin B (4), anomalin A (5) and agonodepside B (6), being 1, 2, 6 not reported in Arthrinium. Fraction F showed excellent absorbance in both UVA and UVB, while compound 6 presented lower absorbance in the UVB region. Fraction F and compound 6 were considered photostable and non-phototoxic on HaCaT cells. Both showed antioxidant activity against UVA induced intracellular ROS production, in keratinocyte monolayers and in reconstructed human skin models (reduction of 34.6% and 30.2% in fluorescence) and both can be considered for use in sunscreens. Fraction F presented higher absorption in UVB and UVA than (6), which can be due to synergism and did not present any irritation potential in HET-CAM assay. The fraction presented the advantage of not needing further purifications, however clinical studies should be performed to confirm its potential.

Arthrinium

Marine Antarctic natural products

Photoprotection

Antioxidant

Reconstructed human skin

The marine environment is characterized by strong fluctuations in abiotic and biotic conditions such as high pressures, salinity, tides, depth and temperatures, for example. As a result, wide biological diversities and unique chemical compounds have emerged, making the marine environment an important source of new bioactive compounds with biotechnological applications. In addition, in the marine environment there is the presence of sessile living organisms, such as sponges, cnidarians, and algae, which have developed chemical defense mechanisms (Li et al., 2020). Surrounded by the Southern Ocean, the Antarctic continent is one of the most primitive and harsh ecosystems on the planet. This is due to low temperatures, low levels of organic nutrients, low water availability, strong winds, and high incidence of UV rays. However, even under these conditions, benthic algal communities, microorganisms thrive. Numerous species of fungi have been described in association with seaweeds in parasitic, saprophytic, or endophytic relationships. These organisms are collectively known as "algicolous fungi'' and are relatively unexplored but hold great metabolic potential. Interest in Antarctic fungi, in particular, has been growing due to their ability to survive in the harsh Antarctic environment, which suggests the presence of unusual and promising biosynthetic pathways that could lead to novel bioactive compounds (Ogaki et al., 2019). These novel active compounds are targeted for application with different approaches, including insecticides, biotechnological insights and finally new cosmetic ingredients. Among the cosmetic application, is it possible to point sunscreens formulation, based on mycosporine-like amino acids (MAAs) contents, that are capable of absorbing the UV rays ranging from 310 to 360 nm. MAAs are colorless, soluble in water and have low molecular weight which can be found in fungi, algae, cyanobacteria, among other marine organisms (Rosic, 2021; Ding et al., 2022). Furthermore, some other natural compounds were reported as having anti-UV activity, such as the phenolic substances. In nature, phenolic compounds are biosynthesized to balance ROS production and present protective effects against DNA damage induced by UV radiation (Bedoux et al., 2014; Ding et al., 2022). Agonodepside B (compound 6) is a depside, a natural product class commonly formed by two or more phenolic units, biosynthetically originated from the polyketide synthase (PKS) route (Kealey et al., 2021; Legaz et al., 2011).

It is well-known that chronic exposure to UV radiation from sunlight has negative health effects, and a growing concern about such effects has caused an increase in the use of formulations containing UV filters (Watkins et al., 2021). In addition to sunscreens, these substances are used in more than 2.000 personal cosmetic products (Dinardo and Downs, 2018; Zhang et al., 2021). The global production of UV filters is estimated to be more than 10.000 tons per year. Inevitably, these products end up being released into the aquatic environment, either directly, such as by swimming in this environment; or indirectly, such as by washing the product off while bathing. This release is increasing concerns about the impact of the presence of these substances in the aquatic environment (Zhang et al., 2021). In vivo and in vitro studies show vast adverse effects caused to the environment and exposed organisms, such as coral reef bleaching and toxicity to the endocrine, neurological, and other systems of resident organisms (Fivenson et al., 2020). In response to these studies concerning environment injury, some regions have banned the use of some UV filters, such as oxybenzone (benzophenone‐3) and octinoxate (ethylhexyl methoxycinnamate). These bans take place in Hawaii, the US Virgin Islands, Key West in Florida, Palau, and Bonaire (Mitchelmore et al., 2021).

There is a need, therefore, for eco-friendly, sustainable and safer UV filters as alternatives to the synthetic compounds available in the cosmetic market (Fivenson et al., 2020). Several organisms, especially those exposed to intense UV radiation, have developed various photo adaptive mechanisms including production of antioxidants and UV absorbing metabolites (Saewan and Jimtaisong, 2015). UV incidence is also highly evident in the Antarctic region. Despite the decrease of ozone-depleting compounds and its consequent recovery, the Antarctic ozone hole continues to occur each year, with the severity of ozone loss strongly modulated by weather conditions, persisting mainly in late spring (Cordero et al. 2022).

Thus, the search for safer, effective and less environmentally aggressive UV filters produced by organisms of Antarctic origin is promising and justified.

Fungal Isolation

Initially, a health specimen of the seaweed Phaeurus antarcticus was collected at Greenwich Island, Antarctica (62°26’46.9” S 59°44’20.3” W) in November/2015 during the Brazilian Antarctic Expedition OPERANTAR XXXIII. The seaweed was rinsed in sterile seawater to remove adherent debris and other contaminants. To the selective isolation of fungal endophytes, the seaweed was cut into small fragments (0,5 – 1,5 cm) and surface disinfected by immersion in 70% ethanol (v/v) for 15 seconds, followed by rinsing in sterile seawater three times. Control plates were used to assess the efficiency of the disinfection procedure. The disinfected fragments were used to make imprints on the agar surface (Control 1) and then inoculated in another petri dish containing an isolation medium for up to 60 days. Control 2 consisted of the inoculation of water drops from the third rinsing procedure (Teixeira et al. 2019). The seaweed was inoculated, aiming the endophytic fungi to grow, in a medium consisted of potato dextrose agar (39 g/L) prepared in sterile seawater supplemented with 200 mg/L of chloramphenicol (Sarasan et al., 2017). The emerging colony was purified by streaking technique and preserved in flasks containing glycerol or mineral oil, in the Laboratory of Marine Environment Organic Chemistry and at -80℃ in Laboratory of Clinical Mycology, both in School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Brazil.

Fungal Identification

To the DNA extraction procedures, the isolated fungal strain was cultivated in potato dextrose broth. The identification was carried based on the sequencing of the internal transcribed sequence (ITS) region of the fungal ribosomal DNA. The polymerase chain reactions (PCR) were performed with Phusion^® High-Fidelity DNA Polymerase (New England BioLabs, Inc.) and primers ITS1 (5′-T C C GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) (White et al. 1990). The PCR products were purified with Wizard® SV Gel and PCR Clean-Up System (Promega) and sequenced using the same primers with ABI3730 DNA Analyzer (Applied Biosystems). Each sequence was analyzed with ChromasPro® Software (ChromasPro 1.7.6, Technelysium Pty Ltd., Tewantin QLD, Australia). The sequences were then tested against the publicly available DNA sequences in the National Institutes of Health (NIH) genetic sequence database (Benson et al. 2013).

Fermentation and Extraction

The fungus was grown in Potato Dextrose Agar medium (PDA NEOGEN®, USA, 39 g/L) for 7 days. After this period, 10 plugs (5 mm diameter) were transferred to each of the 50 Erlenmeyer flasks (500 mL), containing 200 mL of Potato Dextrose Broth (PDB KASVI®, BRAZIL, 27 g/L) medium, prepared in artificial seawater SWBG-11 described by Castenholz, 1988. The flasks remained static and at room temperature for 14 days. Liquid-liquid extraction with ethyl acetate was performed to obtain the extracts, adding 100 mL of the solvent in each Erlenmeyer flask followed by 5 minutes in the ultrasonic bath (75 W). The procedure was performed 3 times for each flask. After the method was completed, the aqueous culture medium was properly discarded. The crude extract (CE) was concentrated in a rotary evaporator under reduced pressure and heat, not exceeding the temperature of 28º C.

Crude extract fractionation and compounds isolation

The CE (1.2g) was fractionated by vacuum liquid chromatography (VLC) method, using a stationary phase silica gel (40-70 mesh, Merck) in a 200 mL glass Buchner funnel (18 mm diameter, 250 mm height). Elution using the organic solvents n-hexane (Hex), ethyl acetate (EtOAc) and methanol (MeOH) with 200 mL of a stepwise polarity gradient yielded nine fractions: VLC A (Hex 100%), VLC B (Hex:EtOAc 9:1), VLC C (Hex:EtOAc 4:1), VLC D (Hex:EtOAc 3:2), VLC E (Hex:EtOAc 2:3), VLC F (Hex:EtOAc 1:4), VLC G (EtOAc 100%), VLC H (EtOAc:MeOH 7.5:2.5) and VLC I (MeOH 100%). Fractions VLC E and F were selected for compounds isolation after chromatographic analyses and bioguided studies.

Fraction VLC E and F were submitted to purification by reverse phase HPLC (LC-6AD model, Shimadzu) coupled with diode array detector (SPD-M10A model, Shimadzu) with a semi-preparative column C-8 (25cm x 10mm, 10μm - Ascentis Supelco, USA). In this isolation, a polarity gradient method changing concentration of MeOH in distilled water: 15% (0 minutes) - 100% (20 – 22 minutes) - 15% (23 – 25 minutes) was used.

In this way, it was possible to obtain from VLC E the compounds 1, 3, and 6, and from VLC F the compounds 2, 4, 5, and 6, respectively.

Structure Identification

The isolated compounds were characterized through spectrometry analyses, such as mass spectrometry (MS) and nuclear magnetic resonance (NMR). The MS data were obtained using a spectrometer of Bruker, microTOF Q II with electrospray ionization and analyzer time of flight type (TOF) in Organic Chemistry Laboratory (FCFRP-USP). The NMR spectroscopy was employed to get data from unidimensional (¹H) and bidimensional (HMBC, HSQC) analyses. The NMR spectra were acquired in Chemistry Department of Faculty of Philosophy, Sciences and Letters at Ribeirão Preto – USP, using a spectrometer model DRX 500, Bruker, Billerica operating at 500 MHz, using deuterated solvents from Sigma-Aldrich: methanol (CD₃OD), dimethyl sulfoxide (DMSO-d₆) and chloroform (CDCl₃). The obtained results were compared with literature data.

Determination of the UV Absorption Spectra

Absorption spectra in the UV region of the CE, fractions and isolated compounds were determined by a spectrophotometer (Agilent 8453) in the 200 to 400 nm range, for the determination of the UV absorption spectra. The samples were diluted in methanol to yield a solution with a concentration of 100 μg/mL. However, the samples that showed higher absorbance in the UVA-UVB region were analyzed in the further studies (photostability, phototoxicity and antioxidant). So, fraction F and the isolated compound 6 were selected to continue our study.

Photostability Test

For the photostability studies, 1 mL of each solution sample was added to glass beakers and then subjected to solvent evaporation until a dried film was obtained. The samples were then submitted or not to UV radiation of 4 mW/cm² emitted from a Philips UVA lamp Actinic BL/10 (Eindhoven, Netherlands) (cumulative dose of 27.5 J/cm²) measured with a Dr. Hönle radiometer (Planegg, Germany) equipped with a UVA sensor. After irradiation, the dried film was resuspended in 1 mL of solvent, and the absorption spectrum of the solutions in the 280 to 400 nm range was analyzed. The area under the curve (AUC), which is the integral of the absorption spectrum of the samples in the UVB (280–320 nm) and UVA (320–400 nm) ranges, were used to calculate the photostability by using the integration function of the MicroCal OriginPro Software (8 SRO, OriginLab Corporation, Northampton, MA, USA). The results of the photostability experiments are expressed as a percentage of the area of irradiated samples related to the area of non-irradiated samples, considered as 100% (Gaspar and Maia Campos, 2006; Tavares et al. 2020).

Toxicity Assessment

Phototoxicity assay (3T3 NRU PT)

The selected fraction (F) and compound 6 (agonodepside B) were submitted to a phototoxicity test based on neutral red (Merck, Darmstadt, Germany) uptake using 3T3 fibroblasts according to Organization for Economic Co-operation and Development (OECD) nº 432 guideline (OECD, 2019). The fibroblasts were cultivated in Dulbecco’s modified Eagle medium (DMEM, Carlsbad, CA, USA) supplemented with newborn calf serum (10%, Carlsbad, CA, USA), glutamine (4 mM), penicillin (100 IU/mL) and streptomycin (100 µg/mL) from Sigma Aldrich (St. Louis, MO, USA). In this test 3T3 Balb/c fibroblasts seeded on two 96-well plates (1 x 10⁴ cells/well) were pre-incubated with eight different concentrations ranging from 6.81 to 100 µg/mL of the samples analyzed for 1h. Norfloxacin (St. Louis, MO, USA) was used as positive control. One plate was then exposed to a UVA irradiation dose of 9 J/cm², using irradiance of 6.8 mW/cm² for 22 min obtained with a solar simulator (Dr. Hönle type SOL-500, Planegg, Germany), while another one was kept in the dark. To evaluate the cyto- and phototoxicity of the samples, cell viability in the presence and absence of radiation was determined by the neutral red uptake method. Data were analyzed using Phototox Software 2.0, which calculated the Mean Photo Effect (MPE). According to the phototoxicity determination guideline recommended by OECD (OECD, 2019) sample with MPE < 0.10 predicts “no phototoxicity”, while MPE > 0.10 and < 0.15 predict “equivocal phototoxicity”; and MPE > 0.15 predicts “phototoxicity”.

Ocular irritation potential (HET-CAM assay)

The Hen's egg test-chorioallantoic membrane (HET CAM) assay was performed according to Luepke and Kemper (1986). Fertilized White Leghorn chicken eggs at the tenth day of incubation at 37 °C under 50–60% relative humidity were used. The eggshell around the air space was removed, the inner membrane was hydrated with a 0.9% NaCl solution and removed carefully, and the chorioallantoic membrane (CAM) was exposed. Three hundred μL of a 0.01% solution (100 μg/mL) of samples (fraction F and compound 6) were applied to the CAM and 20 seconds after, the CAM was rinsed off with 5 mL of 0.9% NaCl solution and monitored for 5 minutes using a stereo microscope (SZT – Led#, Bel Photonics, Brazil), in order to observe the irritant events such as hyperemia, hemorrhage and coagulation. It was given one score for each observed effect, according to the time of appearance (Table 1). The Irritation Score (IS) was obtained by the sum of individual scores of hyperemia, hemorrhage and coagulation. A sample is classified as non-irritant if 0 ≤ IS ≤ 0.9; as slightly irritant if 1.0 ≤ IS ≤ 4.9; as moderate irritant if 5.0 ≤ IS ≤ 8.9 and as severe irritant if 9.0 ≤ IS ≤ 21.

A 1% (w/w) sodium lauryl sulfate (SDS) solution was used as positive control and a 0.9% sodium chloride (NaCl) solution was used as negative control. The test was performed in quadruplicate.

Table 1 Scores given to the irritant events observed in the HET CAM test (adapted from Luepke and Kemper, 1986).

Irritant Event	0 - 30 sec	30 sec – 2 min	2 min – 5 min
Hyperemia	5	3	1
Hemorrhage	7	5	3
Coagulation	9	7	5

Photoprotection against UVA-induced ROS production

HaCat Antioxidant Activity

The quantification of intracellular ROS produced by HaCaT cell exposure to UVA was evaluated using the 2’,7’-dichlorodihydrofluorescein diacetate (DCFH₂-DA, Sigma Aldrich, St. Louis, MO, USA) probe, a non-fluorescent compound that permeates the cell, is hydrolyzed by intracellular esterases and, oxidized by ROS to the fluorescent compound, 2'-7'-DCF (Kalyanaraman et al., 2011). UVA irradiation provokes ROS generation, which can oxidize DCFH to dichlorofluorescein, a fluorescent product, increasing fluorescence (%).

Prior to the beginning of the assay, cell viability was evaluated to exclude the possibility that the decrease in fluorescence intensity obtained in the ROS assay is related to cell death and not to antioxidant activity. The assay was performed under the same conditions (cell type, density and exposure times) as the measurement of intracellular ROS generation in HaCaT monolayers cells.

Cell viability was evaluated using keratinocytes HaCaT (Cell Bank of Rio de Janeiro, Rio de Janeiro, Brazil), whichwere cultivated in DMEM (Gibco, Carlsbad, CA, USA) supplemented with fetal bovine serum (10%, Gibco, Carlsbad, CA, USA), pyruvate (1 mM), penicillin (100 IU/mL) and streptomycin (100 µg/mL) from Sigma Aldrich (St. Louis, MO, USA). Subsequently, they were seeded in 96-well plates (1 x 10⁵ cells/well) and cultured overnight at 37 °C in a 5% CO₂ atmosphere. The cells were treated with the samples selected for this experiment, fraction F and isolated compound 6,as it showed the best UV absorbance among the isolated compounds,using concentrations of 10.0; 5.0 and 2.5 µg/mL. The cells were incubated for 1h. Then, cell viability was determined by neutral red uptake (Rangel et al. 2020; Gaspar, Maia Campos and Liebsch, 2013). Sodium dodecyl sulfate (SDS) was used as a positive cytotoxic control (100 µg/mL). The absorbance of untreated cells was considered as 100% to calculate the percentage of cell viability relative to the samples.

For the evaluation of photoprotection against UVA-induced ROS production, HaCaT cells cultivated in supplemented DMEM were seeded in 96-well plates (1 x 10⁵ cells/well) and cultured overnight at 37 °C in a 5% CO₂ atmosphere. The cells were treated and incubated with sample (compound 6 at 10.0; 5.0 and 2.5 µg/mL) for 1h, then they were incubated with DCFH₂-DA (10 µM) for 30 min, followed by exposure to UVA radiation (Solar simulator Dr. Hönle type SOL-500, Planegg, Germany) with a total dose of 4 J cm^-2, using an irradiance of 6.6 mW/cm for 10 minutes. Quercetin (10 μg/mL) and norfloxacin (100 μg/mL) (Sigma Aldrich, St. Louis, MO, USA) were used as ROS quencher and generator, respectively. The fluorescence was determined with a microplate reader (Synergy^TM HT, BioTek, USA) at 485 nm excitation and 528 nm emission. The fluorescence of untreated irradiated cells was considered to be 100% in order to calculate the relative percentage of samples.

Reconstructed Human Skin Model (RHS) Antioxidant Activity

The assay with in house reconstructed skin models was conducted after approval by the Ethics Committee in Research Involving Human Beings – School of Pharmaceutical Sciences of Ribeirão Preto – USP (CAAE number 31758619.5.0000.5403), using primary human fibroblasts and keratinocytes from foreskin (pooled from three donors). Informed written consent was obtained from all the donors or their parents or legal guardians.

Primarily, the dermal compartment was prepared by seeding 1.14 x 10⁵ normal human fibroblasts with collagen type I (Corning^®) into the insert (0.4 µm pore size; ThinCert^TM, Greiner Bio-One GmbH, Frickenhausen - Germany) and incubated for 20 hours. After that, 3.7 x 10⁵normal human keratinocytes were seeded on the top of the dermal compartment and kept submerged in an in house prepared culture medium for 24 hours, so the cells could form a monolayer. Throughout 7 days, the culture was maintained at the air-liquid interface allowing complete keratinocytes differentiation and stratification, and the culture medium was changed every other day (Pennacchi et al, 2015; Pivetta et al, 2019; Tavares et al, 2020).

After the skin models were fully differentiated on day 10, the measurement of intracellular ROS generation began with the incubation of the models with the DCFH₂-DA (50 µM) for 45 min in the absence of light. After washing with phosphate-buffered saline (PBS), 25 µL of the sample were applied onto the skin models for 1 hour and then, subjected (UV+) or not (UV-) to 10 J.cm^-2 of UVA radiation from a SOL-500 solar simulator (Dr Honle AG, Planegg, Alemanha).

Immediately after irradiation and washing with PBS, the tissues were frozen in liquid nitrogen and the, 8 µm histological sections were obtained in a cryostat. Pictures were obtained in an inverted Ti-S microscope (Nikon Instruments Inc., Holland), 488 nm, using 100 ms of exposure intensity and analyzed by Image J software (Marionnet et al., 2014; Rasmussen et al., 2010). Fluorescence intensity results were normalized to area/pixels and expressed as percent fluorescence compared to untreated irradiated (UV+) control.

Statistical analysis

The results were analyzed by one-way ANOVA followed by Tukey's post-hoc test using Minitab version 18.1, since they presented normal distribution (Anderson-Darling test). p-Values lower than 0.05 were considered to be statistically significant.

Fungal Identification

The fungal isolate, was obtained from the fragments of Phaeurus antarcticus and was later identified as the fungus Arthrinium sp. The results of identification process can be observed in Table 2, as well as the NCBI reference and accession number.

Table 2 Results concerning the fungus identification, NCBI reference and accession.

Code	Score	E-value	Identities	Final identification	Reference NCBI Accession	NCBI Accession
LMC8101	937	0.0	98%	Arthrinium sp.	JQ411349.1	OR412386

Compound’s Identification and Characterization

Six major compounds of fractions VLC E (180.0 mg) and VLC F (141.0 mg) from Arthrinium sp. CE was isolated and identified (Fig. 1, 1-6). The report of compounds 1, 2 and 6 is unprecedented in species of Arthrinium.

Compound 1 (2.0 mg) was obtained as a colorless liquid with λ_max 273 and 324 nm. Its molecular formula was established as C₇H₈O₂ based on high resolution mass spectrometry (HRMS, ESI), m/z 123,0484 [M-H]^-. The full structure was determined by ¹H NMR analyses (500 MHz, CDCl₃, ppm) by observing chemical shifts (δ), δ: 4.66 (2H, s, H-7), 6.76 (1H, dd, J= 7.7, 2.3 Hz, H-6) 6.87 (1H, br s, H-2), 6.92 (1H, d, J= 7.7 Hz, H-4), 7.23 (1H, t, J= 7.7 Hz H-5) and ¹³C NMR (HMBC, HSQC; 500 MHz, CD₃OD, ppm) δ: 142.7 (C, C-1), 113.4 (CH, C-2), 157.1 (C, C-3), 114.0 (CH, C-4), 128.8 (CH, C-5), 117.4 (C, C-6), 63.5 (CH₂, C-7). The identity of the compound was confirmed after data comparison with the literature of Alfaro et al., 2003, proving to be 3-Hydroxybenzyl alcohol (Fig. 1, 1).

Compound 2 (1.0 mg) was obtained as a white powder with [ – 0.63^o and λ_max 244, 238, 277 and 326 nm. Its molecular formula was established as C₁₂H₁₂O₅ based on HRMS (ESI), m/z 235,0756 [M-H]^-. The full structure was determined by ¹H NMR analyses (500 MHz, DMSO-d6, ppm) δ: 1.12 (3H, d, J= 6.2 Hz, H-11), 2.59 (2H, m, H-9), 3.96 (1H, m, H-10), 4.80 (1H, d, J= 4.9 Hz, OH-10), 6.29 (1H, d, J= 2.0 Hz, H-5), 6.35 (1H, d, J= 2.0 Hz, H-7), 6.47 (1H, s, H-4), 10.98 (1H, s, OH-8). The identity of the compound was confirmed after data comparison with the literature of Gremaud et al., 1994, proving to be 6,8-Dihydroxy-3-[(2R)-2-hydroxypropyl]-1H-2-benzopyran-1-one, also known as (-)-orthosporin (Fig. 1, 2).

Compound 3 (4.0 mg) was obtained as a light yellow amorphous solid with λ_max 241 and 311 nm. Its molecular formula was established as C₁₄H₁₀O₅ based on HRMS (ESI), m/z 257,0459 [M-H]^-. The full structure was determined by ¹H NMR analyses (500 MHz, CD₃OD, ppm) δ: 2.76 (3H, s, H-11), 6.09 (1H, d, J= 2.0, H-7), 6.21 (1H, d, J= 2.0, H-5), 6.58 (1H, br s, H-2), 6.59 (1H, br s, H-4) and ¹³C NMR (HMBC, HSQC; 500 MHz, CD₃OD, ppm) δ: 143.2 (C, C-1), 111.3 (C, C-1a), 115.5 (CH, C-2), 157.1 (C, C-3), 100.1 (CH, C-4), 159.7 (C, C-4a), 96.9 (CH, C-5), 164.2 (C, C-6), 97.4 (CH, C-7), 162.7 (C, C-8), 102.3 (C, C-9a), 164.6 (C, C-10a), 23.1 (CH₃, C-11). The identity of the compound was confirmed after data comparison with the literature of Abdel-Lateff et al., 2002, proving to be 3,6,8-trihydroxy-1-methylxanthone, also known as norlichexanthone (Fig. 1, 3).

Compound 4 (1.0 mg) was obtained as a yellow amorphous solid with λ_max 258 and 327 nm. Its molecular formula was established as C₁₄H₁₀O₇based on HRMS (ESI), m/z 289,0519 [M-H]^-. The full structure was determined by ¹H NMR analyses (500 MHz, DMSO-d6, ppm) δ: 2.58 (3H, s, H-11), 6.07 (1H, d, J= 2.0, H-7), 6.30 (1H, d, J= 2.0, H-5), 13.61 (1H, s, OH-8). The identity of the compound was confirmed after data comparison with the literature of Abdel-Lateff et al., 2002, proving to be 2,3,4,6,8-pentahydroxy-1-methylxanthone, also known as anomalin B (Fig. 1, 4).

Compound 5 (2.0 mg) was obtained as a light yellow amorphous solid with λ_max 239, 254, 313 and 359 nm. Its molecular formula was established as C₁₄H₁₀O₆ based on HRMS (ESI), m/z 273,0550 [M-H]^-. The full structure was determined by ¹H NMR analyses (500 MHz, DMSO-d6, ppm) δ: 2.64 (3H, s, H-11), 6.07 (1H, d, J= 2.0, H-7), 6.22 (1H, d, J= 2.0, H-5), 6.67 (1H, s, H-4), 13.65 (1H, s, OH-8). The identity of the compound was confirmed after data comparison with the literature of Abdel-Lateff et al., 2002, proving to be 2,3,6,8-tetrahydroxy-1-methylxanthone, also known as anomalin A (Fig. 1, 5).

Compound 6 (5.5 mg) was obtained as a strong yellow amorphous solid with λ_max 217 and 277 nm. Its molecular formula was established as C₂₄H₂₆O₇ based on HRMS (ESI), m/z 425.1686 [M-H]^-and fragmentation (MS/MS): m/z 425 [M-H]^-, 221, 203. The full structure was determined by ¹H NMR analyses (500 MHz, CDCl₃, ppm) δ: 1.66 (3H, dd, J= 6.8 Hz, 1.0, H-9), 1.79 (3H, dd, J= 6.8, 1.0 Hz, H-9’), 1.92 (3H, br d, J= 1.0 Hz, H-10’), 1.97 (3H, br d, J= 1.0 Hz, H-10), 2.04 (3H, s, H-11’), 2.14 (3H, s, H-11), 5.36 (1H, m, H-8), 5.65 (1H, m, H-8’), 6.21 (1H, s, H-3), 6.38 (1H, s, H-3’), 11.37 (1H, s, OH-6), 11.84 (1H, s, OH-6’) and ¹³C NMR (HMBC, HSQC; 500 MHz, CDCl₃, ppm) δ: 102.7 (C, C-1), 146.2 (C, C-2), 104.4 (CH, C-3), 148.6 (C, C-4), 106.8 (C, C-5), 153.2 (C, C-6), 137.2 (C, C-7), 121.4 (CH, C-8), 13.4 (CH₃, C-9), 18.8 (CH₃, C-10), 7.5 (CH₃, C-11), 109.5 (C, C-1’), 144.3 (C, C-2’), 114.0 (CH, C-3’), 146.2 (C, C-4’), 118.3 (C, C-5’), 158.8 (C, C-6’), 137.9 (C, C-7’), 126.1 (CH, C-8’), 13.8 (CH₃, C-9’), 18.5 (CH₃, C-10’), 8.9 (CH₃, C-11’), 162.9 (C, C-12’) The identity of the compound was confirmed after data comparison with the literature of Cao et al., 2002, proving to be agonodepside B (Fig. 1, 6).

Herein, it was possible to characterize compounds from different classes commonly biosynthesized by endophytic fungi, such as xanthones, isocoumarins, and depsides. This demonstrates the metabolic diversity that Arthrinium has and its biotechnological and pharmaceutical potential.

Isocoumarins (compound 2) mainly derived from the polyketide pathways, are abundant in fungi and have several biological activities, such as antifungal, anti-inflammatory, cytotoxic, and antimicrobial (Pal et al., 2011; Saeed, 2016; Noor et al., 2020). Regardless of the metabolic process, most fungal isocoumarins are always characterized by a C-3 carbon substituent, as we can see in the present study for the compound 2 ((-)-orthosporin). According to Song and co-workers (2017), this is the spontaneous outcome of a biosynthetic intramolecular cyclization, which makes the residual polyketide chain a substituent.

Xanthones (compounds 3-5) are natural polyphenolic products. The biosynthetic pathways of these compounds when produced by fungi are distinct from those produced by plants. Birch and Donovan (1953) suggested at first the biogenesis of xanthones production by fungi. Roberts, in 1961, studied this assembly in more detail, through radiolabeled acetate feeding experiments, concluding that polyketides, from malonyl and acetyl CoA, are the precursors of the fungi’s xanthones (Badiali et al., 2023). The bioactivity of these compounds depends on the nature and/or positions of their substituents, which makes these structures unique with the possibility of binding to a variety of targets and presenting different biological activities, including antimicrobial, antioxidant, and cytotoxic (El-Seedi et al., 2010; Le Pogam and Boustie, 2016).

Finally, agonodepside B (compound 6) is a depside that was isolated for the first time from the terrestrial filamentous fungus F7524 (Cao et al., 2002). Depsides are natural products formed by phenolic units, biosynthetically originated from the condensation of orselinic acid and derivatives of orcinol or two units of orselinic acid through the formation of an ester by the polyketide synthase (PKS) itself or by a separate enzyme (Kealey et al., 2021; Legaz et al., 2011).

Determination of the UV Absorption Spectra

The crude extract (CE) (Fig. 2A) and fractions E-I (Fig. 2B) presented a good absorption of the UVA and UVB region indicating the presence of compounds of interest for photoprotective activity. The VLC F (141.0 mg) and VLC G (72.0 mg) fractions presented absorbance in both UVB (280-320 nm), UVA I and II (320-400 nm) ranges. The fractions H (370.0 mg) and I (155 mg) also presented a good absorption in the UVA II and UVB regions, which were lower than VLC F (mainly in the UVA region). The VLC E fraction presented an UVB absorbance lower than those previously mentioned.

The isolated compounds, however, did not show a high absorbance in the UV region when compared to the studied extract and fractions. This result may be due to synergism, which occurs when substances in mixtures show a better biological activity than when isolated. The compound that showed the best absorbance profile in the UVB-UVA range was agonodepside B (6, Fig. 3).

The use of this substance as an UV filter should not, however, be discarded. The substance may not exert a potent activity when used alone, but it can be studied to be used in combination with other natural products, or with UV filters already on the market, in order to enhance this effect, as shown in the study by Tavares et al. (2020). In this work, the addition of fucoxanthin in a sunscreen formulation increased its photoprotective potential by 72%. Other studies also show the potential of natural products in association with commercial UV filters, enhancing the SPF and maintaining the safety of sunscreens. As another example, Fernandes and collaborators (2015) studied the association of benzophenone-3 with extracts of an antarctic moss (Sanionia uncinata), and found that its aqueous extract significantly increased the SPF of the active compound. In addition, they observed an absence of cytotoxicity (Fernandes et al., 2015; He et al., 2021).

Photostability Studies

The crude extract and the fraction with the highest absorbance in the UVB and UVA I and II regions, as well as agonodepside B (6), were submitted to the photostability study. All samples tested in this study were considered photostable, because their UVA and UVB absorption did not present a high reduction after irradiation. These results are shown in Fig. 4 and Table 1.

Table 3 Remaining percentage of the area under the curve of the irradiated samples compared to non-irradiated samples considered as 100% in the UVA and UVB range of the CE, VLC F, and agonodepside B (6).

Sample	Remaining percentage of the area under the curve		Stability
Sample	UVA	UVB	Stability
CE	87.92%	92.52%	Photostable
VLC F	103.58%	100.88%	Photostable
Agonodepside B (6)	105.35%	94.27%	Photostable

Some UV filters available in the market present higher photo-instability than the fractions and isolated compound 6 of this study. Rangel and co-workers (2020), studied the photostability of some UV filters on the market and the results showed that benzophenone-3 presented approximately 30% of UV absorption reduction, and the broad-spectrum UV filter avobenzone showed a considerable decrease (about 90% reduction) of UV absorption after exposure to UVA radiation (Rangel et al., 2020; Scarpin et al., 2021).

Photochemical reactions can lead to the degradation of the UV filters and, as a consequence, they can loose their ability to protect against UVB-UVA, considerably decreasing the effectiveness of the sunscreen. In addition, these undesirable photoproducts can cause adverse effects, such as allergy and phototoxicity (Scarpin et al. 2021; Nash and Tanner. 2014). Avobenzone, as example, is known to be photo-unstable, due to the keto-enol tautomerization. The enolic form absorbs in the UVA range, and the diketo form absorbs in the UVC range (Pinto da Silva et al. 2014). Some photoallergic reactions of avobenzone have been studied and correlated with its photodegradation products arylglyoxals and benzyls (Scarpin et al. 2021; Afonso et al. 2014; Karlsson et al., 2009).

Toxicity Assessment

Phototoxicity assay (3T3 NRU PT)

This assay is based on a comparison of the cytotoxicity of a chemical when tested in the presence and in the absence of exposure to a non-cytotoxic dose of simulated solar light (OECD, 2019). The results obtained in this assay are presented in Table 3 and indicate that the control norfloxacin was classified as phototoxic, according to the OECD TG 432, showing an MPE > 0.15, and non-cytotoxic due to IC₅₀(–UV) not detected. Agonodepside B (6) and its Fraction F were evaluated at a maximum concentration of 100 µg/mL and were considered non-phototoxic (MPE < 0.10). A negative 3T3 NRU-PT result at sufficiently high concentrations is therefore regarded a sufficient stand-alone indicator for acute photosafety, while a positive result in the 3T3 NRU-PT is always a call for further considerations and probably further testing (Liebsch et al., 2005; Ceridono et al. 2012). However, agonodepside B (6) showed values of IC₅₀(–UV) of 17.8 and 12.8 µg/mL and its Fraction F showed values of IC₅₀(–UV) of 7.1 and 6.2 µg/mL, both considered to be cytotoxic in 3T3 NRU PT assay (Fig. 5, Table 3), which may be due to the presence of xanthones, that are known for its cytotoxicity (El-Seedi et al., 2010; Le Pogam and Boustie, 2016; Bedi, et al., 2021).

Table 4 Results of the phototoxicity assay on 3T3 NRU. IC₅₀ half maximal effective concentration, (− UV) non-irradiated, ND values not determined.

Sample	Run	MPE	IC₅₀(- UV) µg mL^-1	Result
Agonodepside B (6)	1	0.056	17.79	Non-phototoxic/Cytotoxic
Agonodepside B (6)	2	0.081	12.84	Non-phototoxic/Cytotoxic
Fraction F	1	0.014	7.1	Non-phototoxic/Cytotoxic
Fraction F	2	0.027	6.2	Non-phototoxic/Cytotoxic
Norfloxacin	1	0.526	ND	Phototoxic/Non-cytotoxic
Norfloxacin	2	0.511	ND	Phototoxic/Non-cytotoxic

Ocular irritation potential (HET-CAM assay)

The HET CAM assay is a useful test for the screening of eye irritation potential of natural compounds, complementing the in vitro tests for toxicity (Thiesen et al., 2017; Maia Campos et al., 2019; Rangel et al., 2020). As expected, 0.9% NaCl (negative control) did not cause any irritation event, and 1% SDS solution (positive control) caused hemorrhage on the CAM, with an IS of 12 ± 0 and was classified as a severe irritant. The 0.01% Fraction F solution did not induce any irritant events in CAM during the period of observation, and presented a IS of 0 ± 0, being classified as non-irritant. However, 0.01% Compound 6 was classified as a slight irritant due to hyperemia observed in CAM after application, presenting a IS of 1.5 ± 1.7. The results are shown in Fig 6 and Table 4.

Table 5. IS and classification of the effects of the 0.01% Fraction F and 0.01% Compound 6 from Arthrinium sp. in the HET-CAM assay. The 1% SDS and 0.9% NaCl solutions were used as positive and negative controls, respectively.

Sample	Irritation Score	Classification
1% SDS	12 ± 0	Severe Irritant
0.9% NaCl	0 ± 0	Non-irritant
0.01% Fraction F	0 ± 0	Non-irritant
0.01% Compound 6	1.5 ± 1.7	Slight Irritant

Photoprotection against UVA-induced ROS production

HaCat Antioxidant Activity

Although being considered cytotoxic in 3T3 NRU PT assay, probably due to the higher sensibility of the monolayer model of fibroblast cells (TAVARES et al., 2020), Fraction F maintained the cells viability around 100.03% ± 7.8 in the concentration of 10.0 µg/mL, and its isolated compound, agonodepside B (6), proved to be non-cytotoxic for HaCaT cells, with a viability around 97.2% ± 4.7 at the concentration of 10.0 µg/mL. The graph showing the results of the viability test and comparison with untreated cells and cytotoxic control are shown in Fig. 7.

After establishing the safe concentrations to use, the antioxidant potential of Fraction F and agonodepside B (6) in HaCaT keratinocytes was assessed by detecting UVA-induced intracellular ROS using DCFH₂-DA probe. Quercetin was used as an antioxidant control, resulting in a 71% ± 7.8 decrease in ROS generation, and norfloxacin as a pro-oxidant control, resulting in a 39.5% ± 24.5 increase in ROS production.

According to Fig. 8, Fraction F presented an antioxidant activity statistically equal to quercetin (p > 0.05) when tested at 10.0 µg/mL, with a reduction around 66.7% ± 14 in UVA-induced ROS production. On the other hand, lower concentrations of Fraction F, 2.5 and 5.0 µg/mL, did not protect against UVA-induced ROS production (p > 0.05) (3.22% ± 3.6 and 10.9% ± 20.9 fluorescence reduction, respectively). The isolated substance agonodepside B (6) did not present antioxidant activity in the lowest concentrations, 2.5 and 5.0 µg/mL, resulting in an increase of 6.6% ± 4.5 and 10% ± 2.9 in ROS generation, respectively. However, in a similar manner as Fraction F, at the highest concentration tested (10.0 µg/mL), it was able to protect from intracellular UVA-induced ROS production, with a decrease in the fluorescence of 50.4% ± 5.1, being statistically equal to the positive control quercetin (p > 0.05) and different from the untreated control (p < 0.05).

The antioxidant activity of the fraction F can be explained by the presence of phenols, polyphenols derivatives, such as those isolated and identified in our research (2-6). The isolated compound under study agonodepside B (6) is also a polyphenolic structure, which also justifies its antioxidant activity similar to quercetin. Phenolic compounds are capable of donating electrons or hydrogen atoms, thereby neutralizing ROS (Souza et al., 2019, Silva et al., 2022). A wide range of antioxidants from marine fungi have been reported, such as the already mentioned phenolic compounds, and also anthraquinones, xanthones, carotenoids, indole derivatives and alkaloids (Vitale et al., 2020). The use of natural fungal and algal products in cosmetic formulations could present advantages when compared to synthetic antioxidants, as they are derived from sustainable sources, maybe not harm the environment, and also being obtained from large-scale fermentation in case of the fungi growth (Vitale et al., 2020).

Reconstructed Human Skin Model (RHS) Antioxidant Activity

Reconstructed human skin models are composed of a differentiated epidermis, with a corneal stratification and a living dermis. Thus, the concentration of 10 µg/mL of compound agonodepside B (6) and Fraction F were tested to evaluate the protection against UVA-induced intracellular ROS production in the reconstructed human skin model in order to mimic topical application in a physiologically relevant model that consider skin bioavailability of the tested compounds (Roguet et al., 1994; Afaq et al., 2009; Lee et al., 2017).

According to Fig. 9, the results demonstrated that UVA radiation increased ROS generation in the untreated irradiated reconstructed human skin control (NT +UV) (100%) when compared to the untreated and non-irradiated control (NT -UV), while the vehicle (PBS and ethanol 2%) was not capable of reducing ROS generation, with a fluorescence around 85.7%. The Fraction F and compound (6) were able to protect viable epidermis against UVA-induced ROS production, with a reduction of 34.6% and 30.2% in the fluorescence, respectively, both when tested at 10.0 µg/mL. As mentioned, this antioxidant activity can be due to the presence of polyphenolic compounds capable of neutralizing free radicals and other ROS (Souza et al., 2019, Silva et al., 2022), which is in agreement with the protection observed in HaCaT keratinocytes.

The crude extract and fractions F and G from the Antarctic fungus Arthrinium sp. showed a good absorption in both UVA I and II and UVB range (280 - 400 nm). From this extract and fractions, six compounds were isolated and characterized, three of them not yet reported in Arthrinium species (1, 2 and 6). Among these compounds, agonodepside B (6) isolated from fraction E and F showed the best absorbance profile in the UVB-UVA range, and demonstrated to be photostable, non-cytotoxic to HaCaT cells and was classified as a slight irritant in HET-CAM assay; it also did not present any phototoxic potential according to OECD TG 432. Besides that, it was able to protect viable epidermis against UVA-induced ROS production, both in keratinocyte monolayers and in reconstructed human skin models, with a reduction of 30.2% in the fluorescence in 3D skin models. Fraction F presented higher absorption in both UVB (280-320 nm) and UVA I and II (320-400 nm) ranges than agonodepside B (6), which can be due to synergism of other compounds present in the fraction. This fraction was also considered photostable, non-cytotoxic to HaCaT cells, non-irritant in HET-CAM assay and not phototoxic. Furthermore, it was also able to protect viable epidermis against UVA-induced ROS production, both in keratinocyte monolayers (effect similar to quercetin) and in reconstructed human skin models. Thus fraction F of Antarctic fungus Arthrinium sp presents the best photoprotective potential and has the advantage that it does not need further purifications to obtain the proposed safety and efficacy, however other clinical studies should be performed to confirm its photoprotective and antioxidant potential.

Acknowledgements

The authors are thankful to University of São Paulo for providing access to necessary resources, the financial and fellowship support from the Brazilian research funding agencies Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de desenvolvimento Científico e Tecnológico (CNPq). The Department of Biomolecular Sciences and the Núcleo de Pesquisas em Produtos Naturais e Sintéticos – NPPNS are acknowledged.

Funding

This study had financial and logistic support from the Brazilian Antarctic Program PROANTAR/MCTI/CNPq N°64/2013), Brazilian Marine Force, National Institute of Science and Technology (INCT:BioNat), Grant #465637/2014-0, and the State of São Paulo Research Foundation (FAPESP), Grant #2014/50926-0 and 2017/03552-5.

Author contributions

Ana Carolina Jordão: Conceptualization, Methodology, Formal analysis, Investigation, Data Curation, Writing – Original Draft, Review and Editing, Visualization Gustavo Souza dos Santos, Thaiz Rodrigues Teixeira, Ana Júlia Pasuch Gluzezak, Clarissa Bechuate de Souza Azevedo, Ludmilla Tonani and Karina de Castro Pereira: Methodology, Formal analysis, Data Curation, Review and Editing Márcia Regina von Zeska Kress: Conceptualization,Resources, Supervision Lorena Rigo Gaspar: Conceptualization,Resources, Supervision, Review and Editing, Project Administration Pio Colepicolo: Resources, Project administration, Funding Acquisition Hosana Maria Debonsi: Conceptualization, Resources, Review and Editing,Supervision, Project administration, Funding acquisition.

Ethics Approval

All experimental procedures involving humans were performed in accordance with the principles of the Declaration of Helsinki and were approved by the Human Research Ethics Committee of School of Pharmaceutical Sciences of Ribeirão Preto, São Paulo, Brazil (CAAE number 31758619.5.0000.5403). The written informed consent was signed by all the donors or their parents or legal guardian.

Conflict of Interest

The authors declare no competing interests.

Availability of data and material

The raw data supporting the information of this article will be made available by the authors upon request.

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Assessment of the photoprotective potential and structural characterization of secondary metabolites of Antarctic fungus Arthrinium sp

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Abstract

Figures

Introduction

Material and Methods

Fungal Isolation

Fungal Identification

Fermentation and Extraction

Crude extract fractionation and compounds isolation

Structure Identification

Determination of the UV Absorption Spectra

Photostability Test

Toxicity Assessment

Phototoxicity assay (3T3 NRU PT)

Results and Discussion

Fungal Identification

Compound’s Identification and Characterization

Determination of the UV Absorption Spectra

Toxicity Assessment

Phototoxicity assay (3T3 NRU PT)

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1