Roussoelins A and B: two phenols with antioxidant capacity from ascidian-derived fungus Roussoella siamensis SYSU-MS4723

Ascidian-derived microorganisms are a significant source of pharmacologically active metabolites with interesting structural properties. When discovering bioactive molecules from ascidian-derived fungi, two new phenols, roussoelins A (1) and B (2), and ten known polyketides (3–12) were isolated from the ascidian-derived fungus Roussoella siamensis SYSU-MS4723. The planar structure of compounds 1 and 2 was established by analysis of HR-ESIMS and NMR data. The conformational analysis of the new compounds was assigned according to coupling constants and selective gradient NOESY experiments, and absolute configurations were completed by the modified Mosher’s method. Among the isolated compounds, 1, 2, and 9 showed moderate antioxidant capacity. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s42995-020-00066-8) contains supplementary material, which is available to authorized users.


Introduction
Marine organisms have been a significant natural source for the discovery of multiple pharmacologically active molecules with various structures (Blunt et al. 2017(Blunt et al. , 2018Carroll et al. 2019;Jiang et al. 2020;Liu et al. 2019). Among them, about 150 molecules with a wide range of bioactivities have been discovered from ascidian-derived microorganisms (Bugni and Ireland 2004;Chen et al. 2018;Donia et al. 2006). For instance, the lomaiviticins A and B with an intricate dimeric diazobenzofluorene glycoside structure and antitumor activity were discovered from ascidian-derived Actinomycetes Micromonospora lomaivitiensis (He et al. 2001). The ascidian-associated fungus Eurotiomycetes strain 110,162 produced an anti-mycobacterial oxazinin A that contained a unique dimeric structure (Lin et al. 2014b). Another ascidian-derived fungus Trichobotrys effuse 4729 yielded an anti-glioma trichobamide A that was a pyrrocidine alkaloid containing a novel tetrahydro-5H-furo [2,3-b] pyrrol-5-one moiety (Chen et al. 2019b).

Electronic supplementary material
Though 25 genera fungi of 19 families in two phyla have been derived from the ascidian, eight genera have been chemically investigated and the number of reports describing natural products from ascidian-derived fungi is still low. Recently, we focused on bioactive secondary metabolites from ascidian-derived fungi isolated from the South China Sea (Chen et al. 2019a, b;Niaz et al. 2019). As we continue to discover bioactive molecules from ascidian-derived fungi, two new 5-(3-hydroxybutan-2-yl) benzene-1,3-diol, roussoelins A (1) and B (2), together with ten known polyketides (3-12) were obtained from the ascidian-derived fungus Roussoella siamensis SYSU-MS4723 ( Fig. 1), whose secondary metabolites were studied for the first time from a genus of an ascidian-derived fungi. The conformational analysis was assigned according to coupling constants and selective gradient NOESY experiments, and absolute configurations were finally identified by a modified version of Mosher's method (Ohtani et al. 1991). The cytotoxicity, anti-inflammatory, and antioxidant activity of these molecules are reported herein.
The relative configuration of C-2 and C-3 in roussoelin A was established through selective NOESY correlations and coupling constants. A large coupling constant ( 3 J H-2,H-3 = 8.3 Hz) between protons H-2 and H-3 was observed, indicating they should be in an anti conformation (Chlipala et al. 2010;Matsumori et al. 1999). In the analysis of anti conformation of roussoelin A, only two of the six possible relative conformations (blue and red color) for C-2 and C-3 were satisfied with the coupling constant (Fig. 3). A 1D selective gradient NOESY experiment revealed that H 3 -1 and H 3 -10 do not have an NOE correlation (Supplementary Figs. S8, S9), indicating a relative configuration of 2S*,3S*. The absolute configuration of the secondary alcohol was resolved by a modified version of Mosher's method. The (R) and (S)-MTPA chloride reacted with 1, respectively, and esterification occurred at the C-2 hydroxy group to produce the corresponding (S)-MTPA ester (1a) and (R)-MTPA ester (1b). The chemical shifts for H-1, H-3, and H-10 of 1a and 1b were measured as δ H 1.18, 3.09, and 1.27 for 1a and δ H 1.20, 3.04, and 1.24 for 1b, respectively. The observed differences of chemical shifts (∆δ = δ S − δ R ) (Fig. 4) indicated that the C-2 absolute configuration is S. Hence, compound 1 was identified as shown in Fig. 1 and named as roussoelin A.
All isolated compounds were tested for their anti-inflammatory activity in vitro by inhibition of LPS-activated NO production in RAW264.7 cells with the Griess assay and their cytotoxicity using MCF-7 (breast cancer), HepG2 (liver cancer), and A549 (lung cancer) human cell lines. None of them showed inhibition activity or cytotoxicity at 50 μmol/L. Compounds 1-12 were also evaluated using the total antioxidant capacity assay kit with a rapid ABTS method. Only compounds 1, 2, and 9 showed moderate total antioxidant capacity (0.65 of 1; 0.61 of 2; 0.32 of 9) with Trolox as a positive control (Fig. 5). Phenolic compounds (including cinnamic acids, benzoic acids, flavonoids, proanthocyanidins, coumarins, stilbenes, lignans, and lignins) are the most widespread class of metabolites in nature (Pereira et al. 2009). The antioxidant capacity of phenolic compounds 1 and 2 should be attributed to their ability to chelate metal ions involved in the production of free radicals and suggests that chemical protection of symbiotic microbes are benefitial to ascidians screening UV or inhibiting enzymes involved in radical generation (Cos et al. 1998).

General experimental procedures
Optical rotations were measured on an MCP 200 polarimeter (Anton Paar, China). Infrared spectroscopy was performed on a Fourier transformation infrared spectrometer coupled with infrared microscope EQUINOX 55 (Bruker, Germany). 1D and 2D NMR data were measured on Bruker Avance 400 or 600 MHz spectrometers (Bruker, Germany) using tetramethylsilane (TMS) as the internal standard. Electrospray

Fungal material
In this study, the fungus SYSU-MS4723 was isolated from an ascidian Styela plicata, which was collected in the Mirs Bay (22°33′22.1′′N, 114°27′09.3′′E), Shenzhen, Guangdong Province, China, in April 2016. Purified fungus was isolated from ascidian on the base of the standard protocol (Kjer et al. 2010). The strain was identified to be R. siamensis SYSU-MS4723 on the base of morphological characteristics and the ITS region (Raja et al. 2017). The sequence data of the fungal strain have been submitted and deposited at Gen-Bank with accession no. MH465397. The voucher specimen was preserved on potato dextrose agar slants at 4 °C at the School of Marine Sciences, Sun Yat-Sen University.

Cytotoxic assay
All compounds were tested for cytotoxicity against MCF-7 (breast cancer), HepG2 (liver cancer), and A549 (lung cancer) human cancer cell lines. Human cancer cell lines were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The cytotoxicity assay was based on the MTT method according to previously reported procedures (Chen et al. 2016).

Anti-inflammatory assay
All compounds were tested for their anti-inflammatory activity on the basis of previously reported procedures ).

Total antioxidant capacity assay
Total antioxidant capacity assay kit with a rapid ABTS method (Beyotime Institute of Biotechnology, China) was used to evaluate the total antioxidant capacity based on the manufacturer's instructions. Samples were incubated at 25 °C for 6 min and then were recorded at 414 nm using a multimode reader (Thermo Fisher Scientific, USA).
Author contributions SC and LL conceived and designed the experiments; SC and HS performed the experiments; YD, HG, MJ, ZW, HY participated in the experimental process and result discussion. SC analyzed the data and wrote the paper.

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
Animal and human rights statement This article does not contain any studies with human participants or animals performed by the authors.
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