Meroterpenoids and Steroids from the Marine-Derived Fungus Trametes sp. ZYX-Z-16

Marine fungi can metabolize structurally diverse active components, and have become an important source of drug lead molecules. In the present study, the chemical investigation on the EtOAc extract of the fermentation broth of the marine-derived fungus Trametes sp. ZYX-Z-16 led to the isolation of eight meroterpenoids (1–8), including two undescribed ones, together with ten ergostane steroid analogues (9–18). The structures of two new spiromeroterpenoids, asnovolin H (1) and asnovolin I (2), were determined based on 1D, 2D NMR, and HRESIMS spectroscopic data along with ECD spectra calculations. All compounds were tested for antibacterial and α-glucosidase inhibitory activity. Among them, compound 12 showed definite antibacterial activities against Staphylococcus aureus ATCC 6538 (MIC 32 μg/mL) and Bacillus subtilis ATCC 6633 (MIC 16 μg/mL). In addition, compounds 9 and 10 showed superior inhibitory activity, with IC50 values of 104.1 and 111.3 μM, respectively, to the positive control acarbose (304.6 μM).


Introduction
The ocean has a unique and complex ecological environment with low temperature, high temperature, high pressure, high salinity, no light, and low oxygen. The rapidly increasing number of marine natural products (MNPs) with structural novelty and high biological activities each year has greatly stimulated the interest of researchers [1,2]. In recent years, the metabolites of marine microorganisms have become one of the research hotspots of scholars at home and abroad [3]. Marine fungi form different metabolic pathways and adaptation mechanisms in the peculiar marine environment and can produce natural secondary metabolites with unique chemical structures [4]. A great deal of work on mining the active secondary metabolites from marine-derived fungi has been performed. The main types of these metabolites contained alkaloids [5,6], anthraquinones [7], terpenes [8][9][10], polyketones [11], macrolides [12], isocoumarins [13], and sterols [14]. These different types of compounds show diverse biological activities, including antibacterial [15], antifungal [16], antioxidant [17], cytotoxic [8,18], anti-inflammatory [19], enzyme inhibition [20], antituberculosis [21], neuroprotection [22], and antiproliferative [23] activities.
The genus Trametes of fungi is distinguished by a white aerial mycelia. In the present day, researchers have mainly focused on the biodegradable enzymes [24,25] and polysaccharides with cytotoxic activity [26,27] in the macrofungi species of genus Trametes such as T. versicolor and T. robiniophila. The active secondary metabolites from the microfungi species of genus Trametes relatively lack studies. In the course of our ongoing search for structurally polysaccharides with cytotoxic activity [26,27] in the macrofungi species of genus Tra such as T. versicolor and T. robiniophila. The active secondary metabolites from the m fungi species of genus Trametes relatively lack studies. In the course of our ongoing s for structurally new and biologically active metabolites from marine-derived fung fungus Trametes sp. ZYX-Z-16, isolated from conch snails in Silver Island, Xisha, S Sea, China, and presenting complex metabolite profiles revealed by high-pressure l chromatogram, attracted our attention. Subsequent chemical investigation on the E extract of the fermentation broth of this fungus led to the identification of eight merot noids (1)(2)(3)(4)(5)(6)(7)(8), including two new spiromeroterpenoids, asnovolin H (1) and asnovolin together with ten ergostane steroid analogues (9)(10)(11)(12)(13)(14)(15)(16)(17)(18) (Figure 1). Herein, the isol structural elucidation, and bioassays, including antibacterial, antifungal, and α-g sidase inhibitory activities, of these compounds are described.

Structural Elucidation of Compounds
Compound 1 was isolated as a yellow powder, and the molecular formula wa termined as C26H38O7 on the basis of HRESIMS ion peak at m/z [M + Na] + 485.2528 ( 485.2510 for C26H38NaO7) and 13 C NMR data, suggesting eight degrees of unsatur The IR spectrum revealed the presence of hydroxyl group (3475 cm -1 ), double bond cm -1 ), ester carbonyls (1686 cm -1 ), and ketone carbonyl (1735 cm -1 (Table 1) spectra presented 26 carbon signals for eight methyls, four methylenes, five methines (one oxygenated at δ C 69.6), and nine nonprotonated carbons (two olefinic at δ C 183.4 and 110.2, two oxygenated, one ketone at δ C 195.8, and two ester carbonyls at δ C 177.9, 173.3). Three carbonyls and one double bond account for four degrees of unsaturation, indicating that four rings (A-D) are presented in compound 1.

Antifungal Activity
The inhibitory activities of all compounds against five phytopathogenic fungi (Fusarium oxysporum f. sp. cubense, Fusarium spp., Peronophythora litchii, Colletotrichum gloeosporioides, and Hylocereus undatus) were evaluated using the broth microdilution method. Unfortunately, none of the compounds showed definite inhibitory activity.
Meroterpenoids are the main characteristic class of natural products generated by this fungus. Those spiromeroterpenoids with a spiro carbon at (C-9) through an oxygen atom were reported to show bioactivities of suppression of fibronectin expression [28] and antibacterial activity [29]. The other type of meroterpenoids with 6/6/6/6/6 pentacyclic ring framework showed various bioactivities, including insecticidal activity [30,44], lipid droplet formation inhibition [45], protein tyrosine phosphatase 1B inhibitory activity [46], and antiviral activity [42,47]. From the above bioassays of all the isolated meroterpenoids, only compound 5 showed definite inhibitory activity against Staphylococcus aureus ATCC6538. None of the meroterpenoids with 6/6/6/6/6 pentacyclic ring framework had good biological activity in the present experiment.

Fungal Material
The fungus strain Trametes sp. ZYX-Z-16 with white mycelium was isolated from an unidentified sea snail collected from Silver Island, Xisha, South Sea, China, in May 2021. After grinding, the sample (1 g) was diluted to 10 −2 g/mL with sterile H 2 O, 100 µL of which was spread on a PDA medium plate containing chloramphenicol as bacterial inhibitor. A single colony was identified according to its morphological characteristics and ITS4 gene sequences (GenBank accession no. ON386187, see Supplementary Materials). A reference culture of Trametes sp. ZYX-Z-16 was deposited in our laboratory and maintained at −80 • C.

Fermentation, Extraction, and Isolation
The marine fungus Trametes sp. ZYX-Z-16 was cultured in the medium which contained 20 g/L malt, 20 g/L Mannitol, 10 g/L glucose, sodium glutamate 10 g, 3 g/L yeast extract, 1 g/L corn steep liquor, 0.5 g/L KH 2 PO 4 , 0.3 g/L MgSO 4 ·7H 2 O, 10 g/L sea salt, and 1 L H 2 O at pH 6.5. Fungal mycelia were cut and transferred aseptically to 1 L Erlenmeyer flasks, each adding 300 mL of sterilized liquid medium. The flasks were incubated at room temperature (about 27 • C~34 • C) for 30 days.
The whole culture broth (50 L) was harvested and filtered to yield the mycelium cake and liquid broth. The mycelium cake was extracted by tissue crusher using EtOAc for three times. The EtOAc solution was evaporated under reduced pressure. A total of 135 g EtOAc extract was obtained. The extract was extracted between petroleum ether and 90% methanol (1:1) to remove the oil. The secondary metabolites extract (38 g) was subjected to a silica gel VLC column, eluting with a stepwise gradient of petroleum ether-EtOAc (10:1,

Bioassay of Enzyme Inhibition, Antibacterial, and Antifungal Activity
All the compounds were evaluated for their inhibitory effects against α-glucosidase using p-NPG as the substrate, referring to the previous method [48] with acarbose and genistein as the positive control, as well as against acetylcholinesterase using AICI as the substrate, referring to the previous method [49] with Tacrine as the positive control.
Four bacteria (Staphylococcus aureus ATCC6538, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, and Listeria monocytogenes ATCC 1911) were used for antibacterial evaluation for all compounds, referring to the previous method [50] with ampicillin as a positive control. The bacteria inhibitory activity of all compounds was observed after 12 h in a constant temperature incubator at 37 • C. Five phytopathogens (Fusarium oxysporum f. sp. cubense, Fusarium spp., Peronophythora litchii, Colletotrichum gloeosporioides, and Hylocereus undatus) were used for antifungal evaluation for all compounds, referring to the previous broth microdilution method [51] with carbendazim as a positive control. The fungal inhibitory activity of all compounds was observed after 48 h in a constant temperature incubator at 28 • C. The MIC value was defined as the lowest concentration of the test compound at which the microorganism did not demonstrate visible growth. Each test was performed in triplicate.

Computation Section
Conformational search was performed using the iMTD-GC method imbedded in Crest program [52]. Density functional theory calculations were performed using the Gaussian 16 package [53]. The conformers within 5 kcal/mol were optimized at B3LYP/6-31G(d) in gas phase and the conformers with population over 1% were kept. Then, these conformers were further reoptimized at B3LYP/6-311G(d) with IEFPCM solvent model, and frequency analyses of all optimized conformers were also performed at the same level of theory to exclude imaginary frequencies. NMR shielding tensors were calculated with the GIAO method [54] at mPW1PW91/6-31+G(d,p) level with IEFPCM solvent model in methanol. The shielding constants were converted into chemical shifts by referencing to TMS at 0 ppm (δcal = σTMSσcal), where the σTMS (the shielding constant of TMS) was calculated at the same level. For each candidate, the parameters a and b of the linear regression δcal = aδexp + b; the correlation coefficient, R 2 ; the mean absolute error (MAE), defined as Σn |δcal -δexp|/n; and the corrected mean absolute error, CMAE, defined as Σn |δcorr-δexp|/n, where δcorr = (δcal -b)/a, were calculated. DP4+ probability analysis was performed using the shielding tensors [55]. ECD spectra were calculated by the TDDFT methodology at the B3LYP/TZVP, utilizing IEFPCM in methanol. ECD spectra were simulated using SpecDis 1.71 [56].

Conclusions
The metabolites from marine fungi have extensive biological and pharmacological activities and have been an important source of drug lead molecules. The chemical investigation of the marine fungus Trametes sp. ZYX-Z-16 led to the isolation of eight meroter-penoids (1-8), including two undescribed spiro-ones, asnovolins H (1) and I (2), together with eight ergostane steroids (9)(10)(11)(12)(13)(14)(15)(16)(17)(18). The bioassay of enzyme inhibition assay showed that two ergostane steroids (9 and 10) revealed definite inhibition against α-glucosidase with IC 50 values of 104.1 µM and 111.3 µM, respectively. The result of antibacterial assay showed that an ergostane steroid (12) has obvious inhibitory effect on Staphylococcus aureus ATCC6538 (MIC 32 µg/mL) and Bacillus subtilis ATCC6633 (16 µg/mL). This study further deepens the understanding of the structural diversity of meroterpenoids of marine fungi, enriches the marine natural product database, and provides theoretical information for the subsequent utilization and development of marine natural product resources.