Talasteroid, a new withanolide from the marine-derived fungus Talaromyces stollii

Abstract A new withanolide, talasteroid (1), and a known steroid (2), along with eight meroterpenoids (3–10), were obtained from the rice culture of the marine-derived fungus Talaromyces stollii HBU-115. The structure of 1 including its absolute configuration was determined by extensive 1 D and 2 D NMR spectroscopy, and single-crystal X-ray diffraction analysis. Compound 1 represents the first withanolide featuring a 4-substituted 2,3-dimethyl-2-butenolide ring in the side chain. The isolated compounds were evaluated for their antimicrobial and antioxidant activities. Graphic Abstract


Introduction
Steroids are one of the most abundant and structurally unique marine natural products, which could be isolated from marine-derived fungi, sponges, corals, and other marine organisms (Carroll et al. 2020, Carroll et al. 2021. Steroids are a class of compounds containing many types, such as highly oxygenated steroids, spirosteroids, secosteroids, epoxy-and peroxy-steroids, steroid glycosides (Ermolenko et al. 2020). Withanolides belong to the group of naturally occurring steroidal lactones, which contain a C-22/C-26 d-lactone or a C-23/C26 c-lactone in the side chain (Chao et al. 2011). Studies of steroidal metabolites have shown that many steroids with unusual ring systems exhibited antibacterial, antioxidant, antiviral, antitumor, anti-inflammatory and neuroprotective activities (Zubair et al. 2016, Dembitsky et al. 2018, Du et al. 2020. For instance, penijanthoids A and B with a five-membered lactone in the side chain showed significant anti-Vibrio activity, which were isolated from the marine-derived fungus Penicillium janthinellum (Guo et al. 2019). Steroids serve as an important source of drug lead compounds, arousing great interest among chemists.

Results and discussion
Talasteroid (1) was obtained as white crystals. Its molecular formula of C 28 H 40 O 3 was assigned based on the m/z 447.2865 [M þ Na] þ ion in the positive HRESIMS, indicating a hydrogen deficiency index of nine. The IR spectrum indicated the presence of a,b-unsaturated-c-lactone (1744 cm À1 ), carbonyl (1720 cm À1 ), and olefinic bonds (1684 cm À1 ) ( Figure S6). The 1 H and 13 C NMR spectroscopic data of 1 (Table S1) showed 28 carbon signals including a 4-substituted 2,3-dimethyl-2-butenolide ring (d H 1.81, 1.93, 4.78; d C 8.6, 12.1, 81.8, 123.1, 160.3, and 174.9), a trisubstituted double bond (d H 5.19; d C 117.5, 139.3), a carbonyl group (d C 212.0), two methyl singlets, one methyl doublets, and nine methylene groups. The spectroscopic data above revealed 1 had the same core with the known compound ergosta-7,22-dien-3b-ol, which was isolated from fungus Fusarium chlamydosporum (Al-Rabia et al. 2021). Except for the carbonyl group at C-3, and the different side chain with the presence of a lactone moiety skeleton. This assumption was verified by a further spin systems H-17 ( 3) and C-26 (d C 174.9). Detailed analysis of other signals on the HSQC, 1 H-1 H COSY, and HMBC spectra ( Figure S1) allowed the assignment for all carbon and proton resonances of 1. Thus, the planar structure of 1 was established.
In the NOESY spectrum, the correlations between H-5 and H-9, H-9 and H-14, and H-14 and H-17 suggested that all of these protons had the same a-orientation. Additionally, NOESY correlations between H 3 -19 and H-11a, H-11a and H 3 -18, H 3 -18 and H-20, and H-11b and H-14 indicated that these protons had a b-orientation ( Figure S2).
Crystals of 1 were deposited from a mixture of dichloromethane and methanol, and X-ray analysis of the crystal by CuKa radiation revealed that the absolute configurations of 1 were assigned as 5S,9S,10S,13R,14S,17R,20R,23R ( Figure S3).
To verify whether the lactone structure in 1 was natural origin, the fungus T. stollii was re-fermented under the same fermentation conditions. Four solvents including methanol, ethyl acetate, acetone, and dichloromethane were used for the extraction, respectively. All extracts were analyzed by HPLC. It was found that 1 existed in the four extracts, which indicated that 1 with a lactone was likely to be a natural product.

General experimental procedures
Optical rotation was performed in CHCl 3 on a JASCO P-2000 spectrometer. ECD and UV spectra were acquired with the sample dissolved in MeOH by a MOS-450/SFM300 and a Shimadzu UV-3600 spectrophotometers, respectively. IR spectra were measured using KBr pellets on a FTIR-8400 spectrometer. NMR data were recorded on a Bruker AV-600 spectrometer using TMS as the internal standard. The crystallographic data was acquired from a Bruker D8 ADVANCE diffractometer (Cu Ka radiation). HRESIMS spectra were obtained from a spectrometer of Bruker apex-ultra 7.0 T. HPLC separation was performed with a normal phase Viridis TM Silica 2-Ethylpyridine HPLC column (Waters, 10 Â 250 mm, 5 lm), and a C 18 HPLC column (Waters, 10 Â 250 mm, 5 lm) on the Shimadzu LC-20AT system containing a SPD-M20A photodiode array detector.

Fungal material
The fungal strain Talaromyces stollii HBU-115 was isolated from the Bohai Sea (Huanghua, Hebei Province, China, June 2016). On PDA medium, the surface of the colony of this fungus is rough, with the regular circle border and raised middle. While the back of this fungus is yellow and flat in the middle. The fungal strain was deposited in the College of Pharmaceutical Sciences, Hebei University, China, with a Genbank accession number MH389070.

X-Ray crystallography
Crystal data for (1)

Antioxidant assay
The antioxidant activity was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay. (Sharma and Bhat 2009). Compounds 1-10 and ascorbic acid were dissolved in dimethylsulfoxide (DMSO) at the concentration of 1.0, 0.5, 0.2, 0.1 and 0.05 mg/mL, respectively. DPPH was dissolved in ethanol with the concentration of 0.2 mM. Tested samples (100 lL) were added to 100 lL of DPPH, then incubated for 30 min in darkness at room temperature. Finally, the absorbance was tested by a spectrophotometer at 517 nm. Ascorbic acid, DMSO and ethanol were used as positive, negative and blank controls. The DPPH radical scavenging rate (%) was calculated using the equation: DPPH scavenging activity(%) ¼ [(Abs negative control -Abs compound þ Abs blank control)/Abs negative control] x100%

Conclusion
In this study, two steroids and eight meroterpenoids were isolated from the rice solid cultures of marine-derived fungus T. stollii HBU-115 for the first time. Among which, talasteroid (1) was a new withanolide with a 4-substituted 2,3-dimethyl-2-butenolide ring in the side chain. Since the isolated compounds showed weak antioxidant activity, further investigation on the bioactivity of these compounds need to be undertaken in future. Our findings would enrich chemical diversity of T. stollii.